Methods of producing genetically engineered cell compositions and related compositions

Abstract

Provided herein are methods and compositions for producing engineered cells, such as cells expressing recombinant receptors, including methods involving stimulation and / or engineering of input compositions having a defined ratio of naive-like CD4+ T cells to naive-like CD8+ T cells. In particular, the methods can be used to engineer T cells with genetically engineered receptors, such as genetically engineered antigen receptors, such as engineered (recombinant) TCRs and chimeric antigen receptors (CARs), or other recombinant chimeric receptors. Features of the methods include producing more consistent and / or predictable T cell products and / or products with lower toxicity compared to other methods.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 543,363, filed August 9, 2017, entitled “METHODS FOR PRODUCING GENETICALLY ENGINEERED CELL COMPOSITIONS AND RELATED COMPOSITIONS”, and U.S. Provisional Application No. 62 / 596,770, filed December 8, 2017, entitled “METHODS FOR PRODUCING GENETICALLY ENGINEERED CELL COMPOSITIONS AND RELATED COMPOSITIONS”, 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 listing. The sequence listing is provided as a file named 735042013140SEQLIST.txt, created on August 9, 2018, and measuring 35,001 kilobytes in size. Information from the electronic sequence listing is incorporated herein by reference in its entirety. Technical Field

[0005] This disclosure relates to methods and compositions for generating engineered cells, such as cells expressing recombinant receptors, including methods involving stimulating and / or engineering an input composition of naive CD4+ T cells and naive CD8+ T cells in a defined ratio. Specifically, the methods can be used to engineer T cells with genetically engineered receptors, such as genetically engineered antigen receptors, such as 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 products with lower toxicity compared to other methods. Background Technology

[0006] Various methods can be used to prepare and administer cells for therapeutic purposes. For example, various methods can be used to prepare cells (including T cells) for engineered and cell therapies, including methods involving depletion or enrichment of certain subsets. Improved methods are needed, for example, 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 producing a cell composition comprising combining a first cell composition containing naive CD4+ T cells with a second cell composition containing naive CD8+ T cells to produce an input cell composition, wherein the ratio of naive CD4+ T cells to naive CD8+ T cells is between or approximately 0.8:1 and 2.2:1, including end values. In some embodiments, the first cell composition is produced by isolating CD4+ T cells from a biological sample obtained from a subject, and / or the second cell composition is produced by isolating CD8+ T cells from a biological sample obtained from said subject.

[0008] In some embodiments, prior to the combination, the method includes determining the number, number per volume, number per weight, and / or percentage of naïve CD4+ T cells in the first cellular composition and / or the number, number per volume, number per weight, and / or percentage of naïve CD8+ T cells in the second composition. In some cases, prior to the combination, the method includes determining the number, number per volume, number per weight, and / or percentage of naïve CD4+ T cells in a biological sample from a subject and / or the number, number per volume, number per weight, and / or percentage of naïve CD8+ T cells. In some such embodiments, the ratio of naïve CD4+ T cells to naïve CD8+ T cells in the input composition is adjusted or altered compared to the ratio of naïve CD4+ T cells to naïve CD8+ T cells in a biological sample from a subject.

[0009] This document provides a method for producing a cell composition, the method comprising determining the number, number per volume, number per weight, and / or percentage of naïve CD4+ T cells and naïve CD8+ T cells in one or more samples obtained from or derived from a biological sample of a subject; and producing an input composition containing CD4+ T cells and CD8+ T cells, wherein the ratio of naïve CD4+ T cells to naïve CD8+ T cells is between or approximately 2.2:1 and 0.8:1, including end values, wherein the ratio in the input composition is adjusted or altered compared to the ratio of naïve CD4+ T cells to naïve CD8+ T cells in the biological sample of the subject.

[0010] In some embodiments, the method further includes contacting the input composition with an agent containing a nucleic acid molecule encoding a recombinant receptor under certain conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the input composition.

[0011] This article provides a method for producing a cellular composition, the method comprising contacting an input composition containing naïve CD4+ T cells and naïve CD8+ T cells from a biological sample of a subject with an agent containing a nucleic acid molecule encoding a recombinant receptor under certain conditions to introduce the nucleic acid encoding the recombinant receptor into the cells in the composition, wherein the ratio of naïve CD4+ T cells to naïve CD8+ T cells present in the input composition is between or approximately 0.8:1 and 2.2:1, including end values.

[0012] In some embodiments, the method further includes stimulating the cells before, during, and / or after the contact, wherein the stimulation includes incubating the cells in the presence of one or more stimulants, wherein the stimulation leads to cell activation and / or proliferation.

[0013] This document provides a method for producing a cell composition comprising combining a first cell composition containing naive CD4+ T cells with a second cell composition containing naive CD8+ T cells to produce an input cell composition, wherein the ratio of naive CD4+ T cells to naive CD8+ T cells is between or approximately 0.8:1 and 2.2:1, including end values; contacting the input composition with an agent containing a nucleic acid molecule encoding a recombinant receptor under certain conditions to introduce the nucleic acid encoding the recombinant receptor into the cells in the composition; and stimulating the cells before, during, and / or after said contact, wherein the stimulation comprises incubating the cells in the presence of one or more stimulants, wherein the stimulation results in activation and / or proliferation of the cells.

