Methods and compositions for preparing genetically engineered cells

Abstract

Methods for preparing T cells for cell therapy, compositions produced by said methods, and methods for administering said cells to a subject are provided. Specifically, this disclosure relates to the preparation of engineered T cells, such as those expressing genetically engineered receptors, such as genetically engineered antigen receptors, like engineered (recombinant) TCRs and chimeric antigen receptors (CARs), or other recombinant chimeric receptors. The methods are characterized by producing more consistent and / or predictable T cell products and / or lower toxicity compared to other methods. The provided methods include incubating cells under stimulating conditions to induce the expansion or proliferation of naive T cells in the stimulated composition compared to non-naive T cells, which in turn can lead to preferential transduction of cells derived from said naive T cells. The methods may also be characterized by reduced cost, fewer steps, and reduced resource consumption compared to other methods.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 543,359, filed August 9, 2017, entitled “Methods and Compositions for Preparing Genetically Engineered Cells,” the contents of which are incorporated herein by reference in their entirety.

[0003] By referencing and incorporating into the sequence list

[0004] This application is submitted together with an electronic sequence list. The sequence list is provided as a file titled 735042010540SEQLIST.txt, created on July 11, 2018, and is 35,434 bytes in size. Information from the electronic sequence list is incorporated herein by reference in its entirety. Technical Field

[0005] This disclosure relates to methods for preparing T cells for cell therapy, compositions produced by said methods, and methods for administering said cells to a subject. Specifically, this disclosure relates to the preparation of engineered T cells, such as those expressing genetically engineered receptors, such as genetically engineered antigen receptors like engineered (recombinant) TCRs and chimeric antigen receptors (CARs), or other recombinant chimeric receptors. The methods are characterized by producing more consistent and / or predictable T cell products and / or lower toxicity compared to other methods. The provided methods include incubating cells under stimulating conditions to induce the expansion or proliferation of naive T cells in a stimulated composition compared to non-naive T cells, which in turn can lead to preferential transduction of cells derived from said naive T cells. The methods may also be characterized by reduced cost, fewer steps, and reduced resource consumption compared to other methods. Background Technology

[0006] Various methods are available for preparing and administering cells for therapeutic purposes. For example, methods exist for preparing cells (including T cells) for engineered and cell therapies, including methods involving depletion or enrichment of certain subsets. Improved methods are needed, such as to reduce toxicity associated with certain adoptive cell therapy administrations, to improve manufacturing processes, to allow for improved administration, and / or to reduce costs or other resources. Methods, cells, compositions, kits, and systems are provided to meet these needs. Summary of the Invention

[0007] This document provides a method for genetically engineered T cells, the method comprising incubating an input composition for 2 to 6 days under stimulation conditions, the input composition comprising a T cell population containing naive-like T cells and non-naive-like T cells, wherein the stimulation conditions comprise a stimulating agent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules, thereby producing a stimulated composition; and introducing a nucleic acid encoding a genetically engineered recombinant receptor into the stimulated T cell composition, wherein the introduction is performed during at least a portion of the incubation.

[0008] This document provides a method for genetically engineered T cells, the method comprising incubating an input composition under stimulation conditions for 2 to 6 days, the input composition comprising a T cell population containing naive-like T cells and non-naive-like T cells, wherein the stimulation conditions include the presence of a stimulating agent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules, thereby producing a stimulated composition; and the incubation of the input composition under stimulation conditions is performed before, during, and / or after the introduction of a nucleic acid encoding a genetically engineered recombinant receptor. In some embodiments, the incubation is performed for at least 3 days. In some cases, the incubation is performed for at least 4 days. In some embodiments, the incubation is performed for at least 5 days. In some embodiments, the incubation is performed for at least 6 days.

[0009] This document provides a method for stimulating T cells, comprising (a) incubating an input composition containing T cells comprising a subset of naive-like T cells or CD8+ T cells of the same type under stimulating conditions, thereby producing a stimulated composition; and (b) introducing a nucleic acid encoding a genetically engineered recombinant receptor into the stimulated cell composition, wherein the method thereby produces an output composition containing T cells expressing the genetically engineered recombinant receptor. In some embodiments, the T cells comprise naive-like T cells and non-naive-like T cells, wherein the stimulating conditions preferentially induce the expansion or proliferation of the naive-like T cells in the stimulated composition compared to the non-naive-like T cells. In some embodiments, the introduction is performed during at least a portion of the incubation or after the incubation.

[0010] In some embodiments, the initial culture volume of naive-like T cells or a subset thereof of CD8+ T cells is from or from about 0.1 x 10⁻⁶ cells. 8 Up to 5x10 8 One, from or from about 0.1 x 10 8 Up to 4x10 8One, from or from about 0.1 x 10 8 Up to 2x10 8 One, from or from about 0.1 x 10 8 Up to 1x10 8 One, from or from about 1x10 8 Up to 5x10 8 One, from or from about 1x10 8 Up to 4x10 8 One, from or from about 1x10 8 Up to 2x10 8 One, from or from about 2 x 10 8 Up to 5x10 8 One, from or from about 2 x 10 8 Up to 4x10 8 The naïve T cells or a subset of their CD8+ T cells are used as the initial culture volume. In some cases, the initial culture volume of naïve T cells or a subset of their CD8+ T cells is at least or at least about or about 0.5 x 10⁻⁶ cells. 8 0.75x10 8 1 x 10 8 1.5x10 8 1, 2x10 8 One or 4x10 8 The naïve T cells or a subset of their CD8+ T cells are described. In some cases, the initial culture volume of the naïve T cells or a subset of their CD8+ T cells is at least or at least about 2 x 10⁻⁶ cells. 8 The naive-like T cells or a subset of their CD8+ T cells.

[0011] A method for stimulating T cells is provided, the method comprising incubating an input composition containing T cells under stimulation conditions, the T cells containing a culture-initiating amount of naive-like T cells or a subset thereof of CD8+ T cells, i.e., from or from about 1 x 10⁻⁶ T cells. 8 Up to 4x10 8 A subset of naive T cells or CD8+ T cells is used to generate a stimulated composition. In some aspects, the T cells comprise naive T cells and non-naive T cells, wherein the stimulation condition preferentially induces the expansion or proliferation of the naive T cells in the stimulated composition compared to the non-naive T cells.

[0012] In some embodiments, the initial culture volume of naive-like T cells or a subset thereof of CD8+ T cells is at least or at least about or about 2 x 10⁻⁶ cells. 8 The naive-like T cells or a subset thereof. In some respects, the initial culture amount is the amount of naive-like CD8+ T cells.

[0013] In some of these embodiments, the naive T cells or naive CD8+ T cells are surface-positive for T cell activation markers selected from CD45RA, CD27, CD28, and CCR7; and / or surface-negative for markers selected from CD25, CD45RO, CD56, CD62L, and KLRG1; and / or have low expression of CD95; and / or are negative for intracellular expression of cytokines selected from IL-2, IFN-γ, IL-4, and IL-10. In some embodiments, the naive cells or naive CD8+ cells are surface-positive for T cell activation markers selected from CD45RA, CD27, CD28, and CCR7; and / or surface-negative for markers selected from CD45RO, CD56, and KLRG1; and / or have low expression of CD95. In some embodiments, the naive T cells or the naive CD8+ cells are CD45RA+, CD27+, CCR7+, CD62- and / or CD45RO-.

[0014] In some of these embodiments, the non-naive T cells are surface-negative to T cell activation markers selected from CD45RA, CD27, CD28, and CCR7; and / or surface-positive to markers selected from CD25, CD45RO, CD56, CD62L, KLRG1, and perforin; and / or positive for intracellular expression of cytokines selected from IL-2, IFN-γ, IL-4, and IL-10; and / or have high expression of CD95. In some aspects, the non-naive T cells are CD45RA-, CD27-, CCR7-, CD62+, and / or CD45RO+.

[0015] In some embodiments, the cells of the input composition have not undergone and have not experienced a selection step based on endogenous T cell surface markers prior to the incubation, which distinguish between naive and non-naive T cells.

[0016] In some embodiments, the method further includes introducing a genetically engineered recombinant receptor into the stimulated cells, wherein the method thereby produces an output composition comprising T cells expressing the genetically engineered recombinant receptor. In some cases, incubation of the composition under stimulation conditions is performed before, during, and / or after the introduction of the nucleic acid encoding the genetically engineered recombinant receptor.

[0017] In some embodiments, the recombinant receptor is capable of binding to a target antigen that is associated with, specific to, and / or expressed on cells or tissues of 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 cases, the target antigen is a tumor antigen. In some embodiments, the target antigen is selected from 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), and carcinoembryonic antigen (CEA). Hepatitis B surface antigen (CEA), 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), liver glycoside B2, liver glycoside receptor A2 (EPHa2), erb-B2, erb-B3, erb-B4, erbB dimer, type III epidermal growth factor receptor mutant (EGFR vIII), folate-binding protein (FBP), Fc receptor-like protein 5 (FCRL5);Also known as 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, phosphatidylinositol proteoglycan-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, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor α (IL-22R- α), IL-13R-α2 (IL-13Rα2), kinase insertion domain receptor (kdr), κ light chain, Lewis Y, L1 cell adhesion molecule (L1-CAM), L1-CAM CE7 epitope, protein family A containing leucine-rich repeat sequences (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 cell family 2 member D (NKG2D) ligand, melanin A (MART-1), neural cell adhesion molecule (NCAM), oncoemulsification antigen (oncofetal) Antigens, including 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), survival protein, trophoblast glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-associated protein 1 (TRP1, also known as TYRP1 or gp75), tyrosinase-associated protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta isomerase, or DCT), folate receptor-α, 8H9, dual antigens, 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 labels.

[0018] In some embodiments, the recombinant receptor is or contains a functional non-TCR antigen receptor or a TCR or its antigen-binding fragment. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR).

[0019] In some such embodiments, the recombinant receptor comprises an extracellular domain containing an antigen-binding domain, optionally wherein the antigen-binding domain specifically binds to the target antigen. In some cases, the antigen-binding domain is or contains an antibody or an antibody fragment thereof, the antibody fragment optionally being a single-chain fragment. In some aspects, the fragment contains an antibody variable region linked by a flexible linker. In some cases, the fragment contains scFv.

[0020] In some embodiments, the recombinant receptor further comprises a spacer and / or a hinge region. In some aspects, the recombinant receptor comprises an intracellular signaling region. In some examples, the intracellular signaling region comprises an intracellular signaling domain. In some aspects, the intracellular signaling domain is or contains a primary signaling domain, a signaling domain capable of inducing primary activation signals in T cells, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immune receptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain is or contains an intracellular signaling domain or a portion thereof comprising a CD3 chain (optionally a CD3-zeta (CD3ζ) chain).

[0021] In some embodiments, the recombinant receptor further includes a transmembrane domain disposed between the extracellular domain and the intracellular signaling region. In some aspects, the intracellular signaling region further includes a co-stimulatory signaling region. In some embodiments, the co-stimulatory signaling region contains an intracellular signaling domain of a T-cell co-stimulatory molecule or a signaling portion thereof. In some cases, the co-stimulatory signaling region contains an 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 optionally containing 4-1BB, and a cytoplasmic signaling domain derived from an ITAM-containing primary signaling molecule optionally containing a CD3ζ signaling domain, and optionally further comprises a spacer between the transmembrane domain and the scFv; the CAR sequentially comprises an antigen-specific scFv, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule optionally containing 4-1BB signaling domain, and a cytoplasmic signaling domain derived from an ITAM-containing primary signaling molecule optionally containing a CD3ζ signaling domain; or the CAR sequentially comprises an antigen-specific scFv, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule optionally containing a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from an ITAM-containing primary signaling molecule optionally containing a CD3ζ signaling domain.

[0022] In some implementations, the co-stimulatory signal transduction region is located between the transmembrane domain and the intracellular signal transduction region.

[0023] In some such embodiments, the stimulation condition includes incubation with a stimulating agent capable of activating T cells, CD4+ T cells, and / or CD8+ T cells; inducing signaling via the TCR complex; and / or inducing proliferation of T cells, CD4+ T cells, and / or CD8+ T cells. In some embodiments, the stimulation condition includes incubation with a stimulating agent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules. In some cases, the stimulating agent contains a primary agent that specifically binds to a member of the TCR complex, optionally specifically to CD3. In some embodiments, the primary agent is an antibody or antigen-binding fragment.

[0024] In some examples, the stimulant further includes a secondary agent that specifically binds to a T-cell co-stimulatory molecule, optionally wherein the co-stimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS. In some embodiments, the primary agent is an antibody or an antigen-binding fragment. In some embodiments, the primary and secondary agents comprise antibodies, optionally wherein one or more of the stimulants are incubated with anti-CD3 and anti-CD28 antibodies.

[0025] In some embodiments, the primary and / or secondary components are present on the surface of a solid support. In some cases, the solid support is or contains beads. In some embodiments, the diameter of the beads is greater than or greater than about 3.5 μm, but not greater than about 9 μm, or not greater than about 8 μm, or not greater than about 7 μm, or not greater than about 6 μm, or not greater than about 5 μm. In some examples, the diameter of the beads is or is about 4.5 μm. In some aspects, the diameter of the beads is the same as or approximately the same as the size of lymphocytes or antigen-presenting cells.

[0026] In some embodiments, the beads are inert. In some cases, the beads are or contain a polystyrene surface and optionally contain a magnetic or superparamagnetic core.

[0027] In some embodiments, the stimulation conditions include incubating the cells at a specific bead-to-cell ratio, said ratio being from or from about 1:1 to 10:1, from or from about 1:1 to 8:1, from or from about 1:1 to 6:1, from or from about 1:1 to 4:1, from or from about 1:1 to 3:1, from or from about 2:1 to 4:1, from or from about 2:1 to 3:1, from or from about 1:1 to 2:1 The ratio of beads to cells is from about 4:1 to 10:1, from about 4:1 to 8:1, from about 4:1 to 6:1, from about 6:1 to 10:1, from about 6:1 to 8:1, from about 8:1 to 10:1, from about 1:1 to 1:10, from about 1:1 to 1:8, from about 1:1 to 1:6, from about 1:1 to 1:4, from about 1:2 to 1:3. In some examples, the ratio of beads to cells is from about 3:1. In some embodiments, the ratio of beads to cells is from about 1:1.

[0028] This document provides a method for genetically engineered T cells, comprising incubating an input composition for 2 to 6 days under stimulation conditions, the input composition comprising a T cell population containing naive-like T cells and non-naive T cells, wherein the stimulation conditions comprise a stimulation agent comprising an anti-CD3 antibody and a secondary agent attached to beads as an anti-CD28 antibody, wherein the bead-to-cell ratio during the incubation period is from or from about 1:1 to 4:1; and introducing a nucleic acid encoding a genetically engineered recombinant receptor into the stimulated T cell composition, wherein the introduction is performed during at least a portion of the incubation period.

[0029] This document provides a method for genetically engineered T cells, the method comprising incubating an input composition for 2 to 6 days under stimulation conditions, the input composition comprising a T cell population containing naive-like T cells and non-naive T cells, wherein the stimulation conditions comprise a stimulation agent comprising an anti-CD3 antibody and a secondary agent attached to beads as an anti-CD28 antibody, wherein the bead-to-cell ratio during incubation is from or from about 1:1 to 4:1; and the incubation of the input composition under stimulation conditions is performed before, during, and / or after the introduction of a nucleic acid encoding a genetically engineered recombinant receptor.

[0030] In some of these implementations, the T cells are derived from a biological sample, optionally wherein the biological sample is derived from a human subject. In some cases, the biological sample is or contains whole blood, erythrocyte sedimentation rate (ESR) amber layer, peripheral blood mononuclear cell (PBMC) samples, ungraded T cell samples, lymphocyte samples, leukocyte samples, apheresis products, or leukoablation products.

[0031] 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 between approximately 2:1 and approximately 1:5. In some cases, the ratio of CD4+ cells to CD8+ cells is approximately 1:1, 1:2, 2:1, 1:3, or 3:1. In some embodiments, the naive-like T cells comprise naive-like CD4+ T cells and / or naive-like CD8+ T cells.

[0032] In some of these embodiments, the naïve T cells are polyclonal. In some cases, the clonality of the naïve T cells is determined by clonal sequencing, optionally next-generation sequencing, or prototyping analysis.

[0033] In some implementations, the presence, quantity, number, or percentage of naive T cells are detected by flow cytometry.

[0034] In some of these embodiments, the stimulation condition does not include N-acetylcysteine ​​(NAC). In some embodiments, the stimulation condition does not contain IL-15 and / or IL-7. In some embodiments, the stimulation condition causes or induces the death of the non-naive T cells or a subset thereof. In some aspects, the stimulation condition causes activation-induced cell death (AICD) of non-naive T cells or a subset thereof.

[0035] In some embodiments, the method further includes adding a DNA enzyme during the incubation and / or to the stimulated composition.

[0036] In some embodiments, the incubation is performed for 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 the naive T cells in the stimulated composition is increased by more than or greater than about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 50 times, or 100 times compared to the percentage of naive T cells in the input composition. In some embodiments, the ratio of cells derived from the naive T cells to cells derived from the non-naive T cells in the stimulated composition is increased by more than or greater than about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 50 times, or 100 times compared to the ratio of naive T cells to non-naive T cells in the input composition.

[0037] In some embodiments, the stimulated composition contains greater than 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of naive T-like cells derived from the input composition. In some embodiments, the stimulated composition contains less than 10% of cells derived from the non-naive T-like 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 the non-naive T-like cells.

[0038] In some embodiments, a greater percentage of the naive-like T cells in the input composition are induced to proliferate and / or activated compared to the non-naive-like T cells. In some aspects, on days 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 after the start of the incubation, a greater percentage of the naive-like T cells in the input composition are dividing compared to the percentage of non-naive-like T cells in the input composition. In some cases, on days 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 after the start of the stimulation condition, the condition is able to induce a greater percentage of cell proliferation in the human naive-like T cell population compared to human non-naive-like T cells.

[0039] In some of these embodiments, the non-naive-like T cells are selected from effector T (T) cells. EFF ) cells, memory T cells, central memory T cells (T cells) CM ), effect memory T (T EM ) cells and combinations thereof; or the non-naive T cells are multiple T cells, the multiple T cells including effector T (T) cells. EFF ) cells and / or memory T cells, or composed of them, wherein the memory T cells optionally contain central memory T cells (T cells). CM ) and / or effect memory T(T EM )cell.

[0040] In some embodiments, the percentage of naive T cells in the input composition is less than the percentage of engineered cells derived from naive T cells in the stimulated composition. In some embodiments, a greater percentage of the cells incorporating the nucleic acid are naive T cells in the input composition or proliferations derived from naive T cells in the input composition compared to non-naive T cells in the input composition.

[0041] In some of these embodiments, the introduction is performed via transduction. In some embodiments, the nucleic acid contains 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 gamma retroviral vector. In some embodiments, the introduction is performed via transposition containing a transposon of the nucleic acid molecule. In some embodiments, the ratio of naive T cells to non-naive T cells in the stimulated composition is increased by more than or greater than about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, or 100 times compared to the ratio of naive T cells to non-naive 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.

[0042] Output compositions produced by any of the methods provided herein are provided. Pharmaceutical compositions containing said output compositions are also provided. In some embodiments, the pharmaceutical compositions further contain a drug carrier.

[0043] Treatment methods are provided that include administering to a mammalian subject an output composition produced by any of the said methods or any of the said pharmaceutical compositions. In some embodiments, the cells are derived from the subject to whom the cells are administered. Detailed Implementation

[0044] This document provides methods for incubating (e.g., stimulating) cells during the preparation of cells for adoptive cell therapy, as well as compositions and cells produced by said methods. In some embodiments, the cells include T cells that express genetically engineered antigen receptors in some respects. In some embodiments, the genetically engineered antigen receptors include genetically engineered or recombinant T cell receptors (TCRs) and functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs).

[0045] In some embodiments, methods for incubating (e.g., stimulating) T cells are provided. Available methods have used anti-CD3 and anti-CD28 to stimulate T cells. However, currently available methods have not focused on obtaining specific target cell populations through the incubation process and producing a more desirable T cell population and its beneficial outcomes. Available methods also do not result in the elimination of unwanted and specific T cell populations from the input T cell population during incubation, nor do they yield their beneficial outcomes. Furthermore, available methods are not designed to eliminate subsets of the T cell population during the incubation process that will be used to produce an output composition containing T cells expressing genetically engineered recombinant receptors. The available methods also do not describe benefits, including producing a more consistent and / or predictable T cell population that is beneficial for genetic engineering, administration, and / or clinical response.

[0046] In some aspects, the provided embodiments offer a method for producing a more uniform and / or predictable number of T-cell compositions compared to other stimulation methods, and in some aspects, also provide for reducing or eliminating unwanted T-cell populations, such as cells whose presence may make the final product so heterogeneous that efficacy, effectiveness, and safety are difficult to predict. In some embodiments, the provided methods address problems related to the production of genetically engineered T-cells, wherein a large number or majority of such genetically engineered cells are derived from non-immature T-cells. In some aspects, persistent lack, depletion, and / or toxicity-related problems may be associated with adoptive T-cell therapies involving compositions containing genetically engineered cells derived from non-immature T-cells and / or a percentage or number of genetically engineered cells derived from non-immature T-cells in the composition exceeding a certain threshold. In some embodiments, methods for producing genetically engineered T-cell compositions rich in cells derived from immature T-cells may exhibit characteristics related to an increased percentage of healthy cells and / or an overall increase in the number of cells exhibiting increased persistence in the composition, compared to other methods where such immature T-cells are not so enriched.

[0047] Therefore, methods for preparing engineered T cells for adoptive therapy as provided herein include those using conditions that result in preferential expansion, proliferation, or genetic engineering of cells derived from naive T cells (or derived from CD27+, CD45RA+, CCR7+, CD62L-, or CD45RO- T cells) compared to cells derived from non-naive T cells (or derived from CD27-, CD45RA-, CCR7-, CD62L+, or CD45RO+ T cells). In some embodiments, the method involves incubating T cells in an input composition containing naive T cells under stimulating conditions that preferentially produce a stimulated composition containing cells derived from the input composition of naive T cells. In some aspects, the T cell population contains a mixture of both naive and non-naive T cells. In some embodiments, the stimulating conditions preferentially induce a response in non-naive T cells compared to naive T cells. In some cases, the response includes preferential activation of non-naive T cells, which in some embodiments may lead to cell death.

[0048] In some embodiments, the method includes incubating cells under stimulation conditions, said stimulation conditions including incubation with a stimulator capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules. In some cases, the primary agent specifically binds to CD3 and / or the co-stimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS. For example, in some embodiments, the primary agent is or contains anti-CD3, and the secondary agent is or contains anti-CD28. In some cases, the primary and secondary agents contain antibodies and / or are present on the surface of a solid support. In some examples, the solid support is beads.

[0049] In some embodiments, the stimulation conditions include incubating cells of the introduced composition with such a stimulating agent (e.g., anti-CD3 / anti-CD28 beads) in a bead-to-cell ratio of from or from about 1:1 to 10:1, from or from about 1:1 to 8:1, from or from about 1:1 to 6:1, from or from about 1:1 to 4:1, from or from about 1:1 to 3:1, from or from about 4:1 to 10:1, from or from about 4:1 to 8:1, from or from about 4:1 to 6:1, from or from about 6:1 to 10:1, from or from about 6:1 to 8:1, from or from about 8:1 to 10:1, from or from about 1:1 to 1:10, from or from about 1:1 to 1:8, from or from about 1:1 to 1:6, from or from about 1:1 to 1:4, from or from about 1:2 to 1:3. In some specific cases, the ratio of beads to cells is approximately 3:1. In other cases, the ratio is approximately 1:3.

[0050] The method typically also includes a step for genetically engineering the stimulated T-cell composition. Genetic engineering of the stimulated composition can be performed or initiated at any time point after or after the initial incubation and / or concurrently with the incubation. In some embodiments, cells are incubated together with nucleic acids encoding such genetically engineered molecules, thereby introducing the nucleic acids and expressing the genetically engineered molecules in the cells within the composition, resulting in an output composition. Genetically engineered molecules include proteins, such as genetically engineered antigen receptors, including chimeric antigen receptors (CARs) and other recombinant receptors (such as chimeric receptors having both an extracellular ligand-binding portion and an intracellular signaling portion).

[0051] Also provided are culture initiation compositions, stimulated compositions, and output compositions for use in or generated by said methods. Methods relating to administering such compositions and cells to subjects in need, including cancer patients, are also provided. Kits comprising compositions and / or cells generated by any of the methods described herein are also provided.

[0052] The method can be advantageous and can produce more desirable products. In some embodiments, the provided method allows for a more consistent manufacturing process. In some embodiments, the provided method produces cells that result in more uniform transduction and / or expansion in subsequent steps of engineered cell production. In some embodiments, more uniform transduction and / or expansion can produce more predictable T-cell products throughout the manufacturing process. In some cases, the T-cell products can be administered to subjects more consistently. In some contexts, such features can reduce or prevent potential toxicities and / or associated outcomes and symptoms in subjects following adoptive cell therapy.

[0053] All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated herein by reference in their entirety for all purposes, to the extent that each individual publication is incorporated individually by reference. Where the definitions described herein are contrary to or otherwise inconsistent with those described in patents, applications, published applications, and other publications incorporated herein by reference, the definitions described herein shall prevail over those incorporated herein by reference.

[0054] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described. Attached Figure Description

[0055] Figures 1A to 1D Results are depicted from CAR+T cell compositions generated from amplification and non-amplification processes involving bead-based stimulants (beads), bead-based stimulants and incubation in basal medium (bead basal medium) or oligomer stimulants (oligomers). Figure 1AThe percentage of CD4+ and CD8+ T cells that were positive for both CCR7 and CD27 was depicted. Figure 1B The percentage of CCR7+CD27+ cells relative to CD4+CAR+ T cells is depicted. Figure 1C The percentage of CCR7+CD27+ cells relative to CD8+CAR+ T cells is depicted. Figure 1D The percentage of CCR7+CD27+ cells generated from representative donors during the expansion process is shown at different dates during the manufacturing process (including activation on day 1 (d1 AMAT), transduction on day 2 (d2 XMAT), and different times after the start of culture (d4 INOC+2, d6 INOC+4, d7 INOC+5)).

[0056] Figures 2A to 2D It shows the granting of CAR + Kaplan-Meier survival curves of subjects receiving the T-cell combination, divided into those given CD4+. + CAR + T cells ( Figure 2A For progression-free survival, Figure 2C (for the duration of the reaction) neutralization in CD8 + CAR + T cells ( Figure 2B For progression-free survival, Figure 2D The reaction duration contains a certain percentage of CCR7 levels that are above or below a certain threshold. + CD27 + CAR + A group of T-cell compositions.

[0057] Figure 3 The clones of isolated CD4+ and CD8+ T cell compositions before engineering (CMAT) and the T cell clonalness of CD4+ and CD8+ therapeutic CAR+ T cell compositions after engineering (application of the Shannon index) are shown.

[0058] I. Methods for incubating (e.g., stimulating) T cells

[0059] This document provides methods for incubating (e.g., stimulating) cells during the preparation of cells for adoptive cell therapy, as well as compositions and cells produced by said methods. In some embodiments, the cells typically comprise T cells that, in some embodiments, express genetically engineered antigen receptors, such as genetically engineered or recombinant T-cell receptors (TCRs), and functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs).

[0060] In some embodiments, a method for incubating (e.g., stimulating) T cells is provided, comprising incubating, under stimulating conditions, an input composition containing a population of T cells comprising naive-like T cells and non-naive-like T cells, the input composition containing a culture-starting amount of naive-like T cells or a subset thereof of CD8+ T cells, thereby producing a stimulated composition. In some aspects, 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. In some embodiments, the method further includes introducing a nucleic acid encoding a genetically engineered recombinant receptor into the stimulated cell composition, wherein the method thereby produces an output composition comprising T cells expressing the genetically engineered recombinant receptor. In some aspects, the introduction is performed during at least a portion of the incubation or after the incubation. In some embodiments, the cells in the input composition have not and have not undergone a selection step based on endogenous T cell surface markers prior to the incubation, the endogenous T cell surface markers distinguishing between naive-like and non-naive-like T cells.

[0061] In some embodiments of the provided method, the introduction of the nucleic acid encoding the genetically engineered recombinant receptor is performed before, during, and / or after the introduction of the nucleic acid encoding the genetically engineered recombinant receptor. In some embodiments, the method includes incubating the composition under stimulating conditions before the introduction of the nucleic acid encoding the genetically engineered recombinant receptor. In some cases, the method includes incubating the composition under stimulating conditions during the introduction of the nucleic acid encoding the genetically engineered recombinant receptor. In some embodiments, the method includes incubating the composition under stimulating conditions after the introduction of the nucleic acid encoding the genetically engineered recombinant receptor.

[0062] In some embodiments, the stimulating conditions include a surface (such as magnetic beads) having one or more agents attached thereto to bind to a cell surface portion. In one embodiment, the surface has at least one anti-CD3 antibody attached thereto. In another embodiment, the surface has anti-CD3 and / or anti-CD28 antibodies attached thereto. In some embodiments, after approximately incubation, at least a substantial portion of at least one T cell population in the input composition is removed. In one embodiment, the cell-to-bead ratio under the stimulating conditions is from about 50:1 to about 5:1. In some embodiments, the ratio is from about 100:1 to about 1:1. In some embodiments, the ratio is from about 1:1 to 1:50. In some 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 another particular embodiment, the ratio is about 1:3.

[0063] In some embodiments of the methods provided herein, culture conditions preferentially induce the proliferation, stimulation, and / or activation of non-immature T cells compared to naive T cells. In some embodiments, an incubation (e.g., stimulation) method produces a desired output composition containing a desired number of naive T cells derived from the input composition. In some embodiments using the methods for stimulating T cells described herein, a greater percentage of naive T cells are induced to proliferate and / or expand in the cells in the input composition compared to non-immature T cells. In some aspects, the stimulated composition produced by the stimulation methods described herein contains less than 10% cells derived from non-immature 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-immature T cells. In some embodiments, on days 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 after the start of the incubation, 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. In some cases, the stimulation conditions of the method for stimulating T cells induce cell death in a specific cell subset. In a particular example, the stimulation conditions of the method induce activation of non-naive-like T cells, thereby inducing activation-induced cell death (AICD).

[0064] In one aspect, the method preferentially activates non-immature T cells derived from the input composition, thereby inducing cell death in these cells and eliminating T cells from the stimulated composition that originate from the non-immature T cells of the input composition. Simultaneously, the retained desired cells (e.g., those derived from the immature T cells of the input composition) are activated, survive, and stimulated to proliferate, resulting in an activated cell population from which at least a significant portion of the unwanted T cell subset has been eliminated. Furthermore, the incubation (e.g., stimulation) conditions provided by the method described herein restore the polyclonal nature of the T cell population with respect to expressed TCR genes, as indicated by profiling analysis or other methods of quantitative clonality. In some embodiments, the cell signature of the immature T cells derived from the input composition in the output composition is indicated by the expression of a specific marker.

[0065] A. Input composition

[0066] In the provided methods for incubating (e.g., stimulating) T cells, the method includes incubating an input composition containing a T cell population under stimulation conditions. In some aspects, the input composition contains a T cell population, such as containing naive-like T cells and non-naive-like T cells. In some embodiments, the T cell population contains CD4+ and / or CD8+ cells. In some embodiments, the input composition contains 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 a subset of their CD8+ T cells in the input composition. In some aspects, specific markers or identifiers can be used to identify specific subsets of T cells, such as naive-like T cells and / or non-naive-like T cells. In some specific examples, the expression of cell surface markers is used to assess and / or identify subsets of T cells. In some aspects, the clonality of the input composition can be characterized.

[0067] In some aspects, the composition of incubated and / or engineered cells (such as the input composition) has not and has not undergone prior selection of naive T cells. In some embodiments, the method does not include positive or negative selection or enrichment of naive cells in the input composition. In some aspects, the composition of stimulated and / or engineered cells (such as the input composition and / or stimulated composition) has not and has not undergone such selection prior to incubation (e.g., stimulation). In some embodiments, prior to incubation, the cells have not and / or have not undergone a selection step based on the level or presence of T cell surface markers (such as CD27, CD28, CD45RA, CD45RO, CD56, CD62L, CD95, KLRG1, or CCR7) that distinguish naive from non-naive T cells. For example, in some aspects, prior to incubation under stimulation conditions, the cells in the input composition have not undergone selection based on the expression of such markers or on the surface expression of such markers. In some respects, prior to genetic engineering (e.g., incubation with nucleic acids), the cells in the stimulated composition do not undergo selection based on the expression of such a marker or on the surface expression of such a marker. In some respects, the incubation of the input composition is carried out without selection based on the surface expression of the marker to enrich naive T cells.

[0068] In some embodiments, the method includes fewer selection steps compared to other methods, such as not involving the selection of a subset of T cells, and is therefore simpler and associated with cost and / or resource savings compared to multi-step selection methods. In some embodiments, the method does not include enrichment based on the expression of markers specific to memory T cells or subsets thereof and / or naive-like T cells. In some aspects, the composition of incubated (e.g., stimulated) cells (such as the input composition and / or the stimulated composition) has not yet undergone and has not experienced such selection.

[0069] In some examples, the method does not include positive selection based on markers specific to non-naïve T cells or identifying specific T cell subsets (such as CD62L, CCR7, CD27, CD28, CD56, CD3, CD122, CD95, CD25, IL7-Rα, and / or CD127). In some examples, the method does not include positive selection or enrichment based on the expression of CD62L, CCR7, CD27, CD28, CD56, CD3, CD122, CD95, CD25, IL7-Rα, and / or CD127. In some embodiments, the method does not use samples or compositions that have already been enriched or positively selected based on such markers or for enriching specific subtypes.

[0070] In some embodiments, the method does not include an affinity-based selection step designed to separate or distinguish between immature and non-immature T cells. In some examples, the method does not include positive selection based on markers specific to or distinguishing between immature and non-immature 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 samples or compositions that have already been enriched or positively selected based on such markers or for enriching such subtypes.

[0071] cell

[0072] In some embodiments, the methods provided herein include one or more steps for preparing a cell input composition containing a culture starting amount of naive-like T cells, such as in combination with one or more steps including cell stimulation, expansion, proliferation, and / or genetic engineering (e.g., transduction). The cells are typically eukaryotic cells, such as mammalian cells, and are typically human cells. The input composition can be produced or generated by various methods involving the isolation or selection of cells from a biological sample. In some embodiments, the input composition contains CD4+ and / or CD8+ cells derived from a biological sample (e.g., obtained from 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 or is about 1:1, 1:2, 2:1, 1:3, or 3:1. In some embodiments, the input composition containing isolated CD4+ and / or CD8+ T cells contains a mixture of naive-like T cells and non-naive-like T cells.

[0073] In some embodiments, the cells in the input composition are derived from blood, bone marrow, lymph, or lymphoid organs, and are cells of the immune system (such as cells of innate or adaptive immunity), including samples containing bone marrow or lymphoid cells (including lymphocytes, typically T cells). The cells are typically primary cells, such as those isolated directly from the subject and / or isolated from and frozen from the subject. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as the entire T cell population, CD4+ cells, CD8+ cells, and their subsets, such as those defined by: function, activation state, maturity, potential for differentiation, expansion, recycling, localization and / or persistence, antigen specificity, antigen receptor type, presence in a specific organ or compartment, marker or cytokine secretion characteristics, and / or degree of differentiation. Regarding the subject to be treated, the cells may be allogeneic and / or autologous. The methods include off-the-shelf methods. In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and / or engineering them as described herein, and reintroducing them into the same patient before or after cryopreservation.

[0074] T cell and / or CD4+ and / or CD8+ T cell subtypes and subsets include naive T cells (T0). N ) cells, effector T cells (T cells) EFF ), memory T cells and their subtypes (such as stem cell memory T cells (T15)). SCM ), central memory T(T) CM ), effect memory T (T EM The cells may include terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated inertial T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells (such as 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 (Treg). In some embodiments, the cells also contain recombinant FOXP3 or variants thereof.

[0075] In some embodiments, the preparation of engineered cells includes one or more culture and / or preparation steps. Cells used for engineering can be isolated from a sample (such as a biological sample, for example, a biological sample obtained from or derived from a subject). In some embodiments, the subject from whom the cells are isolated is a subject suffering from a disease or condition, requiring cell therapy, or who will receive cell therapy. In some embodiments, the subject is a person requiring a specific therapeutic intervention (such as adoptive cell therapy in which the isolated, processed, and / or engineered cells are used).

[0076] Therefore, in some implementations, the cells are primary cells, such as primary human cells. Samples include tissues, fluids, and other samples taken directly from the subject, as well as samples derived from one or more processing steps (such as separation, centrifugation, genetic engineering (e.g., transduction with a viral vector), washing, and / or incubation). Biological samples can be samples obtained directly from biological sources or processed samples. 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.

[0077] In some respects, the sample from which cells are derived or isolated is blood or a blood-derived sample, or is derived from apheresis or leukoablation products. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, enteroassociated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissues, liver, lungs, stomach, intestines, colon, kidneys, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs and / or cells derived therefrom. In the context of cell therapy (e.g., adoptive cell therapy), samples include those from autologous and allogeneic sources.

[0078] In some embodiments, the cells are derived from cell lines, such as T cell lines. In some embodiments, the cells are obtained from xenogeneic sources, such as mice, rats, non-human primates, or pigs.

[0079] In some embodiments, cell isolation includes one or more preparation steps and / or non-affinity-based cell isolation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse, or remove cells sensitive to a specific reagent. In some examples, cells are isolated based on one or more properties, such as density, adhesion properties, size, sensitivity to a specific component, and / or resistance.

[0080] In some cases, cells from the subject's circulating blood are obtained, for example, via apheresis or leukoablation. In some respects, the sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and / or platelets, and in some respects, cells other than erythrocytes and platelets.

[0081] In some embodiments, blood cells collected from the subject are washed, for example to remove plasma fractions, and the cells are placed in a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution is deficient in calcium and / or magnesium and / or many or all divalent cations. In some aspects, the washing step is performed by a semi-automatic “flow-through” centrifuge (e.g., a Cobe 2991 cell processor, Baxter) according to the manufacturer’s instructions. In some aspects, the washing step is performed by tangential flow filtration (TFF) according to the manufacturer’s instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers (e.g., calcium-free buffers) after washing. ++ / Mg ++ In some embodiments, components of the blood cell sample are removed and the cells are directly resuspended in the culture medium.

[0082] In some implementations, the method includes density-based cell separation methods, such as preparing leukocytes from peripheral blood by lysing red blood cells and centrifuging via a Percoll or Ficoll gradient.

[0083] In some embodiments, the separation method includes separating different cell types based on the expression or presence of one or more specific molecules in the cells, such as surface markers (e.g., surface proteins), intracellular markers, or nucleic acids. In some embodiments, any known separation method based on such markers can be used. In some embodiments, the separation is based on affinity or immunoaffinity. For example, in some aspects, the separation includes separating cells and cell populations based on the expression or expression level of one or more markers (typically cell surface markers) in the cells, for example, by incubating with antibodies or binding partners that specifically bind to such markers, followed typically by a washing step and separating cells that have bound to the antibody or binding partner from those that have not yet bound to the antibody or binding partner.

[0084] Such separation steps can be based on positive selection (where cells that have bound the reagent are retained for further use) and / or negative selection (where cells that have not yet bound to the antibody or binding coupler are retained). In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful when antibodies specifically identifying cell types in a heterogeneous population are unavailable, making it preferable to perform separation based on markers expressed by cells other than the desired population.

[0085] Isolation does not need to result in 100% enrichment or removal of a specific cell population or cells expressing a specific marker. For example, positive selection or enrichment for a specific type of cell (such as those expressing a marker) means increasing the number or percentage of such cells, but does not need to result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion for a specific type of cell (such as those expressing a marker) means reducing the number or percentage of such cells, but does not need to result in the complete removal of all such cells.

[0086] In some examples, multiple rounds of separation steps are performed, where fractions of positive or negative selection from one step undergo another separation step, such as subsequent positive or negative selection. In some examples, a single separation step can simultaneously deplete cells expressing multiple markers, such as by incubating cells with multiple antibodies or binding partners (each antibody or binding partner being specific to the marker targeted for negative selection). Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding partners expressed on various cell types.

[0087] For example, in some respects, specific subsets of T cells (such as cells that are positive for or highly express 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 using positive or negative selection techniques.

[0088] For example, CD3 / CD28 ferrite beads can be used (e.g., M-450CD3 / CD28 T Cell Expander) positively selected CD3 + CD28 + T cells.

[0089] In some embodiments, separation is performed by enriching a specific cell population via positive selection or depleting a specific cell population via negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agents, said antibodies or other binding agents being expressed (labeled) on the cells for positive or negative selection, respectively. + ) or expressed at a relatively high level (marked) 高One or more surface markers specifically bind to it.

[0090] In some implementations, T cells are separated from PBMC samples by negatively selecting markers (such as CD14) expressed on non-T cells (such as B cells, monocytes, or other leukocytes). In some aspects, CD4 is used... + or CD8 + Select the step for separating CD4 + Helper T cells and CD8 + Cytotoxic T cells. These CD4 cells can be identified through positive or negative selection of markers expressed or expressed at relatively high levels on one or more naive, memory, and / or effector T cell subsets. + and CD8 + The group was further divided into subgroups.

[0091] In some implementations, such as by positive or negative selection based on surface antigens associated with the corresponding subgroups, CD8 + Cellular targeting of naive, central memory, effector memory, and / or central memory stem cells is further enriched or depleted. In some implementations, targeting central memory T(T) stem cells is further enriched or depleted. CM Cells are enriched to increase efficacy, such as to improve long-term survival, expansion, and / or transplantation after administration, which is particularly robust in some respects in this subpopulation. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, the combination is enriched with T cells to enhance efficacy, such as to improve long-term survival, expansion, and / or transplantation after administration, which is particularly robust in some respects in this subpopulation. CM CD8 + T cells and CD4 + T-cells further enhance efficacy.

[0092] In the implementation plan, memory T cells are present in CD8. + CD62L of peripheral blood lymphocytes + and CD62L - Two subsets. For example, anti-CD8 and anti-CD62L antibodies can be used to target PBMCs against CD62L. - CD8 + and / or CD62L + CD8 + The fractions are enriched or depleted.

[0093] In some implementations, the central memory T(T) CMCell enrichment is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some respects, it is based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some respects, T-rich cells are enriched. CM CD8 cells + Population segregation was achieved through depletion of cells expressing CD4, CD14, and CD45RA, as well as positive selection or enrichment of cells expressing CD62L. In one aspect, this was observed in central memory T(T) cells. CM Cell enrichment begins with a negative cell fraction selected based on CD4 expression, which then undergoes negative selection based on CD14 and CD45RA expression and positive selection based on CD62L. In some respects, such selection is performed simultaneously, and in others, sequentially in any order. In some respects, it will be used to prepare CD8+. + The same CD4 expression-based selection steps for cell populations or subpopulations are also used to generate CD4. + Cell populations or subpopulations, such that positive and negative fractions from CD4-based isolation are retained and used in subsequent steps of the method, optionally after one or more other positive or negative selection steps.

[0094] In specific cases, PBMC samples or other white blood cell samples are subjected to CD4. + Cell selection was performed, retaining both negative and positive fractions. The negative fraction was then subjected to negative selection based on the expression of CD14 and CD45RA or ROR1 and positive selection based on markers specific to central memory T cells (such as CD62L or CCR7), with the positive and negative selections occurring in any order.

[0095] CD4+ T helper cells were sorted into naive, central memory, and effector cells by identifying cell populations with cell surface antigens. + Lymphocytes can be obtained using standard methods. In some implementations, naïve CD4 cells are used. + T lymphocytes are CD45RO - CD45RA + CD62L -、 CD4 + T cells. In some implementations, central memory CD4... + Cells are CD62L + and CD45RO + In some implementations, the effect is CD4. + Cells are CD62L - and CD45RO - .

[0096] In one example, to enrich CD4 through negative selection + Cellular monoclonal antibody mixtures typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or conjugate is bound to a solid support or matrix (such as magnetic or paramagnetic beads) to allow cell separation for positive and / or negative selection. For example, in some embodiments, immunomagnetic (or affinity magnetic) separation techniques are used to separate or isolate cells and cell populations (reviewed in Methods in Molecular Medicine, Vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In vitro and In vivo, pp. 17-25, edited by SA Brooks and U. Schumacher). Humana Press Inc., Totowa, New Jersey.

[0097] In some aspects, a sample or composition of cells to be separated is incubated together with small magnetizable or magnetically responsive materials, such as magnetically responsive particles or microparticles, like paramagnetic beads (e.g., like Dynalbeads or MACS beads). The magnetically responsive material (e.g., particles) is typically 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 a cell, multiple cells, or cell population that is to be separated (e.g., desired to be negatively or positively selected).

[0098] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material that binds to a specific binding member, such as an antibody or other binding partner. In some aspects, magnetically responsive materials used in magnetic separation methods can be used. Suitable magnetic particles include those described in Molday's U.S. Patent No. 4,452,773 and European Patent Specification EP 452342B, which are hereby incorporated by reference. Colloidal-sized particles, such as those described in Owen's U.S. Patent No. 4,795,698 and Liberti et al.'s U.S. Patent No. 5,200,084, are other examples.

[0099] Incubation is typically carried out under conditions in which antibodies or binding couplers, or molecules (such as secondary antibodies or other reagents) that specifically bind to such antibodies or binding couplers attached to magnetic particles or beads, specifically bind to cell surface molecules (if present on cells within the sample).

[0100] In some aspects, the sample is placed in a magnetic field, and those cells with magnetic responses or magnetizable particles attached thereto are attracted to the magnet and separated from unlabeled cells. For positive selection, cells attracted by the magnet are retained; for negative selection, cells not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, wherein positive and negative fractions are retained for further processing or subjected to further separation steps.

[0101] In some embodiments, the magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In some embodiments, the magnetic particles attach to cells by coating with a primary antibody specific to one or more labels. In some embodiments, cells are labeled with a primary antibody or binding partner instead of beads, and then cell-type-specific secondary antibodies or other binding partners (e.g., streptavidin)-coated magnetic particles are added. In some embodiments, the streptavidin-coated magnetic particles are used in conjunction with a biotinylated primary or secondary antibody.

[0102] In some embodiments, the magnetically responsive particles remain attached to cells that are subsequently incubated, cultured, and / or engineered; in some aspects, the particles remain attached to cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells may include, for example, using competitive unlabeled antibodies, magnetizable particles, or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0103] In some implementations, affinity-based selection is performed using magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, California). Magnetically activated cell sorting (MACS) systems are capable of selecting cells with magnetized particles attached thereto at high purity. In some implementations, MACS operates in a manner where, after the application of an external magnetic field, non-target and target species are eluted sequentially. That is, cells attached to the magnetized particles are held in place, while unattached species are eluted. Then, after this first elution step, the species trapped in the magnetic field and prevented from elution are released in a way that allows them to be eluted and recovered. In some respects, non-target cells are tagged and depleted from the heterogeneous cell population.

[0104] In some embodiments, a system, apparatus, or device is used for separation or partitioning, performing one or more of the isolation, cell preparation, partitioning, processing, incubation, culture, and / or formulation steps of the method. 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 error, and / or contamination. In one example, the system is as described in International PCT Publication No. WO 2009 / 072003 or US 20110003380A1.

[0105] In some embodiments, the system or device performs one or more (e.g., all) of the separation, processing, engineering, and formulation steps in an integrated or stand-alone system and / or in an automated or programmable manner. In some aspects, the system or device includes a computer and / or computer programs communicating with the system or device, allowing a user to program, control, evaluate the results of the processing, separation, engineering, and formulation steps, and / or adjust various aspects of the processing, separation, engineering, and formulation steps.

[0106] In some aspects, separation and / or other steps are performed using the CliniMACS system (Miltenyi Biotec), for example, to automate cell separation at a clinical scale in a closed and sterile system. Components may include an integrated microcomputer, a magnetic separation unit, a peristaltic pump, and various clamping valves. In some aspects, the integrated computer controls all components of the instrument and instructs the system to perform repetitive procedures in a standardized sequence. In some aspects, the magnetic separation unit includes a movable permanent magnet and a support for selecting columns. The peristaltic pump controls the flow rate throughout the tubing assembly and, together with the clamping valves, ensures controlled flow of buffer solution through the system and continuous suspension of cells.

[0107] In some aspects, the CliniMACS system uses antibody-conjugated magnetizable particles provided in a sterile, pyrogen-free solution. In some embodiments, after labeling cells with magnetic particles, the cells are washed to remove excess particles. A cell preparation bag is then connected to a tubing assembly, which in turn connects to a bag containing buffer and a cell collection bag. The tubing assembly consists of pre-assembled sterile tubing (including a pre-column and a separation column) and is for single use only. Upon initiation of the separation procedure, the system automatically applies the cell sample to the separation column. Labeled cells remain within the column, while unlabeled cells are removed through a series of washing steps. In some embodiments, the cell population used with the methods described herein is unlabeled and not retained in the column. In some embodiments, the cell population used with the methods described herein is labeled and retained in the column. In some embodiments, the cell population used with the methods described herein is eluted from the column after removal of the magnetic field and collected in a cell collection bag.

[0108] In some implementations, the CliniMACS Prodigy system (Miltenyi Biotec) is used for separation and / or other steps. In some aspects, the CliniMACS Prodigy system is equipped with a cell processing consortium that allows automated washing and grading of cells via centrifugation. The CliniMACS Prodigy system may also include an onboard camera and image recognition software that determines the optimal cell grading endpoint by identifying 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 may also include an integrated cell culture chamber that enables cell culture protocols such as cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports allow for aseptic removal and replenishment of culture medium, 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.

[0109] In some embodiments, the cell populations described herein are collected and enriched (or depleted) by flow cytometry, wherein 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) by fabrication-scale sorting (FACS). In some embodiments, the cell populations described herein are collected and enriched (or depleted) by using a combination of a microelectromechanical system (MEMS) chip and 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 both cases, cells can be labeled with multiple markers, thereby allowing the separation of well-defined subsets of T cells with high purity.

[0110] In some implementations, antibodies or binding partners are 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 a fluorescently labeled antibody. In some examples, cells are separated by binding to antibodies or other binding partners specific to one or more cell surface markers carried in a fluid stream, such as via fluorescence-activated cell sorting (FACS) (including preparative-scale (FACS)) and / or microelectromechanical systems (MEMS) chips, such as in combination with a flow cytometry detection system. Such methods allow for simultaneous positive and negative selection based on multiple markers.

[0111] In some embodiments, the preparation method includes the step of freezing (e.g., cryopreservation) cells before or after isolation, incubation, and / or engineering. In some embodiments, freezing and subsequent thawing steps remove granulocytes from the cell population and, to some extent, monocytes. In some embodiments, cells are suspended in a freezing solution, for example, after a washing step, to remove plasma and platelets. In some aspects, any of a variety of known freezing solutions and parameters can be used. One example involves using PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing medium. This is then diluted 1:1 with the medium to make the final concentrations of DMSO and HSA 10% and 4%, respectively. The cells are then frozen to -80°C at a rate of 1° / min and stored in the gas phase of a liquid nitrogen tank.

[0112] a. Immature T cells

[0113] In some embodiments, the method includes incubating an input composition containing naive T cells or a certain threshold amount of naive T cells. One aspect of the method of the invention provides an input composition that has been evaluated prior to incubation (e.g., stimulation) 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). In some embodiments, various markers of naive T cells can be utilized to identify naive T cells. In some embodiments, the expression of specific markers of naive T cells can be evaluated. For example, in some cases, naïve T cells are surface-positive for markers including T cell activation markers selected from CD27, CD28, CD45RA, CD62L, and CCR7. In some aspects, naïve T cells are surface-negative for CD56 and / or CD45RO. In some aspects, naïve T cells are surface-negative for CD45RO but cell-surface positive for CD27, CD45RA, and CCR7. In some cases, naïve T cells are negative for intracellular expression of cytokines such as IL-2, IFN-γ, IL-4, and / or IL-10. In some other examples, naïve T cells are negative for the expression of the marker CD25 and / or perforin. In some cases, naïve T cells are CD95... lo .

[0114] In some embodiments, to assess the expression of markers on naive T cells, the method includes detecting the markers by performing in vitro assays. In some examples, the in vitro assays are immunoassays, aptamer-based assays, histological or cytological assays, or mRNA expression level assays. In some cases, the in vitro assays used may be enzyme-linked immunosorbent assays (ELISA), Western blotting, immunophenotyping, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry, surface plasmon resonance (SPR), chemiluminescence assays, lateral immunoassays, inhibition assays, or affinity assays. In some embodiments, the expression of markers on naive T cells is determined by RNA-seq.

[0115] The naïve T cells in the input composition can also be assessed by the clonality of the T cells. In some embodiments, assessing the clonality of the T cell population is an assessment of the clonal diversity of the T cell population. In some embodiments, the naïve T cells are polyclonal (or multiclonal). The polyclonality of the T cell input composition is measured by the magnitude of the population's response to a given antigen. In some aspects, the input composition can be assessed by measuring the number of different epitopes recognized by antigen-specific cells. This can be done using standard techniques for generating and cloning antigen-specific T cells in vitro. In some embodiments, the naïve T cells are polyclonal (or multiclonal), wherein no single clonal population dominates in the naïve T cell population.

[0116] In some aspects, in the context of T cell populations (such as the input composition), polyclonalness is identified as a T cell population exhibiting a wide variety of broad antigen specificities. In some embodiments, polyclonalness involves a T cell population exhibiting high diversity in a TCR library. In some cases, the diversity of the TCR library is due to V(D)J recombination events, which in some respects are triggered by selection events for self and foreign antigens. In some embodiments, a diverse or polyclonal T cell population is one in which analysis indicates the presence of multiple varied or different TCR transcripts or products in the population. In some embodiments, a T cell population exhibiting high or relatively high clonalness is a T cell population with less diversity in the TCR library. In some embodiments, if analysis indicates the presence of several (e.g., two or three) TCR transcripts or products in a T cell population, the T cells are oligoclonal. In some embodiments, if analysis indicates the presence of a single TCR transcript or product in a T cell population, the T cells are monoclonal.

[0117] In some examples, the clonality of cells (such as naive T cells) in the input composition is determined by cloning sequencing, optionally next-generation sequencing, or prototyping analysis. In some aspects, next-generation sequencing methods can be used to evaluate the TCR library using genomic DNA or cDNA from T cells, including sequences encoding complementarity-determining region 3 (CDR3). In some embodiments, whole transcriptome sequencing via RNA-seq can be used. In some embodiments, single-cell sequencing methods can be used.

[0118] In some implementations, polyclonalness (a measure of the hypervariable repertoire of the TCR Vβ, Vα, Vγ, or Vδ chains) can be assessed or determined through spectrogram analysis. A T cell population is considered polyclonal when the Vβ spectrogram of a given TCR Vβ, Vα, Vγ, or Vδ family has multiple peaks (typically five or more main peaks) and, in most cases, a Gaussian distribution. Polyclonalness can also be defined by the generation and characterization of antigen-specific clones against a target antigen.

[0119] In some embodiments, methods for assessing clonality may include various features of the methods described in International Publications WO 2012 / 048341, WO 2014 / 144495, WO 2017 / 053902, WO 2016044227, WO 2016176322, and WO2012048340, each of which is incorporated herein by reference in its entirety. In some embodiments, such methods may be used to obtain sequence information about intracellular target polynucleotides (such as TCRs). Target genes may be obtained from genomic DNA or mRNA of cells from cell samples or cell populations. Cell samples or cell populations may include immune cells. For example, for a target TCR molecule, a gene encoding the TCR chain may be obtained from the genomic DNA or mRNA of immune cells or T cells. In some embodiments, the starting material is RNA from T cells, which consists of a gene encoding the TCR chain.

[0120] In some implementations, 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.

[0121] In some embodiments, the provided method promotes or results in an increase in the polyclonal activity of the T cell population or subset thereof from the input composition. In some embodiments, the provided method promotes or results in an increase in the diversity of the T cell population or subset thereof from the input composition. In some embodiments, the T cells or subset thereof from the composition after incubation or stimulation exhibit reduced or decreased clonality compared to T cells or CD4 or CD8 subset thereof in which the composition was input prior to performing the method. In some embodiments, the degree of reduction in clonality is greater than or greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times.

[0122] In one aspect of the provided method, as described below in the section entitled "Cell Incubation," the T cell population in the input composition is activated or stimulated to induce apoptosis, thereby eliminating a subset from the cell population. Simultaneously, the retained desired cells (e.g., immature-like cells) are activated and stimulated to proliferate, resulting in an activated cell population from which at least a significant portion of the unwanted T cell subsets (such as non-immature-like T cells) have been eliminated. As previously mentioned, the remaining cells can be stimulated / activated as described herein after the cell population has been directly exposed to the pro-apoptotic composition. Furthermore, the subsequent stimulation and activation provided by the present invention restores the polyclonal nature of the T cell population with respect to the expressed TCR genes, as indicated by profiling analysis or sequencing methods.

[0123] Starting amount of culture

[0124] In aspects of the provided method, the input composition includes a culture initiation amount for preferentially activating or expanding naive T cells. In some cases, the culture initiation amount includes or is determined as or based on the number of naive T cells or a subset of their CD8+ T cells in the composition. Thus, in aspects of the provided method, incubation (e.g., stimulation) is performed as long as a threshold or culture initiation amount of naive T cells is present in the input composition, regardless of the total number or percentage of non-naive T cells.

[0125] In some embodiments of the methods provided herein for incubating (e.g., stimulating) cells, the initial amount of naive-like T cells or a subset of their CD8+ T cells is from or from about 0.1 x 10⁻⁶ cells. 8 Up to 5x10 8 One, from or from about 0.1 x 10 8 Up to 4x10 8 One, from or from about 0.1 x 10 8 Up to 2x10 8 One, from or from about 0.1 x 10 8 Up to 1x10 8 One, from or from about 1x10 8 Up to 5x10 8One, from or from about 1x10 8 Up to 4x10 8 One, from or from about 1x10 8 Up to 2x10 8 One, from or from about 2 x 10 8 Up to 5x10 8 One, from or from about 2 x 10 8 Up to 4x10 8 The initial amount of naive-like T cells. In some cases, the initial amount of naive-like T cells or a subset of its CD8+ T cells is at least or at least about 0.5 x 10⁻⁶. 8 0.75x10 8 1 x 10 8 1.5 x 10 8 1, 2x10 8 One or 4x10 8 The initial amount of naive-like T cells. In some cases, the initial amount of naive-like T cells or a subset of its CD8+ T cells is at least or at least about 2 x 10^6 cells. 8 Each cell.

[0126] In some embodiments of the method for incubation under the conditions described herein, the input composition comprising a T cell population containing naive T cells and non-naive T cells contains a culture starting amount of approximately 1 x 10⁻⁶ cells. 8 Up to 4x10 8 A subset of naive T cells or their CD8+ T cells. In some embodiments, the method thereby produces a stimulated composition wherein the stimulation conditions preferentially induce the expansion or proliferation of naive T cells compared to non-naive T cells in the stimulated composition. In some aspects, the initial culture quantity of naive T cells or their CD8+ T cell subset is at least or at least about or about 2 x 10n 8 Cells. In some embodiments, the amounts of naive T cells and non-naive T cells in the input composition are approximately equal. 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 T cells in the input composition. In some aspects, the culture starting amount is determined based on the number of target non-naive T cells in the stimulated composition.

[0127] b. Non-immature T cells

[0128] In some embodiments, the method includes incubating an input composition comprising a population of T cells containing naive-like T cells and non-naive-like T cells. In some embodiments, the non-naive-like T cells include effector T cells (T... EFF ) cells, memory T cells, central memory T cells (T cells) CM), effect memory T (T EM Cells and combinations thereof. One aspect of the method of the present invention provides an input composition that has been evaluated prior to incubation (e.g., stimulation) for cell populations expressing a variety of markers (e.g., 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). In some embodiments, various markers of non-immature T cells can be utilized to identify non-immature T cells. In some embodiments, the expression of specific markers of non-immature T cells can be evaluated. For example, in some cases, non-mature T cells are surface-negative to markers (including T cell activation markers such as CD27, CD28, CD45RA, and CCR7); and in some cases, non-mature T cells are surface-positive to markers (including CD62L). In some aspects, non-mature T cells are surface-positive to CD56 and / or CD45RO. In some aspects, non-mature T cells are surface-positive to CD45RO but cell-surface negative to CD27, CD45RA, and CCR7. In some cases, non-mature T cells are positive for intracellular expression of cytokines such as IL-2, IFN-γ, IL-4, and / or IL-10. In some other examples, non-mature T cells are positive for expression of the marker CD25 and / or perforin. In some cases, non-mature T cells are CD95... hi .

[0129] In some embodiments, to assess the expression of markers on non-naive T cells, the method includes detecting the markers by performing in vitro assays. In some examples, the in vitro assays are immunoassays, aptamer-based assays, histological or cytological assays, or mRNA expression level assays. In some cases, the in vitro assays used may be enzyme-linked immunosorbent assays (ELISA), Western blotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry, surface plasmon resonance (SPR), chemiluminescence assays, lateral immunoassays, inhibition assays, or affinity assays. In some embodiments, the expression of markers on non-naive T cells is determined by RNA-seq.

[0130] The non-immature T cells in the input composition can also be assessed by T cell clonality. In some embodiments, the non-immature T cells are monoclonal. The clonality of the T cell input composition is measured by the magnitude 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 assessed by measuring the number of different epitopes recognized by antigen-specific cells. This can be done using standard techniques for generating and cloning antigen-specific T cells in vitro. In some embodiments, the non-immature T cells exhibit the advantage of a single TCR gene rearrangement pattern. In some examples, the clonality of cells in the input composition (such as non-immature T cells) is determined by clonal sequencing, optionally next-generation sequencing, or profiling analysis.

[0131] In some implementations, assessing the clonality of a T cell population is an evaluation of the clonal diversity of the T cell population. In some implementations, a monoclonal T cell population refers to a T cell population exhibiting low diversity. In the context of a T cell population (such as an input composition), monoclonality refers to a T cell population possessing a single specificity (a measure of the hypervariable library of the TCR Vβ, Vα, Vγ, or Vδ chain) as defined by profilometry. A T cell population is considered monoclonal (or single-specific) when the Vβ, Vα, Vγ, and / or Vδ profilometry of a given TCR Vβ, Vα, Vγ, and / or Vδ family has a single dominant peak. Profilometry identifies rearranged variable genes with a specific size rather than sequence. Therefore, it should be understood that a single peak can represent a population of T cells expressing any one of a limited number of rearranged TCR variable genes (Vβ, Vα, Vγ, or Vδ), which contain any one or a combination of four potential nucleotides (adenine (a), guanine (g), cytosine (c), or thymine (t)) in the linker region. In some embodiments, it may be necessary to clone specific bands and sequence them to determine one or more sequences of one or more rearranged variable genes present in a band representing a specific length.

[0132] In some embodiments, methods for assessing clonality may include various features of the methods described in International Publications WO 2012 / 048341, WO 2014 / 144495, WO 2017 / 053902, WO 2016044227, WO 2016176322, and WO2012048340, each of which is incorporated herein by reference in its entirety. In some embodiments, such methods may be used to obtain sequence information about intracellular target polynucleotides (such as TCRs). Target genes may be obtained from genomic DNA or mRNA of cells from cell samples or cell populations. Cell samples or cell populations may include immune cells. For example, for a target TCR molecule, a gene encoding the TCR chain may be obtained from the genomic DNA or mRNA of immune cells or T cells. In some embodiments, the starting material is RNA from T cells, which consists of a gene encoding the TCR chain. In some implementations, 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.

[0133] Therefore, methods for incubating (e.g., stimulating) T cells when preparing engineered T cells for adoptive therapy include those methods that preferentially induce the expansion and proliferation of cells derived from naive T cells (or derived from CD45RA- or CD45RO+ T cells) compared to those derived from non-naive T cells (or derived from CD45RA- or CD45RO+ T cells). In some embodiments, the naive T cells are CD45RA+, CD45RO-, CD27+, and CCR7+. In some embodiments, the non-naive T cells are CD45RA-, CD45RO+, CD27-, and CCR7-. In some embodiments, the method involves incubating T cells in a culture starting composition under stimulation conditions that preferentially induce the expansion or proliferation of naive T cells compared to non-naive T cells, thereby producing a stimulated composition. When using the provided methods, the evaluation of the input composition using the above-described markings and identifiers can be used as part of the methods.

[0134] B. Cell incubation

[0135] In some embodiments, the provided method includes steps of culturing, incubating, culturing, and / or genetically engineering cells in an input composition (such as an input composition containing a culture starting amount of naive T cells). For example, in some embodiments, a method is provided for incubating and / or engineering a culture starting amount of the provided input composition containing the threshold number of naive T cells. Incubation and / or engineering can be performed in a culture vessel, which is, for example, a unit, chamber, well, column, tube, tube assembly, valve, vial, culture dish, bag, or other container for culturing or cultivating cells.

[0136] In some embodiments, cells are incubated and / or cultured prior to or in conjunction with genetic engineering, as described in any of the methods described in Chapter II. The incubation step may include culturing, raising, stimulating, activating, and / or proliferating. In some embodiments, the composition or cells are incubated in the presence of stimulating conditions or stimulants. Such conditions include those designed to induce cell proliferation, expansion, activation, and / or survival in a population, mimic antigen exposure, and / or initiate cell use for genetic engineering (e.g., for the introduction of recombinant receptors, such as CARs).

[0137] The conditions may include one or more of the following: specific culture medium, temperature, oxygen content, carbon dioxide content, time, agents (e.g., nutrients, amino acids, antibiotics, ions and / or stimulating factors (such as cytokines, chemokines, antigens, binding couplers, fusion proteins, recombinant soluble receptors and any other agents designed to activate cells)).

[0138] In some embodiments, the stimulating condition or stimulant includes one or more agents (e.g., ligands) capable of activating intracellular signaling domains of the TCR complex. In some aspects, the agent opens or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such agents may include antibodies, such as those specific to the TCR, e.g., anti-CD3. In some embodiments, the stimulating condition includes one or more agents (e.g., ligands) capable of stimulating co-stimulatory receptors, e.g., anti-CD28. In some embodiments, such agents and / or ligands may bind to a solid support (e.g., beads) and / or one or more cytokines. Optionally, the amplification method may also include the step of adding anti-CD3 and / or anti-CD28 antibodies to a culture medium (e.g., at a concentration of at least about 0.5 ng / ml).

[0139] The provided methods typically involve the presence, design, and / or use of stimulating conditions that preferentially induce the expansion or proliferation of naive-like T cells relative to non-naive-like T cells, and / or do not preferentially 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 will be activated in a manner that induces cell death. Such preferential conditions are typically used at a stage prior to the introduction of nucleic acids encoding engineered molecules, such as engineered antigen receptors.

[0140] The 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 stimulus (e.g., activation) signal sufficient to activate and / or induce proliferation or division of non-immature T cells or subsets thereof. Immature T cells typically require a minimum signal of TCR / CD3 to reach an activation threshold, e.g., to be fully activated and driven into the cell cycle. This minimum signal is typically higher than the signal required to induce activation / cell cycle entry of non-immature T cells and / or certain subsets thereof. Non-immature T cells typically require a much lower level of TCR / CD3 binding to be activated and enter the cell cycle. In some aspects, stronger signals can cause activation-induced cell death in non-immature cells or certain populations thereof, or increase the level of activation-induced cell death in non-immature cells or certain populations thereof.

[0141] Therefore, in some embodiments, the composition comprises naive and non-naive T cell populations, and conditions are used to induce an activation signal below the activation threshold required for naive T cell activation. In this case, the non-naive T cells are first activated and made susceptible to stimuli that can lead to cell death. For example, under specific stimuli, cell death may be caused by activation-induced cell death.

[0142] In some aspects, the stimulatory conditions induce activation-induced cell death of non-immature cells compared to other conditions (such as standard conditions). Therefore, the present invention provides a method for eliminating at least a substantial portion of any unwanted T cell (e.g., non-immature T cell) subsets from an input composition. For the purposes of the provided method, a substantial portion means at least 70% of the unwanted cell (e.g., non-immature T cell) subsets from the input composition. In some embodiments, a substantial portion means 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and more of the unwanted cell (e.g., non-immature T cell) subsets from the input composition. Various techniques can be used to measure cell clearance (e.g., cell death by non-immature T cells), including but not limited to flow cytometry analysis using various antibodies and / or peptide-MHC tetramers, and functional assays (e.g., proliferation and chromium release assays).

[0143] In some embodiments, an enzyme is added to the input composition to remove residual cellular components through cellular elimination. For example, in some exemplary embodiments, deoxyribonuclease (DNAase) or recombinant human deoxyribonuclease I is added. Add to the input composition. In some aspects, the stimulating conditions include deoxyribonuclease (DNase) or recombinant human deoxyribonuclease I.

[0144] In some cases, the stimulating conditions do not include culture components supplemented to preserve a specific subset of T cells (e.g., non-immature T cells). Therefore, in some cases, removing components from the culture of the stimulating conditions may help eliminate non-immature T cells. In some aspects, stimulating conditions are performed or additionally performed by excluding or reducing the concentration of culture agents known to or potentially reducing AICD and / or promoting the survival of older cells (e.g., non-immature cells). In some cases, the stimulating conditions do not include N-acetylcysteine ​​or include reduced amounts or concentrations of N-acetylcysteine. In some cases, the stimulating conditions do not include a 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) may be included, which additionally aid or promote the removal of non-immature cells.

[0145] In some embodiments, the stimulation and / or amplification time can be between 2 and 15 days, between 2 and 12 days, between 2 and 10 days, between 2 and 8 days, between 2 and 6 days, between 2 and 4 days, between 4 and 12 days, between 4 and 10 days, between 4 and 8 days, between 4 and 6 days, between 6 and 12 days, between 6 and 10 days, between 6 and 8 days, between 8 and 12 days, between 8 and 10 days, or between 10 and 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 more than 14 days. In one embodiment of the provided method, the mixture can be cultured for 30 minutes to several hours (about 3 hours) to about 14 days or any integer value of hour or minute in between. In another embodiment, the mixture can be cultured for 21 days. In one embodiment, the beads and T cells are cultured together for approximately eight days. In another embodiment, the beads and T cells are cultured together for at least, or at least approximately, two to three days. In yet another embodiment, the beads and T cells are cultured together for at least, or at least approximately, two days, or at least, or at least approximately, 48 hours. In some aspects, several stimulation cycles may be required, allowing the T cell culture time to be 60 days or longer.

[0146] Incubation and / or engineering can be carried out in culture vessels, such as units, chambers, wells, columns, tubes, tube assemblies, valves, vials, culture dishes, bags, or other containers used for culturing or cultivating cells. In some embodiments, the composition or cells are incubated in the presence of stimulating conditions or stimulants. Such conditions include those designed to induce cell proliferation, expansion, activation, and / or survival in a population, simulate antigen exposure, and / or induce cells for genetic engineering (e.g., for the introduction of recombinant antigen receptors).

[0147] The provided methods typically involve incubation under a stimulating condition (e.g., incubation with an input composition containing T cells). In the context of T cell stimulation, the stimulating condition typically includes a primary agent capable of binding, linking, crosslinking the TCR complex or a member thereof (such as CD3) and / or inducing activation of intracellular signaling domains via the TCR complex or a member thereof (such as CD3). In some embodiments, the stimulating condition includes a primary agent that specifically binds to a member of the TCR complex and a secondary agent that specifically binds to a T cell co-stimulatory molecule. In some specific examples, the primary agent specifically binds to CD3 and / or the co-stimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS.

[0148] In some embodiments, the stimulating conditions include immobilized anti-CD3 and anti-CD28 antibodies, soluble anti-CD3 antibodies, derivatives of such antibodies, and / or other ligands that bind to the TCR / CD3 complex on T cells. In some aspects, a primary agent opens or initiates the intracellular TCR / CD3 signaling cascade in T cells. Such agents may include binding partners, such as natural ligands, and / or antibodies, including antigen-binding antibody fragments, such as those specific to TCR components (e.g., CD3). Exemplary anti-CD3 antibodies are BC3, OKT3, and G19-4.

[0149] In some embodiments, the stimulation condition further includes incubation with a secondary agent, such as an agent capable of inducing T cell co-stimulatory signals (e.g., signals that combine with primary signals (e.g., TCR / CD3 linkage) leading to T cell proliferation and / or activation) to T cells. Exemplary secondary agents are those that specifically bind, link, or crosslink T cell co-stimulatory molecules or accessory molecules (e.g., CD28, CD137(4-1-BB), OX40, ICOS, CD40, LFA-1, DAP10, and / or CD54) and / or induce intracellular signaling events therethrough. Agents include antibodies (including fragments thereof), natural ligands, and other binding partners. In some embodiments, the secondary agent binds to CD28, such as an anti-CD28 antibody (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.

[0150] In some embodiments, the binding partner or agent that specifically binds to a particular molecule (such as a T-cell stimulating or co-stimulating molecule) includes an antibody specific to such a molecule, including an antigen-binding antibody fragment. In some aspects, the agent contains an antibody, such as an anti-CD3 and / or anti-CD28 antibody, at a concentration of at least or at least about 0.5 ng / mL. In some aspects, the agent includes one or more other binding partners of such a molecule, such as a natural binding partner. The agent may also include natural ligands and complexes, including molecules and / or complexes on antigen-presenting cells and / or superantigens (Staphylococcus enterotoxin A (SEA), Staphylococcus enterotoxin B (SEB), Toxic Shock Syndrome Toxin 1 (TSST-1), endotoxins). Other exemplary agents are those that mimic signaling via primary or co-stimulatory T cell signaling molecules such as mitogens (including PKC activators), phorbol myristate acetate (PMA), phytohemagglutinin (PHA), and / or calcium ion carriers (e.g., ionomycin), lipopolysaccharide (LPS), T cell mitogens, and cytokines. In some aspects, the conditions include incubation with one or more stimulating 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.

[0151] In some embodiments, primary and secondary (co-stimulatory) agents are bound to a solid surface or support (such as particles, e.g., beads). In some embodiments, cells may be incubated and / or contacted with a stimulant capable of activating and / or amplifying T cells. In some aspects, primary and secondary agents are coupled to a solid surface (such as particles, e.g., beads). In some embodiments, the stimulant comprises particles (e.g., beads) conjugated or linked to one or more agents (e.g., biomolecules) capable of activating and / or amplifying cells (e.g., T cells). In some embodiments, the one or more agents are bound to beads. In some embodiments, the beads are biocompatible, i.e., constructed of materials suitable for biological use. In some embodiments, the beads are non-toxic to the cultured cells (e.g., cultured T cells). In some embodiments, the beads can be any particle capable of attaching the agent in a manner that allows interaction between the agent and the cells.

[0152] In some embodiments, the stimulant comprises one or more agents capable of activating and / or amplifying cells (e.g., T cells), said one or more agents being bound to or otherwise attached to the beads, such as bound to or attached to the surface of the beads. In some embodiments, the beads are non-cellular particles. In certain embodiments, the beads may include colloidal particles, microspheres, nanoparticles, magnetic beads, etc. In some embodiments, the beads are agarose beads. In some embodiments, the beads are agarose gel beads.

[0153] In certain embodiments, the stimulant comprises monodisperse beads. In some embodiments, the monodisperse beads comprise a size dispersion in which the standard deviation of their diameters from each other is less than 5%.

[0154] In some embodiments, the beads contain one or more agents, such as agents coupled, conjugated, or linked (directly or indirectly) to the surface of the beads. In some embodiments, agents such as those considered herein may include, but are not limited to, RNA, DNA, proteins (e.g., enzymes), antigens, polyclonal antibodies, monoclonal antibodies, antibody fragments, carbohydrates, lipid lectins, or any other biomolecules with affinity for the desired target. In some embodiments, the desired target is a T-cell receptor and / or a component of a T-cell receptor. In some embodiments, the desired target is CD3. In some embodiments, the desired target is a co-stimulatory molecule, such as CD28. The one or more agents may be attached to the beads directly or indirectly 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 attached to the beads indirectly by another biomolecule (e.g., an anti-biotinylated antibody) that is directly attached to the beads.

[0155] In some embodiments, one or more agents attached to the bead are antibodies. Antibodies may include polyclonal antibodies, monoclonal antibodies (including full-length antibodies having the Fc region of an immunoglobulin), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies, biantibodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab')2, and Fv). In some embodiments, the stimulating agent is an antibody fragment (including an antigen-binding fragment), such as Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragments. It should be understood that any isotype constant region can be used for the antibodies considered herein, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species (e.g., rodent species). In some embodiments, the agent is an antibody that binds to and / or recognizes one or more components of a T-cell receptor. In a particular embodiment, the agent is an anti-CD3 antibody. In some embodiments, the agent is an antibody that binds to and / or recognizes a co-receptor. In some embodiments, the stimulating agent comprises an anti-CD28 antibody.

[0156] In some embodiments, the diameter of the beads is greater than about 0.001 μm, greater than about 0.01 μm, greater than about 0.1 μm, greater than about 1.0 μm, greater than about 10 μm, greater than about 50 μm, greater than about 100 μm, or greater than about 1000 μm but not exceeding about 1500 μm. In some embodiments, the diameter of the beads is about 1.0 μm to about 500 μm, about 1.0 μm to about 150 μm, about 1.0 μm to about 30 μm, about 1.0 μm to about 10 μm, about 1.0 μm to about 5.0 μm, about 2.0 μm to about 5.0 μm, or about 3.0 μm to about 5.0 μm. In some embodiments, the diameter of the beads is about 3 μm to about 5 μm. In some embodiments, the diameter of the beads is at least or at least about or about 0.001 μm, 0.01 μm, 0.1 μm, 0.5 μm, 1.0 μm, 1.5 μm, 2.0 μm, 2.5 μm, 3.0 μm, 3.5 μm, 4.0 μm, 4.5 μm, 5.0 μm, 5.5 μm, 6.0 μm, 6.5 μm, 7.0 μm, 7.5 μm, 8.0 μm, 8.5 μm, 9.0 μm, 9.5 μm, 10 μm, 12 μm, 14 μm, 16 μm, 18 μm, or 20 μm. In some embodiments, the diameter of the beads is or is about 4.5 μm. In some embodiments, the diameter of the beads is or is about 2.8 μm.

[0157] In some implementations, the density of the beads is greater than 0.001 g / cm³. 3 Greater than 0.01 g / cm 3 Greater than 0.05 g / cm 3 Greater than 0.1 g / cm 3 Greater than 0.5 g / cm 3 Greater than 0.6 g / cm 3 Greater than 0.7 g / cm 3 Greater than 0.8 g / cm 3 Greater than 0.9 g / cm 3 Greater than 1g / cm 3 Greater than 1.1 g / cm 3 Greater than 1.2 g / cm 3 Greater than 1.3 g / cm 3 Greater than 1.4 g / cm 3 Greater than 1.5 g / cm 3 Greater than 2g / cm 3 Greater than 3g / cm 3 Greater than 4g / cm 3 or greater than 5g / cm 3 In some implementations, the density of the beads is approximately 0.001 g / cm³. 3 With approximately 100g / cm 3Approximately 0.01 g / cm³ 3 With approximately 50g / cm 3 Approximately 0.1 g / cm³ 3 With approximately 10g / cm 3 Approximately 0.1 g / cm³ 3 With approximately 0.5g / cm 3 Approximately 0.5 g / cm 3 With approximately 1g / cm 3 Approximately 0.5 g / cm 3 With approximately 1.5 g / cm 3 Approximately 1g / cm 3 With approximately 1.5 g / cm 3 Approximately 1g / cm 3 With approximately 2g / cm 3 or about 1g / cm 3 With approximately 5g / cm 3 Between. In some implementations, the density of the beads is approximately 0.5 g / cm³. 3 Approximately 0.6 g / cm³ 3 Approximately 0.7 g / cm³ 3 Approximately 0.8 g / cm³ 3 Approximately 0.9 g / cm³ 3 Approximately 1.0 g / cm³ 3 Approximately 1.1 g / cm³ 3 Approximately 1.2 g / cm³ 3 Approximately 1.3 g / cm³ 3 Approximately 1.4 g / cm³ 3 Approximately 1.5g / cm 3 Approximately 1.6 g / cm³ 3 Approximately 1.7 g / cm³ 3 Approximately 1.8 g / cm³ 3 Approximately 1.9 g / cm³ 3 Or approximately 2.0 g / cm³ 3 In some embodiments, the density of the beads is approximately 1.6 g / cm³. 3 In a particular embodiment, the density of the beads or particles is approximately 1.5 g / cm³. 3 In some embodiments, the particle density is approximately 1.3 g / cm³. 3 .

[0158] In some embodiments, the multiple beads have a uniform density. In some embodiments, uniform density includes a density standard deviation of less than 10%, less than 5%, or less than 1% of the average bead density.

[0159] In some implementations, the surface area of ​​the beads is approximately 0.001 m². 2 / gram of particles (m 2 / g) to about 1,000m2 / g, approximately 0.010m 2 / g to approximately 100m 2 / g, approximately 0.1m 2 / g to approximately 10m 2 / g, approximately 0.1m 2 / g to approximately 1m 2 / g, approximately 1m 2 / g to approximately 10m 2 / g, approximately 10m 2 / g to approximately 100m 2 / g, approximately 0.5m 2 / g to approximately 20m 2 / g, approximately 0.5m 2 / g to approximately 5m 2 / g or approximately 1m 2 / g to approximately 4m 2 Between / g. In some embodiments, the surface area of ​​the particles or beads is approximately 1m². 2 / g to approximately 4m 2 / g.

[0160] In some embodiments, the beads contain at least one material on or near the surface of the beads that can be coupled, linked, or conjugated with a pharmaceutical agent. In some embodiments, the beads are surface-functionalized, i.e., containing functional groups capable of forming covalent bonds with binding molecules (e.g., polynucleotides or polypeptides). In specific embodiments, the beads contain surface-exposed carboxyl, amino, hydroxyl, toluenesulfonyl, epoxy, and / or chloromethyl groups. In specific embodiments, the beads contain surface-exposed agarose and / or agarose gel. In some embodiments, the bead surface contains an attached stimulating agent that can bind or attach binding molecules. In specific embodiments, the biomolecule is a polypeptide. In some embodiments, the beads contain surface-exposed protein A, protein G, or biotin.

[0161] In some embodiments, the beads react in a magnetic field. In some embodiments, the beads are magnetic beads. In some embodiments, the magnetic beads are paramagnetic. In certain embodiments, the magnetic beads are superparamagnetic. In some embodiments, the beads do not exhibit any magnetic properties unless they are exposed to a magnetic field.

[0162] In certain embodiments, the bead comprises a magnetic core, a paramagnetic core, or a superparamagnetic core. In some embodiments, the magnetic core contains a metal. In some embodiments, the metal may be, but is not limited to, iron, nickel, copper, cobalt, gadolinium, manganese, tantalum, zinc, zirconium, or any combination thereof. In some embodiments, the magnetic core comprises a metal oxide (e.g., iron oxide), a ferrite (e.g., manganese ferrite, cobalt ferrite, nickel ferrite, etc.), hematite, and a metal alloy (e.g., CoTaZn). In some embodiments, the magnetic core comprises one or more of a ferrite, a metal, a metal alloy, iron oxide, or chromium dioxide. In some embodiments, the magnetic core comprises elemental iron or a compound thereof. In some embodiments, the magnetic core comprises one or more of magnetite (Fe3O4), maghematite (γFe2O3), or pyrite (Fe3S4). In some embodiments, the core comprises iron oxide (e.g., Fe3O4).

[0163] In some embodiments, the beads contain magnetic, paramagnetic, and / or superparamagnetic cores covered by a surface-functionalized coating (coat or coating). In some embodiments, the coating may contain materials that may include, but are not limited to, polymers, polysaccharides, silica, fatty acids, proteins, carbon, agarose, agarose gel, or combinations thereof. In some embodiments, the polymer may be polyethylene glycol, poly(lactic-co-glycolic acid), polyglutaraldehyde, polyurethane, polystyrene, or polyvinyl alcohol. In some embodiments, the outer coating (coat or coating) comprises polystyrene. In certain embodiments, the outer coating is surface-functionalized.

[0164] In some embodiments, the stimulating agent comprises beads containing a metal oxide core (e.g., an iron oxide core) and a coating, wherein the metal oxide core comprises at least one polysaccharide (e.g., dextran), and wherein the coating comprises at least one polysaccharide (e.g., aminoglucan), at least one polymer (e.g., polyurethane), and silica. In some embodiments, the metal oxide core is a colloidal iron oxide core. In some embodiments, the one or more agents comprise an antibody or an antigen-binding fragment thereof. In specific embodiments, the one or more agents comprise an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the stimulating agent comprises an anti-CD3 antibody, an anti-CD28 antibody, and an anti-biotin antibody. In some embodiments, the stimulating agent comprises an anti-biotin antibody. In some embodiments, the diameter of the beads is from about 3 μm to about 10 μm. In some embodiments, the diameter of the beads is from about 3 μm to about 5 μm. In some embodiments, the diameter of the beads is about 3.5 μm.

[0165] In some embodiments, the stimulating agent comprises one or more agents attached to the beads, the beads comprising a metal oxide core (e.g., an iron oxide core) and a coating (e.g., a protective coating), wherein the coating comprises polystyrene. In some embodiments, the beads are monodisperse superparamagnetic beads comprising a superparamagnetic core (e.g., a core comprising magnetite (Fe3O4) and / or maghemite (γFe2O3)) and a polystyrene coating (coat or coating). In some embodiments, the beads are non-porous. In some embodiments, the beads contain a functionalized surface to which the one or more agents are attached. In some embodiments, the one or more agents are covalently bound to the beads on the surface. In some embodiments, the one or more agents comprise an antibody or an antigen-binding fragment thereof. In some embodiments, the one or more agents comprise anti-CD3 antibodies and anti-CD28 antibodies. In some embodiments, the beads have a concentration of about 1.5 g / cm³. 3 The density and approximately 1m 2 / g to approximately 4m 2 / g surface area. In a particular embodiment; the beads are monodisperse superparamagnetic beads having a diameter of about 4.5 μm and a surface area of ​​about 1.5 g / cm². 3 The density. In some embodiments, the beads have an average diameter of about 2.8 μm and a density of about 1.3 g / cm³. 3 Monodisperse superparamagnetic beads with high density.

[0166] To achieve the separation of different T cell populations, the exposure time to particles can be varied. For example, in a preferred embodiment, by exposing the particles to 3 × 28 beads (e.g., ... M-450 or CD3 / CD28CTS TM The T cells are incubated together for a period of time sufficient to positively select the desired T cells to isolate them. In one embodiment, the incubation period is about 30 minutes. In other embodiments, the incubation period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the incubation period is 10 to 24 hours or longer. In one preferred embodiment, the incubation period is 24 hours. Using a longer incubation time (such as 24 hours) can increase cell yield in order to isolate T cells from cancer patients.

[0167] When coupled to a surface, the agent can be coupled to the same surface (i.e., in the "cis" form) or to a separate surface (i.e., in the "trans" form). Alternatively, one agent can be coupled to a surface while the other is in solution. In one embodiment, the agent providing a co-stimulatory signal is bound to the cell surface, and the agent providing a primary activation signal is in solution or coupled to the surface. In a preferred embodiment, both agents are immobilized on beads, either on the same bead ("cis") or on separate beads ("trans"). For example, the agent providing the primary activation signal is an anti-CD3 antibody, and the agent providing the co-stimulatory signal is an anti-CD28 antibody; and both agents are co-immobilized to the same bead at the same molecular weight. In one embodiment, for CD4+ T cell expansion and T cell growth, a 1:1 ratio of each antibody bound to the bead is used. In some aspects of the invention, a certain ratio of anti-CD3:CD28 antibodies bound to the bead is used such that an increase in T cell expansion is observed compared to 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 amplification observed using a 1:1 ratio. In one embodiment, the CD3:CD28 ratio bound to the beads ranges from 100:1 to 1:100 and all integer values ​​therein. In one aspect of the invention, more anti-CD28 antibody binds to the particles compared to the anti-CD3 antibody, i.e., the CD3:CD28 ratio is less than one. In some embodiments of the invention, the ratio of anti-CD28 antibody bound to the beads to anti-CD3 antibody is greater than 2:1. In one particular embodiment, a 1:200 CD3:CD28 ratio of antibody bound to beads is used. In one particular embodiment, a 1:150 CD3:CD28 ratio of antibody bound to beads is used. 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 other embodiments, a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:45 CD3:CD28 ratio of the antibody bound to the beads is used. In another embodiment, a 1:40 CD3:CD28 ratio of the antibody bound to the beads is used. In another embodiment, a 1:35 CD3:CD28 ratio of the antibody bound to the beads is used. In another embodiment, a 1:30 CD3:CD28 ratio of the antibody bound to the beads is used. In another embodiment, a 1:25 CD3:CD28 ratio of the antibody bound to the beads is used. In another embodiment, a 1:20 CD3:CD28 ratio of the antibody bound to the beads is used. In another embodiment, a 1:15 CD3:CD28 ratio of the antibody bound to the beads is used.In one embodiment, a 1:10 CD3:CD28 ratio of the antibody bound to the beads is used. In another embodiment, a 1:5 CD3:CD28 ratio of the antibody bound to the beads is used. In yet another embodiment, a 1:4 CD3:CD28 ratio of the antibody bound to the beads is used. In yet another embodiment, a 1:3 CD3:CD28 ratio of the antibody bound to the beads is used. In still another embodiment, a 3:1 CD3:CD28 ratio of the antibody bound to the beads is used.

[0168] In some embodiments, an agent (e.g., an antibody) is added to the culture or composition in a soluble form. In some embodiments, the soluble form includes one agent and another agent conjugated to a solid support. In some aspects, where two or more agents are conjugated to one or more solid supports, the two agents are conjugated to the same support or particles, such as anti-CD3 / anti-CD28 beads containing antibodies that recognize CD3 and CD28. In other aspects, the two or more agents are conjugated to a separate support (such as separate beads). For example, in some aspects, anti-CD3 and anti-CD28 beads are added to the culture separately.

[0169] 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 capable of enhancing signaling via the TCR complex, such as anti-CD3 and / or anti-CD28 antibodies. Exemplary antigen-presenting cells are genetically modified cells, such as bone marrow cells (e.g., K562 or U937) engineered to express Fc receptors (such as the CD32 intermediate-affinity or CD64 high-affinity Fc receptor). Such cells may be loaded with anti-CD3 and / or anti-CD28 antibodies recognized by the Fc receptor and incubated with T cells to deliver signals in the culture. Exemplary artificial APCs and the ratios and methods used in their culture conditions are described, for example, in Suhoski et al., Molecular Therapy (2007) 15 5, 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) carrying antigens, such as TCR complexes, or antigens recognized by other receptors on cells to be transduced (such as T cells specific to a particular tumor antigen).

[0170] In some embodiments, preferential expansion or proliferation of naive T cells relative to immature T cells is achieved by using stimulation conditions designed to induce a specific signal intensity, such as a stimulation or activation signal intensity above a certain level, for example, to induce cell death in immature T cells. In some aspects, a stronger signal induces cell death in immature T cells receiving the composition, while a weaker signal, compared to a stronger signal, does not induce cell death in immature T cells receiving the composition and / or maintains the survival of immature T cells. Therefore, in some embodiments, a stronger signal preferentially activates or induces cell death in immature T cells.

[0171] In some aspects, preferential expansion or proliferation of naive T cells relative to non-naive T cells is achieved through a specific bead-to-cell ratio. In some embodiments, any stimulation condition can be employed that favors the expansion or survival of naive T cells relative to non-naive T cells. In some embodiments, the stimulation condition includes incubation in the presence of a bead reagent (e.g., anti-CD3 / anti-CD28 bead reagent) containing primary and / or secondary signals for T cell activation, the bead reagent being provided, for example, in an amount favorable to naive cell proliferation and / or survival and / or preferential induction of activation-induced cell death (AICD) of non-naive 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 drive toward more AICD and an increased percentage of naive cell survival, see, for example, Kalamasz et al., J Immunother. (2004) 27(5):405-418 and U.S. Patent Nos. 7,977,095 and 9,528,088. In one embodiment, the ratio is from about 50:1 to about 5:1. In some embodiments, the ratio is from about 100:1 to about 1:1. In one embodiment, the ratio is at least about 45:1. In some 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 less than 5:1 or less than 3:1 is used. In one particular embodiment, the ratio is about 3:1.

[0172] In one embodiment of the provided method, the bead-to-cell ratio can be customized to obtain a desired T cell phenotype. In a particular embodiment, the bead-to-cell ratio can be altered to selectively expand or remove a subset of T cells. In one embodiment, the specific bead-to-cell ratio used selectively induces cell death in non-naïve T cells or cells derived from non-naïve T cells in the input composition. In other embodiments, the specific bead-to-cell ratio used selectively expands naïve T cells or cells derived from naïve T cells. In some embodiments, the specific ratio can be used whenever the expansion or removal of the desired subset of T cells occurs. Therefore, the compositions and methods described herein can be used to expand or remove specific T cell populations for use in any of the various immunotherapeutic settings described herein.

[0173] Stimulation conditions suitable for incubating (e.g., stimulating) T cells are also provided. Suitable conditions for T cell culture include appropriate culture media (e.g., OpTmizer). TM (Gibco) or minimally essential medium or RPMI 1640 or X-vivo 15 (BioWhittaker) may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), serum alternatives or interleukin-2 (IL-2), insulin or any other additives for cell growth. The medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo20, supplemented with amino acids and vitamins, serum-free or supplemented with adequate amounts of serum (or plasma) or serum alternatives or a defined hormone group and / or one or more cytokines sufficient for T cell growth and expansion. Antibiotics (e.g., penicillin and streptomycin) are included only in the experimental culture and not in the cell culture to be infused into the subject. Target cells are maintained under conditions necessary to support growth (e.g., appropriate temperature (e.g., 37°C) and atmospheric conditions (e.g., air plus 5% CO2)).

[0174] In another implementation, the duration of exposure to stimulants (such as anti-CD3 / anti-CD28 (i.e., 3×28) coated beads) can be modified or customized to achieve the desired T cell phenotype. More helper T cells (TH), typically CD4+ rather than CD8+ cytotoxic or regulatory T cells, may be required, as TH cell expansion can improve or restore overall immunoreactivity. While many specific immune responses are mediated by CD8+ antigen-specific T cells that can directly lyse or kill target cells, most immune responses require the help of CD4+ T cells, for example, those expressing important immunomodulatory molecules such as GM-CSF, CD40L, and IL-2. Where CD4-mediated helper T cells are preferred, methods for maintaining or increasing the CD4:CD8 ratio (as described herein) can have significant benefits. An increase in the number of CD4+ T cells can increase the amount of CD40L expressed by cells introduced into the patient, potentially improving target cell visibility (improved APC function). Similar effects can be seen by increasing the number of infused cells expressing GM-CSF or IL-2 (both of which are primarily expressed by CD4+ T cells). Similarly, in some applications, it may be necessary to utilize regulatory T cell populations (e.g., Autoimmun Rev. Aug. 2002; 1(4):190-7; Curr Opin Immunol. Dec. 2002; 14(6):771-8), which can be generated and expanded using the methods described herein. Alternatively, in cases where less CD4 helper is needed and an increase in the number of CD8+ T cells is desired, XCELLERATE as described herein can also be utilized, for example, by pre-selecting CD8+ cells prior to stimulation and / or culture. TM Methods. It is possible that increased IFN-γ levels or increased target cell lysis are preferred. The timing and type of exposure to stimulants can also be modified to expand T cells with the desired TCR repertoire (e.g., expression of desired Vβ family genes).

[0175] In some implementations, other stimulating conditions may include one or more of the following: specific culture medium, temperature, oxygen content, carbon dioxide content, time, agents (e.g., nutrients, amino acids, antibiotics, ions and / or stimulating factors (e.g., cytokines, chemokines, antigens, binding couplers, fusion proteins, recombinant soluble receptors and any other agents designed to activate cells)).

[0176] In some embodiments, the cells or composition are assessed and / or adjusted during the incubation step. For example, assessment and / or adjustment can occur at any time after the start of incubation or culture, such as during the incubation period. Assessment may include one or more measurements of the composition or container containing cells, such as assessing cell proliferation rate, viability, phenotype (e.g., expression of one or more surface or intracellular markers (e.g., proteins or polynucleotides), and / or assessing the temperature of the composition or container, one or more culture medium components, oxygen or carbon dioxide content, and / or one or more factors, agents, components, and / or cell types (including subtypes), their presence or absence, or their quantity or relative amount. Assessment may also include assessing indicators or predictors of toxicity outcomes, such as those described herein in vitro or ex vivo.

[0177] In some respects, the evaluations are performed automatically, for example using devices as described herein, and / or pre-programmed to be performed at certain points during incubation. In other respects, the evaluation results indicate that adjustments should be made.

[0178] Adjustments may include adjusting any cell culture factors or parameters, such as temperature, the length (time) of the incubation or its steps (incubation duration), adding, supplementing, and / or removing one or more components of the incubated composition, such as culture medium or buffer or its components, agents (e.g., nutrients, amino acids, antibiotics, ions and / or stimulating factors (e.g., cytokines, chemokines, antigens, binding couplers, fusion proteins, recombinant soluble receptors)), or cells, cell types, or cell populations. In some aspects, the removal or addition of various components or other adjustments are performed automatically, for example using devices or systems as described herein. In some embodiments, the system is programmed such that adjustments are automatically initiated based on a readout from a provisional assessment. 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 may be further programmed such that a specific result of such assessment (e.g., a specific ratio of one cell type to another) triggers a specific adjustment, such as adding one or more cell types.

[0179] In some aspects, adjustments are made by adding or removing components in a manner that does not disrupt the closed environment containing the cells and composition, such as by adding or removing via inlet and / or outlet valves designed to add or remove components while maintaining sterility, as in one or more devices or systems as described herein. Various adjustments to the 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. US 20030235908 A1.

[0180] In some implementations, cells are incubated for a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or for 1, 2, 3, 4 weeks or more, or about 1, 2, 3, 4 weeks or more. In some examples, incubation is performed for more than or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days.

[0181] In some respects, incubation is carried out using a variety of techniques, such as those described in the following documents: U.S. Patent No. 6,040,177 to Riddell et al.; Klebanoff et al. (2012) J Immunother. 35(9):651-660; Terakura et al. (2012) Blood. 1:72-82; and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0182] In some embodiments, the stimulation conditions include the addition of feeder cells (such as non-dividing peripheral blood mononuclear cells (PBMCs)) (e.g., such that for each T lymphocyte in the initial population to be expanded, the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells); and incubation of the culture (e.g., for a duration sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells may comprise γ-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with γ 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 T cell population.

[0183] In some embodiments, stimulation conditions include a temperature suitable for the growth of human T lymphocytes, such as at least about 25 degrees Celsius, typically at least about 30 degrees Celsius, and typically about 37 degrees Celsius. Optionally, incubation may also include the addition of non-dividing EBV-transformed lymphoblastoid cells (LCLs) as feeder cells. LCLs can be irradiated with gamma rays in the range of about 6,000 to 10,000 rads. In some aspects, LCL feeder cells are provided in any suitable amount (e.g., an LCL feeder cell to naïve T lymphocyte ratio of at least about 10:1).

[0184] In the implementation scheme, 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 cytomegalovirus antigens by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen.

[0185] Incubation and / or engineering can be carried out in culture containers, such as units, chambers, wells, columns, tubes, tube assemblies, valves, vials, petri dishes, bags, or other containers used for culturing or cultivating cells.

[0186] Also provided are the culture starting compositions (such as those containing T cells (e.g., human primary T cells)) and stimulation conditions (e.g., various agents of concentration / ratio designed to preferentially activate or expand non-naïve T cells) used in the method.

[0187] In some implementations, the cells are engineered, for example, to introduce genetically engineered antigen receptors, and incubation in the presence of one or more stimulants continues during the engineering phase.

[0188] A bead-to-cell ratio ranging from 1:500 to 500:1 and any integer values ​​in between can 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 may bind only a few cells, while larger beads may bind many cells. In some embodiments, the cell-to-particle ratio ranges from 1:100 to 100:1 and any integer values ​​in between, and in other embodiments, the ratio includes 1:50 to 50:1 and any integer values ​​in between. In yet another embodiment, a cell-to-particle ratio ranging from 1:9 to 9:1, and any integer values ​​in between, can also be used to stimulate T cells. As described herein, the ratio of anti-CD3 and anti-CD28 coupled granules to T cells leading to T cell stimulation can vary, 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 / T cells. In one particular embodiment, a preferred beads-to-cell ratio is 3:1. In some cases, the beads-to-cell ratio is 1:3.

[0189] In other embodiments, the particle-to-cell ratio can vary depending on the date of stimulation. For example, in one embodiment, the particle-to-cell ratio is from 1:1 to 10:1 on the first day, and additional particles are added to the cells daily or every other day for up to 10 days, resulting in a final ratio from 1:1 to 1:10 (based on cell counts 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, resulting in a ratio of about 1:10, 1:25, 1:50, or 1:100 on day 5, 1:10, 1:25, 1:50, or 1:100 on day 7, and 1:10, 1:25, 1:50, or 1:100 on day 9. In one particular embodiment, the particle-to-cell ratio is 1:1 on the first day of stimulation and adjusted to 1:5 on days 3 and 5 of stimulation. In another embodiment, particles are added daily or every other day until the final ratio is 1:1 on the first day, and the final ratio is 1:5 on days 3 and 5 of stimulation. In another embodiment, the particle-to-cell ratio is 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In yet another embodiment, particles are added daily or every other day until a final ratio of 1:1 is achieved on the first day, and a final ratio of 1:10 is achieved on the third and fifth days of stimulation. In some respects, a variety of other ratios may be applicable to the invention. In particular, the ratio will vary depending on particle size as well as cell size and type.

[0190] One aspect of the invention relates to the observation that using different bead-to-cell ratios (e.g., anti-CD3 / anti-CD28 bead reagents) can lead to different results regarding the expansion of antigen-specific T cells. Specifically, the bead-to-cell ratio can be varied to selectively expand or remove antigen-specific or antigen-experienced (e.g., memory, effector, or activated) T cells compared to other cell types (e.g., naive or naive-like T cells). In one embodiment, a specific bead-to-cell ratio used selectively removes antigen-specific T cells. Specifically, bead-to-cell ratios (e.g., bead-to-cell ratios of 1:1 or greater and / or high bead-to-cell ratios, 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 removal of antigen-specific T cells. Without being bound by theory, antigen-specific T cells are believed to be sensitive to further stimulation. Therefore, in some embodiments, the strength of the activating signal delivered to T cells by the reagent or composition can influence the expansion and / or death of one or more specific subsets of T cells. In some aspects, selective expansion of memory T cells (antigen-specific T cells) can occur accompanied by a signal (weaker than other signals) via TCR and / or co-receptors; however, in some embodiments, selective removal of memory T cells and / or other antigen-experienced T cells while preserving naive cells can occur after incubation with a reagent delivering a relatively strong signal. In some aspects, the number of ligand-bound CD3 / TCR (and CD28) receptors can particularly determine or contribute to determining the signal strength. Therefore, in some situations, stimulation with a high bead-to-cell ratio can provide a high concentration of stimulating antibodies (i.e., a “strong” signal), leading to overstimulation of antigen-specific T cells, resulting in their death via apoptosis or other mechanisms. Therefore, in this respect, the bead compositions described herein can act as pro-apoptotic compositions in some situations and / or with respect to certain compositions. In some aspects, the signal should not be so strong as to kill naive-like T cells, as also by activation-induced cell death. In some implementations, the ratio of stimulating bead reagents (e.g., anti-CD3 / anti-CD28 bead reagents) is less than 10:1.

[0191] Furthermore, in this regard, in some embodiments, such a reagent or composition (e.g., a surface of an agent having a stimulating cell surface portion attached thereto, such as a bead composition described herein) used as a pro-apoptotic condition for certain cell types is used to amplify the remaining cell population for use in any of the various immunotherapeutic settings described herein. In other embodiments, a specific bead-to-cell ratio is used to selectively amplify naive T cells. Specific ratios can be adjusted to produce desired specific T cell amplification or specific T cell removal. Thus, the compositions and methods described herein can be used to amplify or remove specific T cell populations from the input composition population for use in any of the various immunotherapeutic settings described herein.

[0192] Furthermore, in this regard, in some embodiments, the same reagents or substances or compositions used as in pro-apoptotic compositions for certain cell types (e.g., surfaces of agents having a portion thereon that stimulates the cell surface, such as bead compositions described herein) are used to amplify the remaining cell population for use in any of the various immunotherapeutic settings described herein. Using a lower bead-to-cell ratio provides a stimulatory signal to antigen-specific T cells, which does not overstimulate but rather induces rapid proliferation of these cells. In other embodiments, a specific bead-to-cell ratio is used to selectively amplify antigen-specific T cells. In some respects, any ratio can be used whenever the desired amplification or removal occurs. Therefore, the compositions and methods described herein can be used to amplify or remove specific T cell populations for use in any of the various immunotherapeutic settings described herein.

[0193] Using certain methods, it may be advantageous to maintain long-term stimulation of the T cell population by isolating the T cells from the stimulant after an initial activation and stimulation period of approximately 14 days. The T cell proliferation rate can be monitored periodically (e.g., daily) by, for example, examining the size of the T cells or measuring their volume (e.g., using a Coulter counter). In this regard, the average diameter of resting T cells is approximately 6.8 micrometers, and after initial activation and stimulation, in the presence of the stimulating ligand, the average T cell diameter increases to over 12 micrometers by day 4 and begins to decrease by approximately day 6. When the average T cell diameter decreases to approximately 8 micrometers, the T cells can be reactivated and restimulated to induce further T cell proliferation. Alternatively, the rate of T cell proliferation and the timing of T cell restimulation can be monitored by measuring the presence of cell surface molecules induced on activated T cells (e.g., CD154, CD54, CD25, CD137, CD134).

[0194] To induce long-term stimulation of CD4+ and / or CD8+ T cell populations, it may be necessary to reactivate and restimulate T cells several times with stimulants such as anti-CD3 antibodies and anti-CD28 antibodies (e.g., B-T3, XR-CD28 (Diaclone, Besançon, France)) to generate an increased number of CD4+ or CD8+ cell populations, approximately 10 to 1,000 times the initial T cell population. For example, in one embodiment of the invention, T cells are stimulated 2-3 times as described. In other embodiments, T cells are stimulated 4 or 5 times as described. Using the method of the present invention, a T cell number with an increased polyclonal capacity of approximately 100 to 100,000 times compared to before stimulation can be achieved. Furthermore, T cells expanded by the method of the present invention secrete significant levels of cytokines (e.g., IL-2, IFN-γ, IL-4, GM-CSF, and TNF-α) into the culture supernatant. For example, CD4+ T cells expanded by co-stimulation with anti-CD3 and anti-CD28 secrete high levels of GM-CSF and TNF-α into the culture medium, compared to stimulation with IL-2. These cytokines can be purified from the culture supernatant, or the supernatant can be used directly to maintain cells in culture. Similarly, T cells expanded by the method of the present invention, along with the culture supernatant and cytokines, can be administered to support cell growth in vivo.

[0195] In one implementation, T cell stimulation, for example, with anti-CD3 and anti-CD28 antibodies co-immobilized on beads (3 × 28 beads), is sustained for a period sufficient to allow 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. The method of the present invention renders the cells at the end of the stimulation phase “super-inducible,” as demonstrated by their responsiveness to antigens and their ability to exhibit memory-like phenotypes, as illustrated by the exemplary embodiments. Thus, upon exogenous restimulation with antigens after infusion or in vivo, the activated T cells exhibit robust responses characterized by unique phenotypic properties such as sustained CD154 expression, increased cytokine production, etc.

[0196] In other embodiments of the invention, cells (such as T cells) are combined with drug-coated or conjugated beads, followed by separation of the beads and cells, and then cell culture. In an alternative embodiment, the drug-coated or conjugated beads and cells are cultured together before culture, without separation. In other embodiments, the beads and cells are first concentrated by applying force, resulting in partial attachment of the cell surface, thereby inducing polarization of cell stimulation and / or activation signals.

[0197] For example, when T cells are the target cell population, the cell surface portion can be connected by contacting the prepared T cells with paramagnetic beads (3 × 28 beads) coated with anti-CD3 and anti-CD28 antibodies. In one embodiment, cells (e.g., 10⁴ to 10⁹ T cells) and beads (e.g., in a 1:1 ratio) are used. M-450CD3 / CD28 T paramagnetic beads are combined in a buffer solution, preferably PBS (free of divalent cations such as calcium and magnesium). In some aspects, any cell concentration can be used. For example, target cells may be very rare in the sample and only constitute 0.01% of the sample, or the entire sample (i.e., 100%) may contain the target cells. Therefore, any number of cells is within the scope of this invention. In some embodiments, it may be necessary to significantly reduce the volume of particles and cells mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and particles. For example, in one embodiment, a concentration of approximately 2 billion cells / mL is used. In another embodiment, a concentration greater than 100 million cells / mL is used. In other embodiments, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / mL are used. In yet another embodiment, a cell concentration of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / mL is used. In other embodiments, a concentration of 125 million or 150 million cells / mL can be used. Using high concentrations can lead to increased cell yield, cell activation, and cell expansion. Furthermore, using high cell concentrations allows for more efficient capture of cells that may weakly express the target antigen, such as CD28-negative T cells. Such cell populations may have therapeutic value and are desirable. For example, using high cell concentrations allows for more efficient selection of CD8+ T cells that typically have weak CD28 expression.

[0198] In relevant implementations, it may be necessary to use lower concentrations of cells. By significantly diluting the mixture of T cells and granules, the interaction between granules and cells can be minimized. This selects cells that express high levels of the desired antigens to be bound to the granules. For example, at diluted concentrations, CD4+ T cells express higher levels of CD28 and are more effectively captured and stimulated compared to CD8+ T cells. In one implementation, the cell concentration used is approximately 5 × 10⁻⁶. 6 / mL. In other embodiments, the concentration used can be from about 1×10⁻⁶. 5 / mL to approximately 1×10⁶ / mL and any integer values ​​in between.

[0199] The buffer in which the cells are suspended can be any buffer suitable for a specific cell type. When using certain cell types, the buffer may contain additional components necessary to maintain cell integrity during the process, such as 1%–5% serum. In another embodiment, the cells and beads can be combined in a cell culture medium. The cells and beads can be mixed, for example, by rotation, stirring, or any mixing means, for a period ranging from one minute to several hours. The container of beads and cells is then concentrated by force (such as by placing it in a magnetic field). The culture medium and unbound cells are removed, and cells attached to the beads or other surfaces are washed, for example, by pumping via a peristaltic pump, and then the cells are resuspended in a culture medium suitable for cell culture.

[0200] In one embodiment of the invention, the mixture can be cultured for 30 minutes to several hours (about 3 hours) to about 14 days or any integer value of hours or minutes in between. In another embodiment, the mixture can be cultured for 21 days. In one embodiment of the invention, beads and T cells are cultured together for about eight days. In another embodiment, beads and T cells are cultured together for 2-3 days. As mentioned above, several stimulation cycles may be required, so that the culture time of T cells can be 60 days or longer. Suitable conditions for T cell culture include appropriate culture media (e.g., minimally essential medium or RPMI 1640 or X-vivo 15 (BioWhittaker)) which may contain factors required for proliferation and viability, including serum (e.g., fetal bovine or human serum) or interleukin-2 (IL-2), insulin, or any other additives for cell growth. Culture media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, supplemented with amino acids and vitamins, serum-free or supplemented with adequate amounts of serum (or plasma) or a defined hormone group and / or one or more cytokines sufficient for T cell growth and expansion. Antibiotics (e.g., penicillin and streptomycin) are included only in the experimental culture and not in the cell culture to be infused into the subject. Target cells are maintained under conditions necessary to support growth (e.g., appropriate temperature (e.g., 37°C) and atmospheric conditions (e.g., air plus 5% CO2)).

[0201] In one embodiment of the invention, the bead-to-cell ratio can be customized to obtain a desired T-cell phenotype. In a particular embodiment, the bead-to-cell ratio can be altered to selectively expand or remove antigen-specific (memory) T cells. In one embodiment, the specific bead-to-cell ratio used selectively removes antigen-specific T cells. In other embodiments, the specific bead-to-cell ratio used selectively expands antigen-specific T cells. In some aspects, any ratio can be used whenever the desired expansion or removal of antigen-specific T cells occurs. Therefore, the compositions and methods described herein can be used to expand or remove specific T-cell populations for use in any of the various immunotherapeutic settings described herein.

[0202] In another implementation, the duration of exposure to the stimulant (such as anti-CD3 / anti-CD28 (i.e., 3×28) coated beads) can be modified or customized to achieve the desired T cell phenotype. Alternatively, various selection techniques can be used prior to stimulation to select the desired T cell population. More helper T cells (TH), typically CD4+ rather than CD8+ cytotoxic or regulatory T cells, may be required, as TH cell expansion can improve or restore overall immunoreactivity. While many specific immune responses are mediated by CD8+ antigen-specific T cells that can directly lyse or kill target cells, most immune responses require the help of CD4+ T cells, for example, those expressing important immunomodulatory molecules such as GM-CSF, CD40L, and IL-2. Where CD4-mediated helper is preferred, methods for maintaining or increasing the CD4:CD8 ratio (as described herein) may have significant benefits. An increase in the number of CD4+ T cells can increase the amount of CD40L expressed by cells introduced into the patient, potentially improving target cell visibility (improved 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, in some applications, it may be necessary to utilize regulatory T cell populations (e.g., Autoimmun Rev. Aug. 2002; 1(4):190-7; CurrOpin Immunol. Dec. 2002; 14(6):771-8), which can be generated and expanded using the methods described herein. Alternatively, in cases where less CD4 helper is needed and an increase in the number of CD8+ T cells is desired, XCELLERATE as described herein can also be utilized, for example, by pre-selecting CD8+ cells prior to stimulation and / or culture. TMMethods. It is possible that increased IFN-γ levels or increased target cell lysis are preferred. The timing and type of exposure to stimulants can also be modified to expand T cells with the desired TCR repertoire (e.g., expression of desired Vβ family genes).

[0203] To achieve the separation of different T cell populations, the exposure time to particles can be varied. For example, in a preferred embodiment, by exposing the particles to 3 × 28 beads (e.g., ... Incubation with M-450 for a sufficient period of time to positively select for the desired T cells is used to isolate T cells. In one embodiment, the incubation period is about 30 minutes. In other embodiments, the incubation period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the incubation period is 10 to 24 hours or longer. In one preferred embodiment, the incubation period is or is about 24 hours. Using a longer incubation time (such as at least or at least about 24 hours) can increase cell yield for isolating T cells from cancer patients.

[0204] In some embodiments, the total stimulation and / or amplification time can be between 2 and 15 days, between 2 and 12 days, between 2 and 12 days, between 2 and 8 days, between 2 and 6 days, between 2 and 4 days, between 4 and 12 days, between 4 and 10 days, between 4 and 8 days, between 4 and 6 days, between 6 and 12 days, between 6 and 10 days, between 6 and 8 days, between 8 and 12 days, between 8 and 10 days, or between 10 and 12 days, encompassing all ranges. In some embodiments, cells are incubated and / or incubated with a stimulating agent (e.g., particles as described herein) for at least or at least about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or more than 14 days. When T cell stimulation is performed for a shorter period of time, the number of T cells may not increase, may not increase dramatically, may decrease, or may remain unchanged. However, the population will provide more robust and healthy activated T cells that can continue to proliferate in vivo and / or more closely resemble the pool of natural effector T cells. For example, when T cell stimulation is performed for a shorter period of time, the T cell population may contain a larger 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, or in some embodiments, a total incubation time with the stimulant 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. Since the availability of T cell assistance is often a limiting factor for antibody responses to protein antigens, the ability to selectively expand or selectively infuse a population of CD4+-rich T cells to a subject is extremely beneficial. Other benefits of such enriched populations are evident, as activated helper T cells that recognize antigens presented by B lymphocytes deliver two types of stimulation: physical contact and cytokine production, both of which lead to the proliferation and differentiation of B cells.

[0205] In some cases, the stimulation condition does not include culture components or agents supplemented to preserve a specific subset of T cells (e.g., non-immature T cells). Therefore, in some cases, removing components or agents from the culture of the stimulation condition may help eliminate non-immature T cells. In some embodiments, the stimulation condition does not include agents, such as N-acetylcysteine, that can be used to modulate and / or fine-tune the intensity of TCR / CD3 signaling. In some aspects, such agents are removed or reduced in amounts / ratios that increase the intensity of the activation signal. In some aspects, the stimulation condition is performed or additionally by excluding or reducing the concentration of culture agents known to or potentially reducing AICD and / or promoting the survival of older cells (e.g., non-immature cells). In some cases, the stimulation condition does not include N-acetylcysteine ​​or includes a reduced amount or concentration of N-acetylcysteine. In some cases, the stimulation condition does not include a recombinant IL-7 and / or recombinant IL-15, or includes a reduced amount or concentration of recombinant IL-7 or IL-15. In some implementations, culture additives (e.g., toxins attached to CD45RO) may be included, which additionally help or promote the removal of immature cells.

[0206] C. Stimulated Composition

[0207] Stimulated compositions produced by the aforementioned incubation (e.g., stimulation) method are also provided. In some embodiments, after incubation with the input composition, the cells of the stimulated composition are further engineered, for example, to introduce genetically engineered antigen receptors. In some embodiments, the method further includes incubation in the presence of one or more stimulants during the genetic engineering phase.

[0208] Cells that have not undergone apoptosis and have been stimulated using the incubation (e.g., stimulation) methods described herein can increase the polyclonalness of the remaining T cell population, as measured by the magnitude of the population’s response to a given antigen. The restoration or increase in polyclonalness can be measured by determining the magnitude of the response to a specific target antigen, for example by measuring the number of different epitopes recognized by antigen-specific cells. This can be done using standard techniques for generating and cloning antigen-specific T cells in vitro.

[0209] In some embodiments of the methods provided herein, culture conditions preferentially induce the expansion, proliferation, and / or survival of non-naïve T cells compared to naïve 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 first type or population exhibits greater relative expansion, proliferation, and / or survival compared to the second type or population. This can include situations where the percentage of the first cell type or population that expands, proliferates, and / or survives is greater, and / or the degree of expansion, proliferation, and / or survival of the first population or type relative to the second population or type (e.g., the overall or average degree within the population or type of cells).

[0210] In some embodiments, preferential expansion, proliferation, and / or survival are expressed by comparing the percentage of immature-like cells in the input composition with the percentage of engineered cells in the stimulated composition derived from the immature-like cells in the initial input composition. In some examples, the percentage of engineered cells in the stimulated composition derived from the immature-like cells in the initial input composition is generally greater than the aforementioned percentage under the stimulation conditions described herein. For example, in one embodiment, the percentage of cells in the stimulated composition derived from the immature-like cells in the initial input composition is greater than the percentage of immature cells in the culture starting composition. In one aspect, the input composition (or the T cells therein) comprises, or about 70% immature-like T cells and 30% non-immature-like T cells, and more than 50% (e.g., at least 70%) of the engineered cells in the resulting stimulated composition are derived from the immature-like cells in the input composition. In some cases, using other conditions (such as stimulation with one or more agents that induce a strong signal through the TCR complex) will result in less than 50% (e.g., or about 5%-10%) of the cells in the stimulated composition having a non-immature-like origin. In some implementations, the stimulus condition induces a strong signal that induces activation of non-naive T cells infused with the composition, leading to cell death.

[0211] In some embodiments, the percentage of naive T cells or naive T cells derived from the input composition in the stimulated composition is increased by more than or greater than about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, or 100 times compared to the input composition. In some cases, the ratio of naive T cells derived from the input composition to non-naive T cells derived from the input composition, or the ratio of naive T cells to non-naive T cells in the stimulated composition, is increased by more than or greater than about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, or 100 times compared to the ratio of naive T cells to non-naive T cells in the input composition. In some embodiments, the stimulated composition comprises more than 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of naive T cells derived from the input composition.

[0212] In some embodiments of the method for stimulating T cells described herein, a greater percentage of naive-like T cells are induced to proliferate and / or activated in the cells introduced into the composition compared to non-naive-like T cells. In some aspects, the stimulated composition produced by the stimulation method 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, on days 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 after the start of the incubation, a greater percentage of naive-like T cells in the introduced composition are dividing compared to the percentage of non-naive-like T cells in the introduced composition. In some cases, the stimulation condition induces cell death. In a particular example, the stimulation condition of the method induces activation of non-naive-like T cells, thereby inducing activation-induced cell death (AICD).

[0213] In some embodiments, the method includes a stimulation condition that, on days 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 after incubation begins under the stimulation condition, induces a greater percentage of cell proliferation in the naive-like T cell population compared to non-naive-like T cells.

[0214] In some embodiments, the stimulation condition produces the stimulated composition, wherein the stimulation condition preferentially induces the expansion of naive T cells, resulting in a target amount of T cells derived from the naive T cells introduced into the stimulated composition. In some embodiments, the stimulated composition is further modified to achieve a preferred CD4:CD8 ratio of T cells. In some cases, a specific cell population may be removed from the stimulated composition to achieve the preferred CD4:CD8 ratio.

[0215] In some embodiments, a stimulating condition (e.g., incubation of the input composition under said stimulating condition) preferentially induces the expansion, proliferation, and / or survival of naive T cells or a subset thereof compared to non-naive T cells or a subset thereof. In some aspects, the stimulating condition strongly activates non-naive T cells receiving the input composition, thereby initiating cell death, particularly in non-naive T cells receiving the input composition. In some aspects, this thereby preferentially favors the survival of naive T cells. In some aspects, after administration to a subject of cells and the composition produced by said method, such a stimulating condition results in a reduction in the level of toxicity and / or one or more toxicity-related outcomes or one or more symptoms.

[0216] Whether a particular response has been induced in one population or preferentially induced relative to another population can be measured by a variety of methods. For example, whether cells are induced to enter the cell cycle can be measured by flow cytometry-based methods, which involve assessing cell division using dyes and other reagents (such as CFSE and other interchelating agents), followed by flow cytometry assessment, assessment of tritium (H3)-labeled thymidine and similar reagent incorporation, and / or cell counting. Comparisons can be made by evaluating various pure test populations, such as by comparing naive and non-naive T cell populations separately 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 scattering as measured by flow cytometry). Survival and / or apoptosis can be assessed by various methods, including flow cytometry methods involving the incorporation of various dyes, including propidium iodide, and staining with reagents (e.g., annexin V and similar reagents).

[0217] In some embodiments, the percentage of naive T cells in the input composition is less than the percentage of T cells derived from naive T cells in the stimulated composition. In some embodiments, the method provided herein produces a greater percentage of nucleic acid-introduced cells that are naive T cells in the input composition or that are derived from the proliferation of naive T cells in the input composition compared to non-naive T cells in the input composition. Various modifications 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. US 20030235908 A1.

[0218] In some embodiments, the stimulated composition contains cells that express or are derived from specific markers of naive T cells. For example, the stimulated composition produced by the provided method is derived from a cell population that is surface-positive for T cell activation markers selected from CD27, CD28, CD45RA, and CCR7. In some cases, the stimulated composition produced by the provided method is derived from a cell population that is surface-negative for T cell activation markers (such as CD62L). In some aspects, the cells of the stimulated composition are or are derived from cells that are surface-negative for CD56 and / or CD45RO. In some specific embodiments, the stimulated composition produced by the provided method is derived from a cell population that is CD27+, CD45RA+, CD45RO-, and CCR7+. In some cases, the cells of the stimulated composition are or are derived from cells that are negative for intracellular expression of cytokines (such as IL-2, IFN-γ, IL-4, and / or IL-10). In some other examples, the cells in the stimulated composition are or are derived from cells that are negative for the expression of markers CD25 and / or perforin. In some cases, the cells in the stimulated composition are or are derived from cells that express CD95. lo . cells.

[0219] In some embodiments, the stimulated composition contains cells derived from at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of 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 cells derived from at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of T cells that are surface-negative for CD56 and / or CD45RO. In some aspects, the stimulated composition contains cells derived from at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of T cells that are surface-negative for CD45RO and surface-positive for CD27, CD45RA, and CCR7. In some examples, the stimulated composition contains cells derived from at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of 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 cells derived from at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of T cells that are negative for expression of CD25 and / or perforin. In some cases, the stimulated composition contains cells derived from T cells that are negative for expression of CD95. lo At least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells are T cells.

[0220] In some embodiments, the stimulated composition is more polyclonal (or multiclonal) than the input composition. In some embodiments, the stimulated composition is more diverse than the input composition. In some embodiments, this increase in polyclonalness includes a shift from monoclonal to oligoclonal or polyclonal T cell populations, as measured by the Vβ, Vα, Vγ, or Vδ morphology profile of at least one Vβ, Vα, Vγ, or Vδ family gene. Stimulation and activation of the remaining cells that have survived, expanded, and proliferated using the provided methods can increase the polyclonalness of the remaining T cells in the stimulated composition, as measured by the amplitude of the population's response to a given antigen. The restoration or increase in the polyclonalness of the stimulated composition can be measured by determining the amplitude of the response to a specific target antigen, for example by measuring the number of different epitopes recognized by antigen-specific cells. This can be done using standard techniques for generating and cloning antigen-specific T cells in vitro.

[0221] II. Methods for Genetically Engineered Cells

[0222] In some embodiments, the method further includes introducing a genetically engineered recombinant receptor (e.g., a chimeric receptor, such as a chimeric antigen receptor (CAR)) into a stimulated T cell composition to produce an output composition comprising T cells expressing the genetically engineered recombinant receptor. In some cases, incubation of the input composition under stimulation conditions is performed before, during, and / or after the introduction of the nucleic acid encoding the genetically engineered recombinant receptor. In some examples, the introduction is performed via transduction. In some embodiments, the provided method produces a stimulated composition that can be uniformly 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 gamma retroviral vector. In some embodiments, the method including the introduction is performed in vitro or ex vivo.

[0223] Therefore, the methods provided herein include one or more steps for preparing cells for genetic engineering. In some embodiments, the one or more steps include isolating cells from a biological sample, stimulating the cells with an input composition, and preparing a cell composition to be genetically engineered. Populations of such cells, compositions containing such cells and / or rich in such cells are also provided, for example, wherein cells expressing a recombinant receptor (e.g., a chimeric receptor) constitute at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or a higher percentage of the total cells in the composition or of a certain type of cells (e.g., T cells or CD8+ or CD4+ cells). The compositions include pharmaceutical compositions and formulations for administration (e.g., for adoptive cell therapy). Methods for engineering, producing, or generating such cells are also provided, methods for administering said cells and compositions to a subject (e.g., a patient), and methods for detecting, selecting, isolating, or separating such cells. Therefore, genetically engineered cells expressing a recombinant receptor (e.g., CAR) are provided.

[0224] In some embodiments, the cell includes one or more nucleic acids introduced through genetic engineering, and thereby expresses a recombinant or genetically engineered product of such nucleic acid. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or in a sample obtained from the cell, such as nucleic acids obtained from another organism or cell, for example, said nucleic acid is not typically found in the engineered cell and / or the organism from which such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, such as nucleic acids not found in nature, including nucleic acids comprising a chimeric combination of nucleic acids encoding various domains from a variety of different cell types.

[0225] A. Genetic engineering

[0226] 1. Recombinant antigen receptor

[0227] In some embodiments, engineered cells (such as T cells) are provided that express a CAR specific to a particular antigen (or label or ligand), said particular antigen being, for example, an antigen expressed on the surface of a particular cell type. In some embodiments, the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of a disease or condition (e.g., tumor or pathogenic cells) compared to normal or untargeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or on engineered cells.

[0228] In certain embodiments, the recombinant receptor (such as a chimeric receptor) contains an intracellular signaling region comprising a cytoplasmic signaling domain (also interchangeably referred to as an intracellular signaling domain), such as a cytoplasmic (intracellular) region capable of inducing primary activation signals in T cells, such as a cytoplasmic signaling domain of a T cell receptor (TCR) component (e.g., a cytoplasmic signaling domain of the ζ chain of the CD3-zeta (CD3ζ) chain or a functional variant or signaling portion thereof); and / or the intracellular signaling region contains an immune receptor tyrosine-based activation motif (ITAM).

[0229] In some embodiments, the chimeric receptor also contains an extracellular ligand-binding domain that specifically binds to a ligand (e.g., an antigen). In some embodiments, the chimeric receptor is a CAR containing an extracellular antigen-recognition domain that specifically binds to an antigen. In some embodiments, the ligand (e.g., an antigen) is a protein expressed on the cell surface. 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, like a TCR, is recognized on the cell surface in the context of major histocompatibility complex (MHC) molecules.

[0230] Exemplary antigen receptors (including CARs) and methods for engineering and introducing such receptors into cells include, for example, those described in the following documents: International Patent Application Publications WO2000 / 14257, WO 2013 / 126726, WO2012 / 129514, WO 2014031687, WO 2013 / 166321, WO 2013 / 071154, WO 2013 / 123061; U.S. Patent Application Publications US 2002131960, US 2013287748, US 20130149337; US Patent Nos: 6,451,995, 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 those described in the following documents: Sadelain et al., Cancer Discov. April 2013; 3(4):388-398; Davila et al. (2013) PLoS ONE 8(4):e61338; Turtle et al., Curr. Opin. Immunol., October 2012; 24(5):633-39; Wu et al., Cancer, March 2012, 18(2):160-75. In some respects, antigen receptors include CARs as described in U.S. Patent No. 7,446,190 and those described in International Patent Application Publication No. WO / 2014055668A1. Examples of CARs include those disclosed in any of the aforementioned publications, such as WO 2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, US Patent No. 7,446,190, US Patent 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 WO 2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, US Patent No. 7,446,190 and US Patent No. 8,389,282.

[0231] In some embodiments, the CAR is constructed to be specific to a particular antigen (or marker or ligand), such as an antigen expressed in a specific cell type targeted by adoptive therapy (e.g., a cancer marker) and / or an antigen intended to induce a decay response (e.g., an antigen expressed on normal or disease-free cell types). Therefore, the CAR typically includes one or more antigen-binding molecules in its extracellular portion, 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, the CAR includes one or more antigen-binding portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from a variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb).

[0232] In some embodiments, the antibody or its antigen-binding portion is expressed on the cell as part of a recombinant receptor (such as an antigen receptor). Antigen receptors include functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Typically, a CAR containing an antibody or antigen-binding fragment exhibiting TCR-like specificity against a peptide-MHC complex can also be referred to as a TCR-like CAR. In some embodiments, in some aspects, the extracellular antigen-binding domain of the MHC-peptide complex of the TCR-like CAR is linked to one or more intracellular signaling components via a linker and / or one or more transmembrane domains. In some embodiments, such molecules can typically mimic or approximate signals via a natural antigen receptor (such as a TCR), and optionally, through combination of such receptors with co-stimulatory receptors.

[0233] In some implementations, recombinant receptors (such as chimeric receptors (e.g., CARs)) include a ligand-binding domain that binds (e.g., specifically) to an antigen (or ligand). The antigens targeted by chimeric receptors include those expressed in the context of a disease, condition, or cell type targeted via adoptive cell therapy. These diseases and conditions include proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including blood cancers, immune system cancers such as lymphoma, leukemia, and / or myeloma, such as B-type leukemia, T-type leukemia, and myeloid leukemia, lymphoma, and multiple myeloma.

[0234] In some embodiments, the antigen (or ligand) is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen (or ligand) is selectively expressed or overexpressed on diseased or symptom-related cells (e.g., tumor or pathogenic cells) compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or on engineered cells.

[0235] In some implementations, the CAR contains an antibody or antigen-binding fragment (e.g., scFv) that specifically recognizes antigens (such as intact antigens) expressed on the cell surface.

[0236] In some implementations, the antigens are 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), and type III epidermal growth factor receptor mutant (EGFR). vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), liver glycoprotein B2, liver glycoprotein receptor A2 (EPHa2), estrogen receptor, Fc receptor-like protein 5 (FCRL5);Also known as 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, glycoprotein 100 (gp100), phosphatidylinositol proteoglycan-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 (H MW-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 insertion domain receptor (kdr), κ light chain, L1 cell adhesion molecule (L1-CAM), L1-CAM CE7 epitope, Protein 8 family member A containing leucine-rich repeat sequences (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE- A10, mesothelin (MSLN), c-Met, mucin cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer cell family 2 member D (NKG2D) ligand, melanin A (MART-1), neural cell adhesion molecule (NCAM), oncoemulsification antigen, melanoma preferred expression 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), survival protein, trophoblast glycoprotein (TPBG, also known as 5T) 4) Tumor-associated glycoprotein 72 (TAG72), tyrosinase-associated protein 1 (TRP1, also known as TYRP1 or gp75), tyrosinase-associated protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta isomerase, or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor 1 (WT-1), pathogen-specific or pathogen-expressed antigens, or antigens associated with universal tags, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV, or other pathogens. In some embodiments, the receptor-targeted antigens include antigens associated with B-cell malignancies, such as any of many 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.

[0237] In some embodiments, the antigen is or includes pathogen-specific antigens or antigens expressed by pathogens. In some embodiments, the antigen is a viral antigen (such as viral antigens from HIV, HCV, HBV, etc.), a bacterial antigen, and / or a parasitic antigen.

[0238] In some embodiments, the antigen or antigen-binding domain is CD19. In some embodiments, the scFv contains VH and VL derived from an antibody or antibody fragment specific to CD19. In some embodiments, the antibody or antibody fragment binding 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 U.S. Patent Publication No. US 2016 / 0152723.

[0239] In some implementations, scFv is derived from FMC63. FMC63 typically refers to a mouse monoclonal IgG1 antibody against human-derived Nalm-1 and Nalm-16 cells expressing CD19 (Ling, NR et al. (1987). Leucocytetyping III. 302). The FMC63 antibody comprises CDRH1 and H2 as shown in SEQ ID NO: 38 and 39, CDRH3 as shown in SEQ ID NO: 40 or 54, CDRL1 as shown in SEQ ID NO: 35, CDR L2 36 or 55, and CDR L3 sequence 37 or 34, respectively. The FMC63 antibody contains a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 41. H ) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO:42. L In some embodiments, the svFv 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 FMC63 variable heavy chain region shown in SEQ ID NO:41 and the FMC63 variable light chain region shown in SEQ ID NO:42. In some embodiments, the variable heavy chain and the variable light chain are connected by a connector. In some embodiments, the connector is as shown in SEQ ID NO:56. In some embodiments, the scFv comprises V in sequence. H , connectors and V L In some implementations, scFv contains V in sequence. L , connectors and VH In some embodiments, svFc is encoded by the nucleotide sequence shown in SEQ ID NO:57 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:57. In some embodiments, scFv comprises the amino acid sequence shown in SEQ ID NO:43 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:43.

[0240] In some implementations, scFv is derived from SJ25C1. SJ25C1 is a mouse monoclonal IgG1 antibody against human-derived Nalm-1 and Nalm-16 cells expressing CD19 (Ling, NR et al. (1987). Leucocyte typing III. 302). The SJ25C1 antibody contains CDRH1, H2, and H3 sequences shown in SEQ ID NO:47-49, and CDRL1, L2, and L3 sequences shown in SEQ ID NO:44-46, respectively. The SJ25C1 antibody contains a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO:50. H ) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO:51. L In some embodiments, the svFv 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 SJ25C1 variable heavy chain region shown in SEQ ID NO:50 and the SJ25C1 variable light chain region shown in SEQ ID NO:51. In some embodiments, the variable heavy chain and the variable light chain are connected by a connector. In some embodiments, the connector is as shown in SEQ ID NO:52. In some embodiments, the scFv comprises V in sequence. H , connectors and V L In some implementations, scFv contains V in sequence. L , connectors and V HIn some embodiments, scFv comprises the amino acid sequence shown 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 with SEQ ID NO:53.

[0241] In some embodiments, the antigen or antigen-binding domain is BCMA. In some embodiments, the scFv contains VH and VL derived from an antibody or antibody fragment specific to BCMA. In some embodiments, the antibody or antibody fragment binding to BCMA is or contains VH and VL of an antibody or antibody fragment described in International Patent Application Publications WO 2016 / 090327 and WO 2016 / 090320.

[0242] In some embodiments, the antigen or antigen-binding domain is GPRC5D. In some embodiments, the scFv contains VH and VL derived from an antibody or antibody fragment specific to GPRC5D. In some embodiments, the antibody or antibody fragment binding GPRC5D is or contains VH and VL of an antibody or antibody fragment described in International Patent Application Publications WO 2016 / 090329 and WO 2016 / 090312.

[0243] In some embodiments, the CAR contains a TCR-like antibody, such as an antibody or antigen-binding fragment (e.g., scFv), which specifically recognizes intracellular antigens (such as tumor-associated antigens) present on the cell surface as MHC-peptide complexes. In some embodiments, the antibody recognizing the MHC-peptide complex or its antigen-binding portion may be expressed on the cell as part of a recombinant receptor (such as an antigen receptor). Antigen receptors include functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Generally, a CAR containing an antibody or antigen-binding fragment exhibiting TCR-like specificity against the peptide-MHC complex may also be referred to as a TCR-like CAR.

[0244] The term "major histocompatibility complex" (MHC) refers to a protein, typically a glycoprotein, containing a polymorphic peptide-binding site or binding groove, which in some cases can complex with peptide antigens of polypeptides (including peptide antigens processed by cellular machinery). In some cases, MHC molecules can be displayed or expressed on cell surfaces, including as peptide-binding complexes, i.e., MHC-peptide complexes, for presenting antigens with conformations recognizable by antigen receptors on T cells (such as TCRs or TCR-like antibodies). Typically, MHC class I molecules are heterodimers with a membrane spanning an α chain, and in some cases, non-covalently associated β2-microglobulins with three α domains. Typically, MHC class II molecules consist of two transmembrane glycoproteins, α and β, both of which typically span the membrane. MHC molecules may include an effective portion of the MHC containing an antigen-binding site or a site for binding peptides, as well as a sequence necessary for recognition by a suitable antigen receptor. In some embodiments, MHC class I molecules deliver peptides derived from the cytosol to the cell surface, wherein the MHC-peptide complex is generated by T cells (such as typically CD8+). + T cells (but in some cases CD4+ T cells) recognize it. In some implementations, MHC class II molecules deliver peptides derived from the vesicle system to the cell surface, wherein these peptides are typically recognized by CD4+ T cells. + T cell recognition. Typically, MHC molecules are encoded by a set of linked loci, collectively known as H-2 in mice and human leukocyte antigens (HLA) in humans. Therefore, human MHC is often also referred to as human leukocyte antigens (HLA).

[0245] The term "MHC-peptide complex" or "peptide-MHC complex," or variations thereof, refers to a complex or association between a peptide antigen and an MHC molecule, typically formed through nonvalent 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 cell surface. In some embodiments, the MHC-peptide complex can be specifically recognized by an antigen receptor (such as a TCR, a TCR-like CAR, or its antigen-binding moiety).

[0246] In some embodiments, the peptide (such as a peptide antigen or epitope) of the polypeptide can associate with an MHC molecule, for example, for recognition by an antigen receptor. Typically, the peptide is derived from or based on fragments of longer biomolecules (such as polypeptides or proteins). In some embodiments, the peptide is typically about 8 to about 24 amino acids in length. In some embodiments, the peptide is from or from about 9 to 22 amino acids in length for recognition in MHC class II complexes. In some embodiments, the peptide is from or from about 8 to 13 amino acids in length for recognition in MHC class I complexes. In some embodiments, upon recognition of the peptide in the context of an MHC molecule (such as an MHC-peptide complex), an antigen receptor (such as a TCR or TCR-like CAR) generates or triggers an activation signal to T cells, thereby inducing a T cell response, such as T cell proliferation, cytokine production, cytotoxic T cell responses, or other responses.

[0247] In some implementations, TCR-like antibodies or antigen-binding moieties may be generated (see, for example, US Publication Nos. US 2002 / 0150914; US 2003 / 0223994; US 2004 / 0191260; US 2006 / 0034850; US 2007 / 00992530; US 20090226474; US 20090304679; and International PCT Publication No. WO 03 / 068201).

[0248] In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to MHC-peptide complexes can be generated by immunizing the 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 capable of binding to MHC, such as a tumor antigen, for example a universal tumor antigen, myeloma antigen, or other antigens as described below. In some embodiments, the host is then given an effective amount of the immunogen to induce an immune response, wherein the immunogen remains in its three-dimensional form for a period of time sufficient to induce an immune response against the three-dimensional representation of the peptide in the binding groove of the MHC molecule. Serum collected from the host is then measured to determine whether the desired antibody recognizing the three-dimensional representation 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 between the MHC-peptide complex and an individual MHC molecule, an individual target peptide, and a complex of MHC and an unrelated peptide. The desired antibody can then be isolated.

[0249] In some embodiments, antibodies or their antigen-binding moieties that specifically bind to MHC-peptide complexes can be generated using antibody library display methods (such as phage antibody libraries). In some embodiments, phage display libraries in the form of mutant Fab, scFv, or other antibodies can be generated, for example, wherein members of said library are mutated at one or more residues of one or more CDRs. See, for example, U.S. Publication Nos. US 20020150914, US 2014 / 0294841; and Cohen CJ. et al. (2003) J Mol. Recogn. 16:324-332.

[0250] The term "antibody" is used in the broadest sense in this article and includes both polyclonal and monoclonal antibodies, including complete antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, and variable heavy chains (V) capable of specifically binding antigens. H This term encompasses single-chain antibody fragments (including single-chain variable fragments (scFv)) and single-domain antibody fragments (e.g., sdAb, sdFv, nanobodies). The term covers genetically engineered and / or otherwise modified forms of immunoglobulins, such as intracellular antibodies, peptide bodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugated antibodies, multispecific (e.g., bispecific) antibodies, biantibodies, triantibodies and tetraantibodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass its functional antibody fragment. The term also covers complete or full-length antibodies, including antibodies of any class or subclass (including IgG and its subclasses, IgM, IgE, IgA, and IgD).

[0251] In some embodiments, antigen-binding proteins, antibodies, and their antigen-binding fragments specifically recognize the antigen of a full-length antibody. In some embodiments, the heavy and light chains of the antibody may be full-length or may be antigen-binding portions (Fab, F(ab')2, Fv, or single-chain Fv fragments (scFv)). In other embodiments, the constant region of the antibody heavy chain is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly from, for example, IgG1, IgG2, IgG3, and IgG4, and more particularly IgG1 (e.g., human IgG1). In yet another embodiment, the constant region of the antibody light chain is selected from, for example, κ or λ, particularly κ.

[0252] The antibodies provided include antibody fragments. An "antibody fragment" is a molecule distinct from the complete antibody, containing a portion of the antigen bound to the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; and variable heavy chains (V...). H ) region, single-chain antibody molecules (such as scFv) and single-domain V H Single antibody; and multispecific antibody formed from antibody fragments. In a particular embodiment, the antibody is a single-chain antibody fragment containing a variable heavy chain region and / or a variable light chain region, such as scFv.

[0253] The term "variable region" or "variable domain" refers to the structural domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains of the heavy and light chains of natural antibodies (V1 and V2, respectively) are... H and V L They typically have similar structures, with each domain containing four conserved frame regions (FRs) and three core regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single V H or V L The structural domain is sufficient to confer antigen-binding specificity. Furthermore, V-terminal molecules derived from antibodies that bind to antigens can be used. H or V L The domains are separated to isolate antibodies that bind to the specific antigen, in order to screen for complementary V antibodies. L or V H Library of structural domains. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0254] A single-domain antibody is an antibody fragment containing all or part of the variable domain of the heavy chain or all or part of the variable domain of the light chain. In some embodiments, the single-domain antibody is a human single-domain antibody. In some embodiments, the CAR contains an antibody heavy chain domain that specifically binds to an antigen, such as a cancer marker or a cell surface antigen of a cell or disease to be targeted (e.g., tumor cells or cancer cells), as described herein or any target antigen known in the art.

[0255] Antibody fragments can be prepared using various techniques, including but not limited to proteolytic digestion of intact antibodies and production via recombinant host cells. In some embodiments, the antibody is a recombinantly generated fragment, such as a fragment containing an arrangement not naturally occurring (e.g., those having two or more antibody regions or chains linked by synthetic linkers (e.g., peptide linkers), and / or a fragment that can be generated without enzymatic digestion of naturally occurring intact antibodies. In some embodiments, the antibody fragment is an scFv.

[0256] A “humanized” antibody is an antibody in which all or substantially all of the CDR amino acid residues are derived from a non-human CDR and all or substantially all of the FR amino acid residues are derived from human FR. A humanized antibody may optionally include at least a portion of the antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization to generally reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in the humanized antibody are replaced by corresponding residues from a non-human antibody (e.g., an antibody from which CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0257] Therefore, in some embodiments, chimeric antigen receptors (including TCR-like CARs) include an extracellular portion containing an antibody or antibody fragment. In some embodiments, the antibody or fragment includes scFv. In some aspects, chimeric antigen receptors include an extracellular portion containing an antibody or fragment and an intracellular signaling region. In some embodiments, the intracellular signaling region includes an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or includes a primary signaling domain, a signaling domain capable of inducing primary activation signals in T cells, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immune receptor tyrosine-based activation motif (ITAM).

[0258] In some embodiments, the recombinant receptor (such as a CAR, or its antibody portion) further includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or a variant or modified form thereof, such as a hinge region (e.g., the IgG4 hinge region) and / or C H 1 / C Land / or the Fc region. In some embodiments, the recombinant receptor also includes a spacer and / or a hinge region. In some embodiments, the constant region or a portion thereof is human IgG (such as IgG4 or IgG1). In some aspects, said portion of the constant region serves as a spacer region between an antigen recognition component (e.g., scFv) and a transmembrane domain. The length of the spacer can provide increased cellular reactivity after antigen binding compared to the absence of a spacer. In some examples, the length of the spacer is 12 amino acids or about 12 amino acids or no more than 12 amino acids. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids (and including any integer between the endpoints of any of the listed ranges). In some embodiments, the spacer region has about 12 or fewer amino acids, about 119 or fewer amino acids, or about 229 or fewer amino acids. Exemplary spacers include a single IgG4 hinge, an IgG4 hinge connected to both CH2 and CH3 domains, or an IgG4 hinge connected to the CH3 domain. Exemplary spacers include, but are not limited to, those described in the following documents: Hudecek et al. (2013) Clin. Cancer Res., 19:3153 or International Patent Application Publication No. WO 2014031687. In some embodiments, the spacer has the sequence shown in SEQ ID NO:1 and is encoded by the sequence shown in SEQ ID NO:2. In some embodiments, the spacer has the sequence shown in SEQ ID NO:3. In some embodiments, the spacer has the sequence shown in SEQ ID NO:4.

[0259] In some embodiments, the constant region or portion is IgD. In some embodiments, the spacer has the sequence shown in SEQ ID NO:5. In some embodiments, the spacer has an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any one of SEQ ID NO:1, 3, 4 and 5. In some embodiments, the spacer has the sequences shown in SEQ ID NO:23-31. In some embodiments, the spacer has an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any one of SEQ ID NO:27-31, 58, 59.

[0260] Antigen recognition domains are typically linked to one or more intracellular signaling components (e.g., signaling components that mimic activation via an antigen-receptor complex (e.g., a TCR complex) (in the case of a CAR) and / or signaling components that mimic signals via another cell surface receptor). Therefore, in some embodiments, an antigen-binding component (e.g., an antibody) is linked to one or more transmembrane and intracellular signaling regions. In some embodiments, the transmembrane domain is fused to an extracellular domain. In one embodiment, a transmembrane domain naturally associated with a domain of the receptor (e.g., a CAR) is used. In some cases, transmembrane domains are selected or modified by amino acid substitution to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0261] In some embodiments, the transmembrane domain is derived from a natural or synthetic source. When the source is natural, the domain is derived in some respects from any membrane-binding or transmembrane protein. The transmembrane region includes those derived from (i.e., containing at least the following transmembrane regions): 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, and CD154. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some respects, the synthetic transmembrane domain primarily contains hydrophobic residues such as leucine and valine. In some respects, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain. In some embodiments, the connection is achieved through a linker, a spacer, and / or one or more transmembrane domains.

[0262] Intracellular signal transduction regions include those that mimic or approximate signals via natural antigen receptors, via combinations of such receptors and co-stimulatory receptors, and / or via co-stimulatory receptors alone. In some embodiments, short oligopeptide or polypeptide linkers are present, such as linkers with a length between 2 and 10 amino acids (e.g., linkers containing glycine and serine, such as glycine-serine duplexes), and a connection is formed between the transmembrane domain of the CAR and the cytoplasmic signal transduction domain.

[0263] Receptors (e.g., CARs) typically include at least one or more intracellular signaling components. In some embodiments, the receptor includes an intracellular component of the TCR complex, such as the TCR CD3 chain, for example, the CD3ζ chain, which mediates T cell activation and cytotoxicity. Therefore, in some aspects, ROR1-binding antibodies are linked to one or more cell signaling modules. In some embodiments, the cell signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the receptor (e.g., CAR) also includes a portion of one or more additional molecules, such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

[0264] In some embodiments, upon CAR conjugation, the cytoplasmic domains or intracellular signaling regions of the CAR activate at least one of the normal effector functions or responses of immune cells (e.g., T cells engineered to express the CAR). For example, in some contexts, the CAR induces T cell functions 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 co-stimulatory molecule (e.g., if it transduces effector function signals) is used instead of the complete immune stimulation chain. In some embodiments, the intracellular signaling region (e.g., comprising one or more intracellular domains) includes a cytoplasmic sequence of a T cell receptor (TCR) and, in some respects, also includes co-receptors (which function in parallel with such receptors in the natural context to initiate signal transduction upon antigen receptor conjugation) and / or any derivatives or variants of such molecules, and / or any synthetic sequence having the same functional capabilities.

[0265] In the context of a natural TCR, complete activation typically requires not only signal transduction via the TCR but also co-stimulatory signals. Therefore, in some embodiments, components for generating secondary or co-stimulatory signals are also included in the CAR to promote complete activation. In other embodiments, the CAR does not include components for generating co-stimulatory signals. In some aspects, an additional CAR is expressed in the same cell and provides components for generating secondary or co-stimulatory signals.

[0266] In some respects, T cell activation is described as being mediated by two classes of cytoplasmic signaling sequences: those sequences that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those sequences that act in a non-antigen-dependent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences). In some respects, CARs include one or both of these signaling components.

[0267] In some aspects, a CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. The primary cytoplasmic signaling sequence that functions in a stimulatory manner may contain a signaling motif (referred to as an immune receptor tyrosine-based activation motif, or ITAM). Examples of primary cytoplasmic signaling sequences containing ITAMs include those derived from the TCR or CD3ζ, FcRγ, or FcRβ. In some embodiments, one or more cytoplasmic signaling molecules in the CAR contain a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3ζ.

[0268] In some implementations, the CAR includes the signal transduction region and / or transmembrane portion of a co-stimulatory receptor (such as CD28, 4-1BB, OX40, DAP10, and ICOS). In some aspects, the same CAR includes both the signal transduction region and the co-stimulatory component.

[0269] In some embodiments, the signal transduction region is included within a CAR, while the co-stimulatory component is provided by another CAR that recognizes another antigen. In some embodiments, the CAR includes an activating or stimulating CAR and a co-stimulatory CAR expressed on the same cell (see WO 2014 / 055668).

[0270] In some embodiments, the intracellular signaling region includes a CD28 transmembrane and signaling domain connected to the intracellular domain of CD3 (e.g., CD3-ζ). In some embodiments, the intracellular signaling region includes a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domain connected to the intracellular domain of CD3ζ.

[0271] In some implementations, the CAR encompasses one or more (e.g., two or more) co-stimulatory domains and activation domains (e.g., primary activation domains) in the cytoplasm. Exemplary CARs include intracellular components of CD3-ζ, CD28, and 4-1BB.

[0272] In some contexts, CARs are referred to as first-generation, second-generation, and / or third-generation CARs. In some respects, first-generation CARs are CARs that provide only CD3 chain-induced signaling upon antigen binding; in some respects, second-generation CARs are CARs that provide both this signaling and co-stimulatory signals, such as CARs that include intracellular signaling domains from co-stimulatory receptors (e.g., CD28 or CD137); and in some respects, third-generation CARs are CARs that include multiple co-stimulatory domains from different co-stimulatory receptors.

[0273] In some embodiments, the chimeric antigen receptor includes an extracellular portion containing an antibody or fragment described herein. In some aspects, the chimeric antigen receptor includes an extracellular portion containing an antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment includes scFv or a single-domain V. H The antibody contains an intracellular domain that includes ITAM. In some aspects, the intracellular signaling domain includes a signaling domain of the ζ chain of the CD3-zeta (CD3ζ) chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain disposed between the extracellular domain and the intracellular signaling region.

[0274] In some aspects, the transmembrane domain contains the transmembrane portion of CD28. The extracellular domain and the transmembrane domain may be directly or indirectly connected. In some embodiments, the extracellular domain and the transmembrane domain are connected by a spacer (as described herein). In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T-cell co-stimulatory molecule, such as between the transmembrane domain and the intracellular signaling domain. In some aspects, the T-cell co-stimulatory molecule is CD28 or 4-1BB.

[0275] In some embodiments, the CAR contains an antibody (e.g., an antibody fragment), a transmembrane domain (which is or contains a transmembrane portion of CD28 or a functional variant thereof), and an intracellular signaling domain containing a signaling portion of CD28 or a functional variant thereof and a signaling portion of CD3ζ or a functional variant thereof. In some such embodiments, the receptor also includes a spacer containing a portion of an Ig molecule (such as a human Ig molecule) (e.g., an Ig hinge, such as an IgG4 hinge), such as a hinge-only spacer.

[0276] In some embodiments, the transmembrane domain of the receptor (e.g., CAR) is the transmembrane domain of human CD28 or a variant thereof, such as the 27-amino acid transmembrane domain of human CD28 (accession number: P10747.1), or a transmembrane domain containing the amino acid sequence shown 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 higher sequence identity with SEQ ID NO:8; in some embodiments, the transmembrane domain containing a portion of the recombinant receptor contains the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it.

[0277] In some implementations, the chimeric antigen receptor contains an intracellular domain of a T-cell co-stimulatory molecule. In some aspects, the T-cell co-stimulatory molecule is CD28 or 4-1BB.

[0278] In some embodiments, the intracellular signaling domain comprises the intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as its 41-amino acid domain, and / or such a domain having LL to GG substitutions at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular signaling domain may comprise the amino acid sequence shown 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 higher sequence identity with SEQ ID NO: 10 or 11. In some embodiments, the intracellular region comprises an intracellular costimulatory signaling domain of 4-1BB or a functional variant or portion thereof, such as a cytoplasmic domain of 42 amino acids of human 4-1BB (accession number Q07011.1) or a functional variant or portion thereof, such as the amino acid sequence shown 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 higher sequence identity with SEQ ID NO:12.

[0279] In some embodiments, the intracellular signaling region comprises a human CD3 chain, optionally a CD3ζ stimulatory signaling domain, or a functional variant thereof, such as a cytoplasmic domain of 112 amino acids of isotype 3 of human CD3ζ (accession number: P20963.2) or a CD3ζ signaling domain as described in U.S. Patent Nos. 7,446,190 or 8,911,993. In some embodiments, the intracellular signaling region comprises the amino acid sequence shown 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 higher sequence identity with SEQ ID NO: 13, 14, or 15.

[0280] In some aspects, the spacer contains only the hinge region of IgG, such as only the hinge of IgG4 or IgG1, as shown in SEQ ID NO:1, which is a hinge-only spacer. In other embodiments, the spacer is associated with C H 2 and / or C H 3. Ig hinges connected by structural domains, such as the IgG4 hinge. In some embodiments, the spacer is connected to C... H 2 and C H 3. Ig hinges connected by structural domains, such as the IgG4 hinge, as shown in SEQ ID NO:3. In some embodiments, the spacer is only connected to C.H 3. A domain-connected Ig hinge, such as the IgG4 hinge, as shown in SEQ ID NO:4. In some embodiments, the spacer is or contains a glycine-serine-rich sequence or other flexible linker, such as known flexible linkers.

[0281] 2. Chimeric autoantibody receptor (CAAR)

[0282] In some embodiments, the recombinant receptor expressed by engineered cells used in conjunction with the provided methods, uses, articles, and compositions is a chimeric autoantibody receptor (CAAR). In some embodiments, the CAAR is specific for autoantibodies. In some embodiments, cells expressing CAAR (such as T cells engineered to express CAAR) can be used to specifically bind to and kill cells expressing autoantibodies, but not cells expressing normal antibodies. In some embodiments, cells expressing CAAR can be used to treat autoimmune diseases associated with the expression of self-antigens, such as autoimmune diseases. In some embodiments, cells expressing CAAR 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 interventions. In some embodiments, cells expressing CAAR can be used to effectively target and kill pathogenic B cells in autoimmune diseases by using antigen-specific chimeric autoantibody receptors to target disease-causing B cells. In some embodiments, the recombinant receptor is a CAAR, as described in any of the U.S. Patent Application Publication No. US 2017 / 0051035.

[0283] In some embodiments, the CAAR includes an autoantibody-binding domain, a transmembrane domain, and an intracellular signaling region. In some embodiments, the intracellular signaling region includes an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or includes a primary signaling domain, a signaling domain capable of inducing primary activation signals in T cells, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain including an immune receptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling region includes a secondary or co-stimulatory signaling region (a secondary intracellular signaling region).

[0284] In some embodiments, the autoantibody-binding domain contains an autoantigen or a fragment thereof. The selection of the autoantigen can depend on the type of autoantibody being targeted. For example, the autoantigen may be selected because it recognizes autoantibodies on target cells (such as B cells) associated with a specific disease state (e.g., an autoimmune disease, such as an autoantibody-mediated autoimmune disease). In some embodiments, autoimmune diseases include pemphigus vulgaris (PV). Exemplary autoantigens include desmosome core glycoprotein 1 (Dsg1) and Dsg3.

[0285] 3. Multi-target

[0286] In some embodiments, the cells used in conjunction with the provided methods, uses, articles, and compositions include cells employing a multi-targeting strategy. In some embodiments, the cells express a multi-chain chimeric antigen receptor (CAR) or express two or more genetically engineered receptors, each of which recognizes the same or different antigens and typically includes different intracellular signaling components. Such multi-targeting strategies are described, for example, in the following literature: International Patent Application Publication No. WO 2014055668 A1 (which describes a combination of activating and co-stimulatory CARs, e.g., targeting two different antigens that are present individually on off-target (e.g., 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) (which describes cells expressing activating and repressive CARs, such as those cells in which the activating CAR binds to an antigen expressed simultaneously on normal or disease-free cells and cells of the disease or condition to be treated, and the repressive CAR binds to another antigen expressed only on normal cells or cells that are not intended to be treated).

[0287] For example, in some embodiments, the cell includes a receptor expressing a first genetically engineered antigen receptor (e.g., CAR or TCR), which typically induces activation or stimulation signals to the cell upon specific binding to an antigen (e.g., a first antigen) recognized by the first receptor. In some embodiments, the cell also includes a second genetically engineered antigen receptor (e.g., CAR or TCR), such as a chimeric co-stimulatory receptor, which typically induces co-stimulatory signals to immune cells upon specific binding to a second antigen 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.

[0288] In some embodiments, a first and / or second genetically engineered antigen receptor (e.g., CAR or TCR) is capable of inducing activation signals to the cell. In some embodiments, the receptor includes an intracellular signaling component containing ITAM or an ITAM-like motif. In some embodiments, activation induced by the first receptor involves changes in signal transduction or protein expression in the cell, leading to the initiation of an immune response (e.g., ITAM phosphorylation) and / or the initiation of an ITAM-mediated signal transduction cascade, the formation of an immune synapse and / or the clustering of molecules near the bound receptor (e.g., CD4 or CD8), the activation of one or more transcription factors (e.g., NF-κB and / or AP-1), and / or the induction, proliferation, and / or survival of gene expression of factors such as cytokines.

[0289] In some embodiments, the first and / or second receptors include intracellular signaling domains or regions of co-stimulatory receptors (such as CD28, CD137 (4-1BB), OX40, and / or ICOS). In some embodiments, the first and second receptors include intracellular signaling domains of different co-stimulatory receptors. In one embodiment, the first receptor contains a CD28 co-stimulatory signaling region, and the second receptor contains a 4-1BB co-stimulatory signaling region, or vice versa.

[0290] In some implementations, the first and / or second receptors include both an intracellular signal transduction domain containing an ITAM or ITAM-like motif and an intracellular signal transduction domain of a co-stimulatory receptor.

[0291] In some embodiments, the first receptor contains an intracellular signaling domain comprising ITAM or an ITAM-like motif, and the second receptor contains an intracellular signaling domain of a co-stimulatory receptor. The co-stimulatory signal, combined with an activation signal induced in the same cell, is a co-stimulatory signal that leads to an immune response, such as a robust and sustained immune response, including increased gene expression, secretion of cytokines and other factors, and T cell-mediated effector functions (such as cell killing).

[0292] In some embodiments, neither the connection of the first receptor alone nor the connection of the second receptor alone induces a robust immune response. In some aspects, if only one receptor is connected, the cell becomes resistant to the antigen or unresponsive to the antigen, or is suppressed, and / or not induced to proliferate or secrete factors or perform effector functions. However, in some such embodiments, when multiple receptors are connected, such as upon encountering cells expressing the first and second antigens, the desired response is achieved, such as full immune activation or stimulation, for example, as indicated by the secretion, proliferation, persistence, and / or performance of immune effector functions (such as cytotoxic killing of target cells).

[0293] In some implementations, two receptors induce activation and inhibition signals to the cell, respectively, such that the binding of one receptor to its antigen activates the cell or induces a response, while the binding of a second inhibitory receptor to its antigen induces a signal that inhibits or attenuates the response. An example is a combination of an activating CAR and an inhibitory CAR or iCAR. For instance, such a strategy can be used where the activating CAR binds to an antigen expressed in a disease or symptom but also on normal cells, and the inhibitory receptor binds to a separate antigen expressed on normal cells but not on cells with the disease or symptom.

[0294] In some implementations, a multi-targeting strategy is used where antigens associated with a specific disease or condition are expressed transiently (e.g., after stimulation and genetic engineering) or permanently on disease-free cells and / or on the engineered cells themselves. In such cases, specificity, selectivity, and / or efficacy can be improved by requiring the connection of two separate and individually specific antigen receptors.

[0295] In some embodiments, the multiple antigens (e.g., first and second antigens) are expressed on the targeted cells, tissues, or diseases or conditions (e.g., on 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 also typically expressed on cells that do not require targeting with cell therapy (e.g., normal or disease-free cells or tissues) and / or on the engineered cells themselves. In such embodiments, specificity and / or efficacy are achieved by requiring the connection of multiple receptors to achieve a cellular response.

[0296] 4. T cell receptors

[0297] In some implementations, engineered cells (such as T cells) are provided that express peptide epitopes or T cell receptors (TCRs) that recognize target polypeptides (such as antigens of tumor, viral, or autoimmune proteins) or T cell epitopes, or their antigen-binding portions.

[0298] In some embodiments, a "T cell receptor" or "TCR" is a molecule or its antigen-binding moiety containing variable α and β chains (also referred to as TCRα and TCRβ, respectively) or variable γ and δ chains (also referred to as TCRα and TCRβ, respectively), and said molecule or its antigen-binding moiety is capable of specifically binding to peptides bound to MHC molecules. In some embodiments, the TCR is in αβ form. Typically, TCRs present in αβ and γδ forms are generally similar in structure, but T cells expressing them can have different anatomical locations or functions. TCRs can be found on the cell surface or in a soluble form. Typically, TCRs are found on the surface of T cells (or T lymphocytes), where they are usually responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0299] Unless otherwise stated, the term "TCR" should be understood to encompass both the complete TCR and its antigen-binding moiety or fragment. In some embodiments, the TCR is a complete or full-length TCR, including TCRs in αβ or γδ form. In some embodiments, the TCR is an antigen-binding moiety that is less than a full-length TCR but binds to a specific peptide bound to an MHC molecule, such as an MHC-peptide complex. In some cases, the antigen-binding moiety or fragment of the TCR may contain only a portion of the structural domains of the full-length or complete TCR, but is still capable of binding to peptide epitopes (such as MHC-peptide complexes) that bind to the complete TCR. In some cases, the antigen-binding moiety contains variable domains of the TCR (such as variable α-chains and variable β-chains of the TCR) sufficient to form a binding site for binding to a specific MHC-peptide complex. Typically, the variable chains of the TCR contain complementary determinant regions involved in the recognition of peptides, MHC, and / or MHC-peptide complexes.

[0300] In some embodiments, the variable domains of the TCR contain hypervariable loops or complementarity-determining regions (CDRs), which are typically major contributors to antigen recognition and binding capacity and specificity. In some embodiments, the CDRs of the TCR, or combinations thereof, form all or substantially all of the antigen-binding sites of a given TCR molecule. Individual CDRs within the variable region of the TCR chain are typically separated by framework regions (FRs), which generally exhibit 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 major CDR responsible for antigen binding or specificity, or, among the three CDRs on a given TCR variable region, is the most important for antigen recognition and / or for interaction with the processed peptide moiety of the peptide-MHC complex. In some contexts, CDR1 of the α-chain can interact with the N-terminal portion of certain antigenic peptides. In some contexts, CDR1 of the β-chain can interact with the C-terminal portion of the peptide. In some contexts, CDR2 has the strongest role in the interaction or recognition of the MHC portion of the MHC-peptide complex, or is the main responsible CDR. In some embodiments, the variable region of the β-chain may contain other hypervariable regions (CDR4 or HVR4), which are typically involved in superantigen binding rather than antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

[0301] In some embodiments, the TCR may also contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, for example, Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd ed., Current Biology Publications, 4:33, 1997). In some aspects, each strand of the TCR may have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail located at the C-terminus. In some embodiments, the TCR associates with an invariant protein of the CD3 complex involved in mediating signal transduction.

[0302] In some implementations, the TCR chain contains one or more constant domains. For example, the extracellular portion of a given TCR chain (e.g., the α chain or the β chain) may contain two immunoglobulin-like domains adjacent to the cell membrane, such as variable domains (e.g., Vα or Vβ; typically amino acid positions 1 to 116 based on Kabat numbering, Kabat et al., “Sequences of Proteins of Immunological Interest”, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th edition) and constant domains (e.g., the α chain constant domain or Cα, typically chain positions 117 to 259 based on Kabat numbering; or the β chain constant domain or Cα). β Typically, this is based on the Kabat chain position 117 to 295. For example, in some cases, the extracellular portion of a TCR formed by two chains contains two proximal constant membrane domains and two distal variable membrane domains, each of which contains a CDR. The constant domains of the TCR may contain short linker sequences 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.

[0303] 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 (like CD3) and their subunits. For example, a TCR containing a constant domain and a transmembrane region can anchor the protein in the cell membrane and associate with the invariant subunits of the CD3 signaling apparatus or complex. The intracellular tails of the CD3 signaling subunits (e.g., the CD3γ, CD3δ, CD3ε, and CD3ζ chains) contain one or more tyrosine-based activation motifs or ITAMs involved in the signaling ability of the TCR complex.

[0304] In some embodiments, the TCR can be a heterodimer of two chains, α and β (or optionally γ and δ), or it can be a single-chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β chains or γ and δ chains) linked by one or more disulfide bonds.

[0305] In some embodiments, the TCR can be generated from one or more known TCR sequences (such as sequences of the Vα and Vβ chains), the substantially full-length coding sequences of which are readily available. Methods for obtaining the full-length TCR sequence (including the V chain sequence) from a cellular source can be used. In some embodiments, the nucleic acid encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of the TCR-encoding nucleic acid from one or more given cells or isolated from said one or more given cells, or by synthesis of a publicly available TCR DNA sequence.

[0306] In some embodiments, the TCR is obtained from a biological source, such as from cells (e.g., from T cells (e.g., cytotoxic T cells)), T cell hybridomas, or other publicly available sources. In some embodiments, the T cells can be obtained from cells isolated in vivo. In some embodiments, the TCR is a thymus-selective TCR. In some embodiments, the TCR is a novel epitope-restricted TCR. In some embodiments, the T cells can be cultured T cell hybridomas or clones. In some embodiments, the TCR, its antigen-binding portion, or its antigen-binding fragment can be synthesized based on knowledge of the TCR sequence.

[0307] In some embodiments, the TCR is generated from TCRs identified or selected by screening candidate TCR libraries against a target polypeptide antigen or its target T cell epitopes. The TCR library can be generated by amplifying a library of Vα and Vβ 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, the TCR library can be generated from CD4+ or CD8+ cells. In some embodiments, the TCR can be amplified from T cells derived from normal or healthy subjects, i.e., a normal TCR library. In some embodiments, the TCR can be amplified from T cells derived from diseased subjects, i.e., a diseased TCR library. In some embodiments, the gene libraries of Vα and Vβ are amplified using degenerate primers, such as by RT-PCR amplification in samples obtained from humans (e.g., T cells). In some embodiments, the scTv library can be assembled from a natural Vα and Vβ library, wherein the amplified products are cloned or assembled to be separated by adapters. Depending on the source of the subject and cells, the library can be HLA allele-specific. Alternatively, in some embodiments, the TCR library can be generated by mutagenesis or diversification of parental or scaffold TCR molecules. In some aspects, the TCR undergoes directed evolution, such as α or β chain evolution, as performed by mutagenesis. In some aspects, specific residues within the CDR 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 assess CTL activity against the peptide. In some aspects, TCRs present on antigen-specific T cells can be selected, for example, by binding activity, such as specific affinity or affinity for the antigen.

[0308] In some embodiments, the TCR or its antigen-binding portion is a modified or engineered TCR or its antigen-binding portion. 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 through display methods, including but not limited to yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) ProcNatl 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 method involves engineering or modifying a known parental or reference TCR. For example, in some cases, wild-type TCRs can be used as templates to generate mutagenic TCRs, in which one or more residues of the CDR are mutated and mutants with the desired altered properties (such as higher affinity for the desired target antigen) are selected.

[0309] In some embodiments, the peptide used to generate or produce the target TCR is known or readily identifiable. In some embodiments, the peptide suitable for generating the TCR or antigen-binding motif can be determined based on the presence of an HLA restriction motif in the target peptide (as described below). In some embodiments, available computer prediction models are used to identify the peptide. In some embodiments, such models for predicting MHC class I binding sites include, but are not limited to, ProPred1 (Singh and Raghava (2001) Bioinformatics 17(12):1236-1237) and SYFPEITHI (see Schuler et al. (2007) Immunoinformatics Methods in Molecular Biology, 409(1):75-93 2007). In some embodiments, the MHC restriction epitope is HLA-A0201, which is expressed in approximately 39%-46% of all Caucasians and therefore represents a suitable choice of MHC antigen for the preparation of TCRs or other MHC-peptide-binding molecules.

[0310] HLA-A0201 binding motifs and proteasome and immunoproteasome cleavage sites can be predicted using computer-aided models. For predicting MHC class I binding sites, such models include, but are not limited to, ProPred1 (described in more detail in Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17(12):1236-1237 2001) and SYFPEITHI (see Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction. In Immunoinformatics Methods in Molecular Biology, Vol. 409(1):75-93 2007).

[0311] In some embodiments, the TCR or its antigen-binding portion may be a recombinantly derived natural protein or a mutant form thereof, wherein one or more properties (such as binding characteristics) have been altered. In some embodiments, the TCR may be derived from one of several animal species, such as human, mouse, rat, or other mammals. The TCR may be cell-bound or in a soluble form. In some embodiments, for the purposes of the provided methods, the TCR is in a cell-bound form expressed on the cell surface.

[0312] In some embodiments, the TCR is a full-length TCR. In some embodiments, the TCR is an antigen-binding moiety. In some embodiments, the TCR is a dimer TCR (dTCR). In some embodiments, the TCR is a single-chain TCR (sc-TCR). In some embodiments, the dTCR or scTCR has the structure described in WO 03 / 020763, WO 04 / 033685, and WO 2011 / 044186.

[0313] In some embodiments, the TCR contains a sequence corresponding to a transmembrane sequence. In some embodiments, the TCR does indeed contain a sequence corresponding to a cytoplasmic sequence. In some embodiments, the TCR is capable of forming a TCR complex with CD3. In some embodiments, any TCR (including dTCR or scTCR) can be linked to a signal transduction domain, thereby generating an active TCR on the T cell surface. In some embodiments, the TCR is expressed on the cell surface.

[0314] In some embodiments, the dTCR contains a first polypeptide (wherein a sequence corresponding to the variable region sequence of the TCR α chain is fused at the N-terminus to a sequence corresponding to the extracellular sequence of the TCR α chain constant region) and a second polypeptide (wherein a sequence corresponding to the variable region sequence of the TCR β chain is fused at the N-terminus to a sequence corresponding to the extracellular sequence of the TCR β chain constant region), the first and second polypeptides being linked by a disulfide bond. In some embodiments, the bond may correspond to a natural interchain disulfide bond present in the native dimer αβTCR. In some embodiments, an interchain disulfide bond is not present in the native TCR. For example, in some embodiments, one or more cysteine ​​residues may be incorporated into the extracellular sequence of the constant region of the dTCR polypeptide pair. In some cases, both natural and non-natural disulfide bonds may be required. In some embodiments, the TCR contains a transmembrane sequence to anchor to the membrane.

[0315] In some embodiments, dTCR contains a TCRα chain comprising a variable α domain, a constant α domain, and a first dimerizing motif attached to the C-terminus of the constant α domain; and a TCRβ chain comprising a variable β domain, a constant β domain, and a first dimerizing motif attached to the C-terminus of the constant β domain, wherein the first and second dimerizing motifs readily interact to form a covalent bond between an amino acid of the first dimerizing motif and an amino acid of the second dimerizing motif, thereby linking the TCRα chain and the TCRβ chain together.

[0316] In some implementations, the TCR is a scTCR. Typically, scTCRs can be generated using methods known to those skilled in the art, see, for example, Soo Hoo, WF et al., PNAS (USA) 89,4759 (1992); Wülfing, C. and Plückthun, A., J. Mol. Biol. 242,655 (1994); Kurucz, I. et al., PNAS (USA) 90 3830 (1993); International Publication PCT Nos. WO 96 / 13593, WO 96 / 18105, WO 99 / 60120, WO 99 / 18129, WO 03 / 020763, WO 2011 / 044186; and Schlueter, CJ et al., J. Mol. Biol. 256,859 (1996). In some embodiments, the scTCR contains introduced non-natural interchain disulfide bonds to promote TCR chain association (see, for example, International Publication PCT No. WO 03 / 020763). In some embodiments, the scTCR is a non-disulfide-linked truncated TCR, wherein an isoleucine zipper fused to its C-terminus promotes chain association (see, for example, International Publication PCT No. WO 99 / 60120). In some embodiments, the scTCR contains a TCRα variable domain covalently linked to a TCRβ variable domain via a peptide linker (see, for example, International Publication PCT No. WO 99 / 18129).

[0317] In some embodiments, the scTCR contains a first segment (consisting of an amino acid sequence corresponding to the variable region of the TCRα chain), a second segment (consisting of an amino acid sequence corresponding to the variable region of the TCRβ chain fused to the N-terminus of an amino acid sequence corresponding to the extracellular sequence of the constant domain of the TCRβ chain), and a linker sequence (which links the C-terminus of the first segment to the N-terminus of the second segment).

[0318] In some embodiments, the scTCR contains a first segment (consisting of an α-chain variable region sequence fused to the N-terminus of an α-chain extracellular constant domain sequence) and a second segment (consisting of a β-chain variable region sequence fused to the N-terminus of a sequenced β-chain extracellular constant and transmembrane sequence) and optionally a linker sequence (which connects the C-terminus of the first segment to the N-terminus of the second segment).

[0319] In some embodiments, the scTCR comprises a first segment (which consists of a TCR β chain variable region sequence fused to the N-terminus of the β chain extracellular constant domain sequence) and a second segment (which consists of an α chain variable region sequence fused to the N-terminus of the α chain extracellular constant and transmembrane sequence) and optionally a linker sequence (which connects the C-terminus of the first segment to the N-terminus of the second segment).

[0320] In some embodiments, the linker connecting the first and second TCR segments of the scTCR can be any linker capable of forming a single polypeptide chain while maintaining TCR binding specificity. In some embodiments, the linker sequence can, for example, have the formula -P-AA-P-, where P is proline and AA represents an amino acid sequence, wherein 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 this binding. Thus, in some cases, the linker is long enough to span the distance between the C-terminus of the first segment and the N-terminus of the second segment, or vice versa, but not so long as to block or reduce the binding of the scTCR to the target ligand. In some embodiments, the linker can contain from or from about 10 to 45 amino acids, such as 10 to 30 amino acids or 26 to 41 amino acid residues, such as 29, 30, 31, or 32 amino acids. In some embodiments, the linker has the formula -PGGG-(SGGGG)5-P-, where P is proline, G is glycine, and S is serine (SEQ ID NO: 23). In some implementations, the connector has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO:24).

[0321] In some embodiments, the scTCR contains covalent disulfide bonds that link 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, interchain disulfide bonds are absent in the native TCR. For example, in some embodiments, one or more cysteine ​​residues may be incorporated into the extracellular sequence of the constant region of the first and second segments of the scTCR polypeptide. In some cases, both native and non-native disulfide bonds may be required.

[0322] In some embodiments of dTCRs or scTCRs containing introduced interchain disulfide bonds, native disulfide bonds are absent. In some embodiments, one or more native cysteine ​​residues forming native interchain disulfide bonds are substituted with another residue, such as serine or alanine. In some embodiments, the introduced disulfide bonds can be formed by mutating non-cysteine ​​residues on the first and second segments to cysteine. Exemplary non-native disulfide bonds in TCRs are described in the published International PCT No. WO 2006 / 000830.

[0323] In some embodiments, the TCR or its antigen-binding fragment exhibits affinity for the target antigen at an equilibrium binding constant, which is between or between about 10⁻⁵ and 10⁻¹² M and all individual values ​​and ranges therewith. In some embodiments, the target antigen is an MHC-peptide complex or ligand.

[0324] In some implementations, 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.

[0325] In some embodiments, the vector may be from the following series: pUC (Fermentas LifeSciences), pBluescript (Stratagene, La Jolla, California), pET (Novagen, Madison, Wisconsin), pGEX (Pharmacia Biotech, Uppsala, Sweden), or pEX (Clontech, Palo Alto, California). In some cases, phage vectors such as λG10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 may also be used. In some embodiments, plant expression vectors may 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.

[0326] In some embodiments, the recombinant expression vector may be prepared using standard recombinant DNA techniques. In some embodiments, the vector may contain regulatory sequences (such as transcription and translation start and stop codons) that are specific to the type of host (e.g., bacteria, fungi, plants, or animals) to which the vector is introduced, depending on whether the vector is DNA-based or RNA-based. In some embodiments, the vector may contain a non-natural promoter that is operatively linked to a nucleotide sequence encoding a TCR or antigen-binding moiety (or other MHC-peptide-binding molecule). In some embodiments, the promoter may be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and promoters found in long terminal repeat sequences of mouse stem cell viruses. Other known promoters are also envisioned.

[0327] In some embodiments, to generate a vector encoding a TCR, total cDNA isolated from T cell clones expressing the target TCR is amplified by PCR and cloned into an expression vector. In some embodiments, the α and β chains are cloned into the same vector. In some embodiments, the α and β chains are cloned into different vectors. In some embodiments, the resulting α and β chains are incorporated into a retroviral (e.g., lentiviral) vector.

[0328] B. Nucleic Acids and Vectors

[0329] Also provided are one or more polynucleotides (e.g., nucleic acid molecules) encoding a recombinant receptor, a vector for genetically engineering cells to express the receptor, and a method for generating the 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), such as a transgenic TCR.

[0330] In some cases, the nucleic acid sequence encoding a recombinant receptor (e.g., a chimeric antigen receptor (CAR)) contains a signal sequence encoding a signal peptide. Non-limiting exemplary examples of signal peptides include, for example, the GMCSFRα chain signal peptide shown in SEQ ID NO:26 and encoded by the nucleotide sequence shown in SEQ ID NO:25, or the CD8α signal peptide shown in SEQ ID NO:18.

[0331] In some cases where a nucleic acid molecule encodes two or more different polypeptide chains, each of the polypeptide chains may be encoded by a separate nucleic acid molecule. For example, two separate nucleic acids are provided, and each can be separately transferred to or introduced into a cell for expression.

[0332] In some embodiments, such as those in which the polynucleotide contains first and second nucleic acid sequences, the coding sequences encoding each different polypeptide chain can be operatively 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 polycistronic (bicistronic or tricistronic, see, for example, U.S. Patent No. 6,060,273). In some embodiments, the transcription unit can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows co-expression of the gene product via information from a single promoter. Alternatively, in some cases, a single promoter can direct the expression of RNA containing two or three genes separated from each other in a single open reading frame (ORF) by sequences encoding self-cleaving peptides (e.g., 2A sequences) or protease recognition sites (e.g., furin proteases). Thus, the ORF encodes a single polypeptide, which is processed into a single protein during translation (in the case of 2A) or post-translational. In some cases, the peptide (such as T2A) can cause ribosome skipping (ribosome jumping) of the peptide bond at the C-terminus of the 2A element, resulting in separation between the 2A sequence terminus and the next downstream peptide (see, for example, de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)). Various 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 viruses such as foot-and-mouth disease virus (F2A, e.g., SEQ ID NO:22), equine rhinitis A virus (E2A, e.g., SEQ ID NO:21), *Tetrasomy β* virus (T2A, e.g., SEQ ID NO:6 or 17), and porcine swine vesiculovirus-1 (P2A, e.g., SEQ ID NO:19 or 20), as described in U.S. Patent Publication No. 20070116690.

[0333] In some implementations, the extrinsic marker gene may be combined with engineered cell therapies to allow for the detection or selection of cells, and in some cases, to promote cell suicide. Exemplary alternative markers may include truncated forms of cell surface peptides, such as non-functional forms that do not transduce or cannot transduce signals, or signals that are typically transduced by the full-length form of the cell surface peptide, and / or truncated forms that are not internalized or cannot be internalized. Exemplary truncated cell surface peptides 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, the exemplary tEGFR sequence shown in SEQ ID NO:7 or 16), or prostate-specific membrane antigen (PSMA) or modified forms thereof. tEGFR may contain the antibody cetuximab. Or other therapeutic anti-EGFR antibodies or epitopes recognized by binding molecules, which can be used to identify or select cells engineered with tEGFR constructs and encoded exogenous proteins, 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. April 2016; 34(4):430-434. In some aspects, markers (e.g., alternative markers) include all or part (e.g., truncated forms) of CD34, NGFR, CD19, or truncated CD19 (e.g., truncated nonhuman CD19) or epidermal growth factor receptor (e.g., tEGFR).

[0334] In some embodiments, the marker is or comprises a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP) (e.g., hyperfolded 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, including species variants, monomeric variants, and codon-optimized and / or enhanced variants of the fluorescent protein. In some embodiments, the marker is or comprises an enzyme (e.g., luciferase), the lacZ gene from *E. coli*, alkaline phosphatase, secretory embryonic 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.

[0335] In some embodiments, the marker is a selection marker. In some embodiments, the selection marker is or comprises a polypeptide that confers resistance to a foreign agent or drug. In some embodiments, the selection marker is an antibiotic resistance gene. In some embodiments, the selection marker is an antibiotic resistance gene that confers antibiotic resistance to mammalian cells. In some embodiments, the selection marker is or comprises a puromycin resistance gene, a hygromycin resistance gene, a blastcin resistance gene, a neomycin resistance gene, a genimycin resistance gene, or a bleomycin resistance gene, or a modified form thereof.

[0336] In some embodiments, the nucleic acid encoding the tag is operatively linked to a polynucleotide encoding an adapter sequence (such as a cleavable adapter sequence, e.g., T2A). For example, the tag and optionally the adapter sequence can be any of those disclosed in PCT Publication No. WO2014031687. For example, the tag can be a truncated EGFR (tEGFR) optionally linked to an adapter sequence, such as the T2A cleavable adapter sequence. Exemplary polypeptides of truncated EGFR (e.g., tEGFR) comprise the amino acid sequence shown in SEQ ID NO:7 or 16, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with SEQ ID NO:7 or 16. In some cases, truncated epidermal growth factor receptors (EGFRt), such as those shown in SEQ ID NO:7 or 16, can be co-expressed with the target transgene (CAR or TCR) in the transduced cells (see, for example, U.S. Patent No. 8,802,374). EGFRt may contain the antibody cetuximab. Or other therapeutic anti-EGFR antibodies or binding molecules that recognize epitopes that can be used to identify or select cells engineered with an EGFRt construct and another recombinant receptor (such as a chimeric antigen receptor (CAR)), and / or to eliminate or isolate cells expressing said receptor. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. April 2016; 34(4):430-434.

[0337] Vectors or constructs containing such nucleic acids and / or polynucleotides are also provided. In some embodiments, the vector or construct contains one or more promoters operatively linked to a nucleic acid encoding a recombinant receptor to drive its expression. In some embodiments, the promoter is operatively linked to one or more nucleic acid molecules or polynucleotides. Therefore, vectors, such as those containing any of the polynucleotides provided herein, are also provided.

[0338] In some cases, the vector is a viral vector, such as a retroviral vector, for example a lentiviral vector or a gamma retroviral vector. Compositions containing such vectors or combinations of vectors are also provided. In some embodiments, the group or combination of vectors is used together for cell engineering. In some embodiments, the first and second vectors in the group are introduced sequentially, simultaneously or in any order, into the cells for engineering.

[0339] In some implementations, the vector includes viral vectors, such as retroviruses or lentiviruses, nonviral vectors, or transposons, such as the Sleeping Beauty transposon system; vectors derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV); lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors; and retroviral vectors derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen lesion-forming virus (SFFV), or adeno-associated virus (AAV).

[0340] C. Vectors and methods used for genetic engineering

[0341] Various methods for introducing genetically engineered components, such as recombinant receptors (e.g., CARs or TCRs), are well known. Exemplary methods include those for transferring nucleic acids encoding the receptor, including via viruses (e.g., retroviruses or lentiviruses), transduction, transposons, and electroporation. In some embodiments, surface glycan expression is evaluated in compositions of cells collected immediately before, during, or after the genetic engineering process. In some embodiments, surface glycan expression is evaluated and compared in cell compositions at the respective stages of the process for introducing the genetically engineered component. In some embodiments, the compositions are engineered or have been engineered to express the same recombinant receptor, but by different methods of introducing genetic material.

[0342] In some implementations, gene transfer is accomplished by first stimulating cells, such as by combining them with stimuli that induce responses (e.g., proliferation, survival, and / or activation), as measured by the expression of cytokines or activation markers, then transducing the activated cells and expanding them in culture to a quantity sufficient for clinical use.

[0343] In some embodiments, recombinant infectious viral particles (e.g., vectors derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV)) are used to transfer recombinant nucleic acids into cells. In some embodiments, recombinant lentiviral vectors or retroviral vectors (e.g., gamma-retroviral vectors) are used to transfer recombinant nucleic acids into T cells (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-Camino et al. Mol Ther Nucl Acids,2,e93 (2013); Park et al., Trends Biotechnol.,November 29(11):550-557 (2011)). In some embodiments, the virus is adeno-associated virus (AAV).

[0344] In some embodiments, the retroviral vector has a long terminal repeat (LTR) sequence, such as retroviral vectors derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), and spleen lesion-forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. Retroviruses are generally amphiphilic, meaning they can infect host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol, and / or env sequences. Several illustrative retroviral systems have been described (e.g., U.S. Patent Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman, BioTechniques, 7:980-990 (1989); Miller, AD Human Gene Therapy, 1:5-14 (1990); Scarpa et al. Virology, 180:849-852 (1991); Burns et al. Proc. Natl. Acad. Sci. USA, 90:8033-8037 (1993); and Boris-Lawrie and Temin, Cur. Opin. Genet. Develop., 3:102-109 (1993)).

[0345] Methods of lentiviral transduction are known. Exemplary methods are described in, for example, the following literature: Wang et al., J. Immunother., 35(9):689-701 (2012); Cooper et al., Blood. 101:1637-1644 (2003); Verhoeyen et al., Methods Mol Biol., 506:97-114 (2009); and Cavalieri et al., Blood., 102(2):497-505 (2003).

[0346] In some embodiments, recombinant nucleic acids are transferred to T cells via electroporation (see, for example, Chicaybam et al., PLoS ONE 8(3):e60298(2013) and Van Tedeloo et al., Gene Therapy 7(16):1431-1437(2000)). In some embodiments, recombinant nucleic acids are transferred to T cells via transposition (see, for example, Manuri et al., Hum Gene Ther 21(4):427-437(2010); Sharma et al., Molec Ther Nucl Acids 2,e74(2013); and Huang et al., Methods Mol Biol 506:115-126(2009)). Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-promoted microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)).

[0347] Other methods and vectors for transferring nucleic acids encoding recombinant products are those described, for example, in International Patent Application Publication No. WO2014055668 and U.S. Patent No. 7,446,190.

[0348] In some implementations, cells (e.g., T cells) can be transfected during or after amplification, for example with a T-cell receptor (TCR) or a chimeric antigen receptor (CAR). Such transfection, for example, can be performed using any suitable retroviral vector to introduce the gene for the desired receptor. The genetically modified cell population can then be evacuated from the initial stimulus (e.g., a CD3 / CD28 stimulus) and subsequently stimulated with a second type of stimulus, for example, via a de novo introduced receptor. This second type of stimulus can include an antigenic stimulus in the form of a peptide / MHC molecule, a homologous (crosslinked) ligand of the gene-introduced receptor (e.g., a natural ligand of the CAR), or any ligand (e.g., an antibody) that binds directly within the framework of the new receptor (e.g., by recognizing a constant region within the receptor). See, for example, Cheadle et al., Methods Mol Biol. 907:645-66 (2012); or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine, Vol. 65:333-347 (2014).

[0349] In some cases, vectors that do not require cell activation (e.g., T cells) can be used. In some such cases, cells can be selected and / or transduced prior to activation. Therefore, cells can be engineered before or after cell culture, and in some cases, simultaneously with or during at least a portion of the culture.

[0350] In some respects, cells are further engineered to promote the expression of cytokines or other factors. Additional nucleic acids (e.g., genes for introduction) include those for improving therapeutic efficacy, such as by promoting the viability and / or function of the transferred cells; genes for providing genetic markers for selecting and / or evaluating cells (e.g., for assessing in vivo survival or localization); and genes for improving safety, such as by making cells susceptible to negative selection in vivo, as described in: Lupton SD et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications of Lupton et al., PCT / US91 / 08442 and PCT / US94 / 05601, which describe the use of bifunctional selection fusion genes obtained by fusing a dominant positive selection marker with a negative selection marker. See, for example, Riddell et al., U.S. Patent No. 6,040,177, columns 14-17.

[0351] D. Characteristics of the output composition

[0352] In certain embodiments, the methods provided herein produce or generate cell compositions containing genetically engineered cells, such as output compositions. In some embodiments, the output composition is a cell composition produced by some or all of the steps for genetically engineering cells. In some embodiments, the output composition is produced by a process of genetically engineering cells from an input cell composition. In some embodiments, the process includes one or more steps for activating, transducing or transfecting, amplifying, and / or harvesting cells (e.g., cells obtained from an input cell composition). In some embodiments, the output cell composition contains genetically engineered cells. In certain embodiments, the cells of the output composition have undergone all steps of the genetic engineering process.

[0353] In some embodiments, the output composition contains cells comprising one or more nucleic acids introduced through genetic engineering, and thereby expressing recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., not typically present in cells or samples obtained from cells, such as nucleic acids obtained from another organism or cell, for example, said nucleic acids are not typically found in the engineered cells and / or the organism from which such cells are derived. In some embodiments, the nucleic acids are not naturally occurring, such as nucleic acids not found in nature, including nucleic acids comprising chimeric combinations of nucleic acids encoding various domains from a variety of different cell types.

[0354] In some embodiments, the exported composition contains genetically engineered cells. In a particular embodiment, the exported cell composition contains engineered T cells. In some embodiments, the engineered T cells include engineered CD4+ T cells and engineered CD8+ T cells. In a particular embodiment, the exported composition contains or includes at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% of engineered T cells. In some embodiments, the engineered cells express a recombinant receptor. In certain embodiments, the output composition contains or includes at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100%, or about 100%, of T cells expressing the recombinant receptor. In some embodiments, the recombinant receptor is a TCR or a CAR. In a particular embodiment, the recombinant receptor is a CAR.

[0355] III. Compositions and Formulations

[0356] This document provides compositions or formulations containing cells prepared according to the incubation (e.g., stimulation) methods described herein. In some embodiments, the compositions and methods described herein can be used to eliminate at least a portion of a cell population, such as non-immature T cells, from a cell population. Compositions comprising cell populations that no longer contain unwanted cells or have a significantly reduced number of unwanted cells (such as cells derived from non-immature T cells) in the stimulated composition are also provided; and their uses. The compositions and methods provided herein are also used for selectively expanding cell populations from which unwanted subsets have been removed for therapeutic purposes. This document also provides stimulated compositions resulting from the output or enrichment of T cells produced by any of the methods described herein for stimulating T cells.

[0357] In some embodiments, cells generated using any of the incubation (e.g., stimulation) methods described herein (such as cells genetically engineered with recombinant receptors (e.g., CAR-T cells)) are provided as compositions (including pharmaceutical compositions and formulations, such as unit dosage form compositions comprising a number of cells administered at a given dose or fraction thereof). Pharmaceutical compositions and formulations typically include one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition includes at least one additional therapeutic agent.

[0358] In some embodiments, a cell composition is generated or manufactured for cell therapy purposes. In some embodiments, the cell composition is a pharmaceutical composition or formulation. Such compositions can be used according to provided methods, for example, to evaluate their release for use in the prevention or treatment of diseases, conditions, and disorders, or in methods for detection, diagnosis, and prognosis.

[0359] The term "pharmaceutical formulation" refers to a preparation in a form that allows for the bioactivity of the active ingredient contained therein and that contains no additional components that would have unacceptable toxicity to a subject to be given the formulation. In some embodiments, the methods provided herein can be used to compare the expression of surface glycans in cell compositions composed of identical engineered cells but with different pharmaceutical formulations.

[0360] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical formulation that are non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In certain embodiments, the methods provided herein can be used to compare the expression of surface glycans in cell compositions composed of identical engineered cells but with different pharmaceutically acceptable carriers.

[0361] In some embodiments, T-cell therapies (such as engineered T cells (e.g., CAR T cells)) are formulated using pharmaceutically acceptable carriers. In some aspects, the choice of carrier depends in part on the specific cells and / or the method of administration. Therefore, a variety of suitable formulations exist. For example, the pharmaceutical composition may contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. Preservatives or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10). (Residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0362] In some aspects, the composition includes a buffer. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffer or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administerable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st edition (May 1, 2005).

[0363] The formulation may include an aqueous solution. The formulation or composition may also contain more than one active ingredient that can be used for the prevention or treatment of a specific indication, disease, or condition by cell therapy, including one or more active ingredients whose activities are complementary to those of the cells and / or whose respective activities do not adversely affect each other. Such active ingredients are suitably combined in amounts effective for the intended purpose. Therefore, in some embodiments, the pharmaceutical composition also includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vincristine, etc.

[0364] In some embodiments, the pharmaceutical composition contains cells in an amount effective in treating or preventing the disease or condition (e.g., a therapeutically effective amount or a preventatively effective amount). In some embodiments, therapeutic or preventative efficacy is monitored by periodically evaluating the treated subjects. For repeated administration over several days or longer, treatment is repeated until the desired suppression of disease symptoms is achieved, depending on the condition. However, other dosing regimens may be useful and can be determined. The required dose can be delivered by a single bolus injection, by multiple bolus injections, or by continuous infusion.

[0365] Cells can be formulated for administration using standard administration techniques, formulations, and / or devices. Formulations and devices, such as syringes and vials, are provided for storing and administering the composition. Regarding the cells, administration can be autologous or allogeneic. For example, immune-reactive cells or progenitor cells can be obtained from a subject and administered to the same subject or different but compatible subjects. Peripheral blood-derived immune-reactive cells or their progeny (e.g., in vivo, ex vivo, or in vitro derived) can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When administering therapeutic compositions (e.g., pharmaceutical compositions containing genetically modified immune-reactive cells), they are typically formulated into injectable unit-dose forms (solutions, suspensions, emulsions).

[0366] Preparations include those intended for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the drug or cell population is administered parenterally. As used herein, the term "parentereal" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the drug or cell population is administered to the subject via peripheral systemic delivery through intravenous, intraperitoneal, or subcutaneous injection.

[0367] In some embodiments, the composition is provided as a sterile liquid formulation (e.g., an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition), which can be buffered to a selected pH in some respects. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are slightly more convenient to administer, particularly by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact time with a particular tissue. Liquid or viscous compositions may contain a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0368] Sterile injectable solutions can be prepared by incorporating cells into a solvent, such as by mixing with a suitable carrier, diluent, or excipient (e.g., sterile water, physiological saline, glucose, dextrose, etc.). The composition may also be lyophilized. The composition may contain excipients such as wetting agents, dispersants or emulsifiers (e.g., methylcellulose), pH buffers, gelling or viscosity-enhancing additives, preservatives, flavoring agents, pigments, etc., depending on the desired route of administration and formulation. In some respects, standard texts can be consulted to prepare suitable formulations.

[0369] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Antimicrobial activity can be ensured by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, etc.). Prolonged absorption of injectable drug forms can be achieved by using agents with delayed absorption (e.g., aluminum monostearate and gelatin).

[0370] Preparations intended for internal administration are typically sterile. Sterility can be readily achieved, for example, through filtration via a sterile filter membrane.

[0371] For the prevention or treatment of a disease, the appropriate dosage may depend on the type of disease to be treated, the type of one or more agents, the type of cell or recombinant receptor, the severity and course of the disease, whether the agent or cell is administered for preventive or therapeutic purposes, prior therapy, the subject's clinical history and response to the agent or cell, and the attending physician's decision. In some embodiments, the composition is appropriately administered to the subject once or in a series of treatments.

[0372] Methods and uses of cells and compositions (such as those present in the output compositions described herein) in the treatment of diseases, conditions, and disorders expressing antigens recognized by recombinant receptors (e.g., CARs) are also provided herein. Treatment methods comprising administering to a subject an output or enriched output composition produced by any of the incubation (e.g., stimulation) methods described herein. In some embodiments, the methods include generating genetically engineered T cells using any of the methods described herein, and administering the genetically engineered T cells generated by the methods described herein.

[0373] Methods for administering engineered cells and compositions are provided, as well as uses of such engineered cells and compositions for treating or preventing diseases, conditions, and disorders, including cancer. In some embodiments, cell-based therapies are or include administering cells, such as immune cells, like T cells, that target molecules expressed on the surface of a lesion (such as a tumor or cancer). The provided methods and uses include methods and uses for adoptive cell therapy. In some embodiments, the methods include administering engineered cells or compositions containing said cells (such as cells from the exported composition as described) to a subject, tissue, or cell, such as a subject, tissue, or cell that has, is at risk of having, or is suspected of having said disease, condition, or disorder. In some embodiments, cells, populations, and compositions are administered to a subject suffering from a specific disease or condition that requires treatment, for example, by adoptive cell therapy (such as adoptive T-cell therapy). In some embodiments, cells or compositions are administered to a subject, such as a subject who has, or is at risk of having, said disease or condition, to improve one or more symptoms of said disease or condition, such as by reducing tumor burden in cancer expressing antigens recognized by engineered T cells.

[0374] In some aspects, the disease or condition being treated can be any disease or condition in which the expression of the antigen is associated with, specific to, the cells or tissues of the disease, disorder, or condition, and / or expressed on, the cells or tissues of the disease, disorder, or condition, and / or involved in the etiology of the disease, disorder, or disorder, such as causing, aggravating, or otherwise contributing to such disease, disorder, or disorder. Exemplary diseases and conditions may include those associated with malignant tumors or cell transformations (e.g., cancer), autoimmune or inflammatory diseases, or infectious diseases, such as those caused by bacteria, viruses, or other pathogens. Exemplary antigens have been described above, including antigens associated with a variety of diseases and conditions that can be treated. In certain embodiments, immunomodulatory peptides and / or recombinant receptors (e.g., chimeric antigen receptors or TCRs) specifically bind to antigens associated with the disease or condition. In some embodiments, the subject suffers from a disease, disorder, or condition, optionally cancer, tumor, autoimmune disease, disorder, or condition, or infectious disease.

[0375] In some implementations, the disease, disorder, or condition includes tumors associated with various cancers. In some implementations, the cancer can be any cancer located in the subject's body, such as, but not limited to, cancers of the head and neck, breast, liver, colon, ovary, prostate, pancreas, brain, cervix, bone, skin, eye, bladder, stomach, esophagus, peritoneum, or lung. For example, anticancer agents can be used to treat colon cancer, cervical cancer, central nervous system cancer, breast cancer, bladder cancer, anal cancer, head and neck cancer, ovarian cancer, endometrial cancer, small cell lung cancer, non-small cell lung cancer, neuroendocrine cancer, soft tissue cancer, penile cancer, prostate cancer, pancreatic cancer, stomach cancer, gallbladder cancer, or esophageal cancer. In some cases, the cancer can be a blood cancer. In some implementations, the disease, disorder, or condition is a tumor, such as a solid tumor, lymphoma, leukemia, hematologic malignancy, metastatic tumor, or other cancer or tumor type. In some implementations, the disease, disorder, or symptom is selected from colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, ovarian cancer, epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and / or mesothelioma.

[0376] Diseases, conditions, and disorders include tumors, including solid tumors, hematologic malignancies, and melanoma, and include both localized and metastatic tumors; infectious diseases, such as infections with viruses or other pathogens, such as HIV, HCV, HBV, CMV, HPV, and parasitic diseases; and autoimmune and inflammatory diseases. In some implementations, a disease, disorder, or condition is a tumor, cancer, malignancy, ulcer, or other proliferative disease or disorder. These diseases include, but are not limited to, leukemia and lymphoma, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), anaplastic large cell lymphoma (ALCL), follicular lymphoma, refractory follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM). B-cell malignancies are selected from acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL).

[0377] In some implementations, the disease or condition is an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial and protozoan infections, immunodeficiency, cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, and BK polyomavirus. In some implementations, the disease or condition is an autoimmune or inflammatory disease or condition, such as arthritis (e.g., rheumatoid arthritis (RA)), type 1 diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, asthma, and / or transplant-related diseases or conditions.

[0378] In some implementations, the antigen associated with the disease or disorder is selected from orphan tyrosine kinase receptor ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2 / neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22, mesothelin, CEA, hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), liver glycoprotein receptor A2 (EPHa2), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, type III epidermal growth factor receptor mutant (EGFR vIII), folate-binding protein (FBP), FCRL5, and Fc receptor-like protein 5 (FCRL5).Also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor, ganglioside GD2, ganglioside GD3, G protein-coupled receptor 5D (GPCR5D), HMW-MAA, IL-22R-α, IL-13R-α2, kinase insertion domain receptor (kdr), κ light chain, leucine-rich repeat-containing protein 8 family member A (LRRC8A), Lewis Y, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, melanoma preferential expression antigen (PRAME), survival protein, TAG72, B7-H6, IL-13 receptor α2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250 / CAIX, human leukocyte antigen A1 (HLA-A1), MAGE A1, HLA-A2, NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7 / 8, αvβ6 integrin (avb6 integrin), 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, natural killer cell family 2 member D (NKG2D) ligand, CD44v6, dual antigens, cancer-testis antigen, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, prostate stem cell antigen (PSCA), NKG2D, cancer-testis antigen cancer / testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), MART-1, glycoprotein 100 (gp 100), Carcinoembryonic antigen (CEA), ROR1, trophoblast glycoprotein (TPBG, also known as 5T4), TAG72, VEGF-R2, Her2 / neu, estrogen receptor, progesterone receptor, liver glycoside B2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms tumor 1 (WT-1), cyclins, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD138, pathogen-specific antigens and antigens associated with universal tags, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens.

[0379] In some embodiments, the antigen or ligand is a tumor antigen or cancer marker. In some embodiments, the antigen or ligand antigen is or includes 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), and type III epidermal growth factor receptor mutant (EGFR). vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), liver glycoprotein B2, liver glycoprotein receptor A2 (EPHa2), estrogen receptor, Fc receptor-like protein 5 (FCRL5);Also known as 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, glycoprotein 100 (gp100), phosphatidylinositol proteoglycan-3 (GPC3), G protein-coupled receptor 5D (GPCR5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, human high molecular weight melanoma-associated antigen (HMI). 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 insertion domain receptor (kdr), κ light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, Protein 8 family member A containing leucine-rich repeat sequences (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE -A10, mesothelin (MSLN), c-Met, mucin cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer cell family 2 member D (NKG2D) ligand, melanin A (MART-1), neural cell adhesion molecule (NCAM), oncoemulsification antigen, melanoma preferred expression 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), survival protein, trophoblast glycoprotein (TPBG, also known as 5T) 4) Tumor-associated glycoprotein 72 (TAG72), tyrosinase-associated protein 1 (TRP1, also known as TYRP1 or gp75), tyrosinase-associated protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome δ isomerase, or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor 1 (WT-1), pathogen-specific or pathogen-expressed antigens, or antigens associated with universal tags, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV, or other pathogens.

[0380] In some implementations, the disease or condition is a B-cell malignancy. In some implementations, the B-cell malignancy is leukemia or lymphoma. In some aspects, the disease or condition is acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma (NHL), or diffuse large B-cell lymphoma (DLBCL). In some cases, the disease or condition is NHL (e.g., or including NHL as aggressive NHL), diffuse large B-cell lymphoma (DLBCL) NOS type (de novo and converted from indolent), primary mediastinal large B-cell lymphoma (PMBCL), T-cell / histocyte-rich large B-cell lymphoma (TCHRBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), and / or follicular lymphoma (FL), optionally grade 3B follicular lymphoma (FL3B). In some embodiments, recombinant receptors (such as CARs) specifically bind to antigens expressed in cells associated with a disease or condition or in a lesion environment associated with B-cell malignancies. In some embodiments, the receptor-targeted antigen includes antigens associated with B-cell malignancies, such as any of many known B-cell markers. In some embodiments, the receptor-targeted antigen is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igκ, Igλ, CD79a, CD79b, or CD30.

[0381] In some implementations, the disease or condition is myeloma, such as multiple myeloma. In some aspects, recombinant receptors (such as CARs) specifically bind to antigens expressed in cells associated with the disease or condition or in the lesion environment associated with multiple myeloma. In some implementations, the receptor-targeted antigens include antigens associated with multiple myeloma, such as GPRC5d or BCMA.

[0382] In some embodiments, the antigen is a pathogen-specific antigen or an antigen expressed by a pathogen. In some embodiments, the antigen is a viral antigen (such as viral antigens from HIV, HCV, HBV, etc.), a bacterial antigen, and / or a parasitic antigen.

[0383] In some embodiments, immune cells express T-cell receptors (TCRs) or other antigen-binding receptors. In some embodiments, immune cells express recombinant receptors, such as transgenic TCRs or chimeric antigen receptors (CARs). In some embodiments, the cells are autologous to the subject. In some embodiments, the cells are allogeneic to the subject.

[0384] Methods for administering engineered cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, adoptive T-cell therapy methods are described in, for example, U.S. Patent Application Publication No. 2003 / 0170238 by Gruenberg et al.; U.S. Patent No. 4,690,915 by Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85. See, for example, Themeli et al., (2013) Nat Biotechnol. 31(10):928-933; Tsukahara et al., (2013) Biochem Biophys Res Commun 438(1):84-9; Davila et al., (2013) PLoS ONE 8(4):e61338.

[0385] In some implementations, cell therapy (e.g., adoptive T-cell therapy) is performed via autologous transfer, wherein cells are isolated and / or otherwise prepared from a subject receiving the cell therapy or from a sample derived from such a subject. Thus, i...

Claims

1. A method for genetically engineered T cells, the method comprising: (a) The input composition, comprising T cells, comprising a T cell population including naive T cells and non-naive T cells, is incubated under stimulation conditions for 2 to 6 days, wherein the stimulation conditions preferentially induce the expansion or proliferation of naive T cells compared to the non-naive T cells, wherein: (i) The cells of the input composition have not and have not undergone a selection step based on endogenous T cell surface markers prior to the incubation, which distinguish between immature and non-immature T cells; (ii) The stimulation conditions include the presence of a stimulating agent capable of activating one or more intracellular signal transduction domains of one or more components of the TCR complex and / or one or more intracellular signal transduction domains of one or more co-stimulatory molecules, thereby producing a stimulated T cell composition; and (iii) The naive-like T cells are present in the input composition at a culture starting amount of at least 2.0 x 10⁻⁶. 8 10 immature T cells, wherein the immature T cells are CD27+ and CCR7+; and (b) During or after incubation of the input composition under stimulating conditions, a nucleic acid encoding a genetically engineered recombinant receptor is introduced into T cells of the T cell population; The method thereby produces an output composition comprising T cells expressing the genetically engineered recombinant receptor.

2. A method for genetically engineered T cells, the method comprising incubating an infused composition under stimulation conditions for 2 to 6 days, wherein: The cells of the input composition have not undergone a selection step based on endogenous T cell surface markers prior to the incubation, which distinguish between naive and non-naive T cells; The input cell composition comprises a T cell population, which includes naive T cells and non-naive T cells; and the stimulation condition preferentially induces the expansion or proliferation of the naive T cells compared to the non-naive T cells in the stimulated composition. The naïve T cells are present in the input composition at a naïve T cell culture starting amount of at least 2.0 x 10⁻⁶ cells. 8 1 immature T cell, wherein the immature T cell is CD27+ and CCR7+; The stimulation condition includes the presence of a stimulating agent comprising a primary agent that specifically binds to CD3 and a secondary agent that specifically binds to CD28; and During or after incubation of the input composition under stimulating conditions, a nucleic acid encoding a genetically engineered recombinant receptor is introduced into T cells of the T cell population. The method described therein produces an output composition comprising T cells expressing the genetically engineered recombinant receptor.

3. The method according to claim 1 or claim 2, wherein the incubation lasts for at least 3 days.

4. The method according to claim 1 or claim 2, wherein the incubation lasts for at least 4 days.

5. The method according to claim 1 or claim 2, wherein the incubation lasts for at least 5 days.

6. The method according to claim 1 or claim 2, wherein the incubation lasts for at least 6 days.

7. The method of claim 1 or claim 2, wherein the introduction is performed during at least a portion of the incubation.

8. The method according to claim 1 or claim 2, wherein the introduction is performed after the incubation.

9. The method according to claim 1 or claim 2, wherein the initial culture volume of naive T cells is from 2 x 10⁻⁶ cells / year. 8 Up to 5x 10 8 indivual.

10. The method according to claim 1 or claim 2, wherein the initial culture volume of naive T cells is at least 4 x 10⁻⁶. 8 The naive-like T cells mentioned above.

11. The method according to claim 1 or claim 2, wherein the naive-like T cells: It is surface negative for markers selected from CD25, CD45RO, CD56, CD62L, and KLRG1; Low expression of CD95; or The cells showed negative intracellular expression of cytokines selected from IL-2, IFN-γ, IL-4, and IL-10.

12. The method according to claim 1 or claim 2, wherein the non-immature T cells: It was surface negative for T cell activation markers selected from CD45RA, CD27, CD28 and CCR7; and surface positive for markers selected from CD25, CD45RO, CD56, CD62L, KLRG1 and perforin. Positive intracellular expression of cytokines selected from IL-2, IFN-γ, IL-4, and IL-10; or It exhibits high expression of CD95.

13. The method of claim 1 or claim 2, wherein the stimulation condition further includes incubation with IL-2.

14. The method of claim 1 or claim 2, wherein the recombinant receptor comprises a TCR or an antigen-binding fragment thereof.

15. The method according to claim 1 or claim 2, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

16. The method according to claim 1 or claim 2, wherein the stimulating agent comprises a primary agent as an anti-CD3 antibody and a secondary agent as an anti-CD28 antibody present on the surface of the bead.

17. The method of claim 16, wherein the diameter of the bead is greater than 3.5 μm but not greater than 9 μm.

18. The method of claim 16, wherein the diameter of the bead is greater than 3.5 μm but not greater than 5 μm.

19. The method of claim 16, wherein the bead has a diameter of 4.5 μm.

20. The method of claim 16, wherein the stimulation condition comprises incubating the cells at a bead-to-cell ratio, the ratio being from 1:1 to 10:

1.

21. The method of claim 16, wherein the stimulation condition comprises incubating the cells with beads at a bead-to-cell ratio of 1:1 to 4:

1.

22. The method according to claim 1 or claim 2, wherein the T cells are derived from a biological sample, the biological sample comprising whole blood, erythrocyte sedimentation rate (ESR) chromatogram (ESR) layer, or apheresis product.

23. The method according to claim 22, wherein the biological sample is a whole blood sample, an erythrocyte sedimentation rate (ESR) sample, or apheresis product.

24. The method of claim 22, wherein the biological sample comprises leukapheresis products.

25. The method of claim 23, wherein the biological sample is a product of leukapheresis.

26. The method of claim 22, wherein the biological sample comprises a leukocyte sample.

27. The method of claim 26, wherein the biological sample is a leukocyte sample.

28. The method of claim 22, wherein the biological sample comprises a peripheral blood mononuclear cell (PBMC) sample.

29. The method of claim 28, wherein the biological sample is a peripheral blood mononuclear cell (PBMC) sample.

30. The method of claim 22, wherein the biological sample comprises a lymphocyte sample.

31. The method of claim 30, wherein the biological sample is a lymphocyte sample.

32. The method of claim 22, wherein the biological sample comprises an ungraded T cell sample.

33. The method of claim 32, wherein the biological sample is an ungraded T cell sample.

34. The method according to claim 1 or claim 2, 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.

35. The method according to claim 1 or claim 2, 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.

36. The method according to claim 1 or claim 2, wherein the stimulation condition does not include N-acetylcysteine ​​(NAC).

37. The method according to claim 1 or claim 2, wherein the stimulation condition does not include IL-15 and / or IL-7.

38. The method of claim 1 or claim 2, wherein the stimulation condition leads to activation-induced cell death (AICD) of the non-naive T cells or a subset thereof.

39. The method of claim 1 or claim 2, wherein the percentage of cells derived from the naive T cells in the stimulated composition is increased by more than 1.5 times compared to the percentage of naive T cells in the input composition.

40. The method of claim 1 or claim 2, wherein the ratio of cells derived from the immature T cells to cells derived from the non-immature T cells in the stimulated composition is increased by more than 1.5 times compared to the ratio of immature T cells to non-immature T cells in the input composition.

41. The method of claim 1 or claim 2, wherein the stimulated composition comprises more than 75% naive T cells derived from the input composition.

42. The method of claim 1 or claim 2, wherein the stimulated composition comprises less than 10% cells derived from the non-naïve T cells.

43. The method of claim 1 or claim 2, wherein the stimulated composition comprises less than 9% cells derived from the non-naïve T cells.

44. The method of claim 1 or claim 2, wherein the ratio of naive T cells to non-naive T cells in the stimulated composition is increased by more than 1.5 times compared to the ratio of naive T cells to non-naive T cells in the input composition.

45. The method according to any one of claims 1 or 2, wherein the stimulated composition is more polyclonal (or multiclonal) than the input composition.

46. ​​The method according to claim 1 or claim 2, wherein it is performed in vitro or ex vivo.

47. An output composition produced by the method according to claim 1 or claim 2.

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