[0014] In some of these embodiments, naïve CD4+ cells and / or naïve CD8+ cells are surface-positive for markers selected from CD45RA, CD27, CD28, CD62L, and CCR7; and / or surface-negative for markers selected from CD25, CD45RO, CD56, 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, naïve CD4+ cells and / or naïve 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 aspects, naïve CD4+ cells and / or naïve CD8+ cells are surface-positive for CD45RA and CCR7. In some implementations, immature CD4+ cells and immature CD8+ cells are surface-positive for CD45RA, CD27, and CCR7, and surface-negative for CD45RO.

[0015] In some of these embodiments, the number, number per volume, number per weight, and / or percentage of naïve CD4+ T cells and / or the number, number per volume, number per weight, and / or percentage of naïve CD8+ T cells are determined by flow cytometry. In some aspects, the ratio of naïve CD4+ T cells to naïve CD8+ T cells has been adjusted to the ratio of naïve CD4+ T cells to naïve CD8+ T cells in a biological sample from the subject.

[0016] In some implementations, the biological sample is or is obtained from a blood, plasma, or serum sample. In some aspects, the biological sample is or includes whole blood samples, erythrocyte sedimentation rate (ESR) amber layer samples, peripheral blood mononuclear cell (PBMC) samples, ungraded T cell samples, lymphocyte samples, leukocyte samples, apheresis products, or leukoablation products. In some cases, the biological sample is or is obtained from an apheresis sample or leukoablation sample. In some implementations, the subject is a human subject.

[0017] In some of these embodiments, the input composition contains naïve CD4+ cells and naïve CD8+ cells in a ratio of or approximately between 0.8:1 and 2.0:1, 0.8:1 and 1.6:1, 0.8:1 and 1.4:1, 0.8:1 and 1.2:1, or 1.0:1 and 1.2:1 (each including end values). In some embodiments, the input composition comprises naïve CD4+ cells and naïve CD8+ cells in a ratio of or approximately 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, or 1.0:1. In some embodiments, the input composition contains naïve CD4+ cells and naïve CD8+ cells in a ratio of or approximately 1.1:1.

[0018] In some of these embodiments, the input composition contains from or from about 1 x 10 7 Up to 5x 10 9 Total cells or total T cells, from or from approximately 5 x 103 7 Up to 1x 10 9 Total cells or total T cells, from or from approximately 1 x 103 8 Up to 5x 10 8 Total cells or total T cells, or from or from approximately 2 x 103 8 Up to 5x 10 8 A live population of total cells or total T cells, or any of the foregoing. In some cases, the input composition contains at least or at least about 1 x 10⁻⁶ cells. 8 2 x 10 8 3 x 10 8 4 x 10 8 Or 5 x 10 8 A live population of total cells or total T cells, or either of the foregoing.

[0019] In some embodiments, the one or more stimulators are capable of activating T cells, CD4+ T cells, and / or CD8+ T cells; inducing signaling via the TCR complex; and / or inducing the proliferation of T cells, CD4+ T cells, and / or CD8+ T cells. In some aspects, the one or more stimulators contain a primary agent that binds to a member of the TCR complex, optionally a primary agent that specifically binds to CD3. In some cases, the one or more stimulators also contain a secondary agent that specifically binds to a T cell co-stimulatory molecule. In some examples, the co-stimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS.

[0020] In some embodiments, the primary and secondary agents contain antibodies, optionally wherein the one or more stimulants include incubation with anti-CD3 and anti-CD28 antibodies. In some embodiments, the one or more stimulants are present on the surface of a solid support (optionally a bead). In some embodiments, the one or more stimulants are present on the surface of a bead, and the bead is a paramagnetic bead. In some aspects, the one or more stimulants are selected from CD3-binding molecules; CD28-binding molecules; recombinant IL-2; recombinant IL-15; and recombinant IL-7, vaccines containing antigens specifically recognized by antigen receptors, and anti-idiotype antibodies or combinations thereof that specifically bind to antigen receptors.

[0021] In some of these implementations, incubation lasts for 2 to 15 days, 2 to 12 days, 2 to 12 days, 2 to 8 days, 2 to 6 days, 2 to 4 days, 4 to 12 days, 4 to 10 days, 4 to 8 days, 4 to 6 days, 6 to 12 days, 6 to 10 days, 6 to 8 days, 8 to 12 days, 8 to 10 days, or 10 to 12 days. In some cases, incubation lasts for at least or approximately at least 4 days, 6 days, 8 days, 10 days, or 12 days.

[0022] In some of these embodiments, the agent containing nucleic acid molecules is a viral vector or a transposon. In some cases, the agent containing nucleic acid molecules is a viral vector, and said viral vector is a retroviral vector. In some examples, the viral vector is a lentiviral vector or a gamma retroviral vector.

[0023] In some implementations, the recombinant receptor is capable of binding to a target antigen associated with a disease, disorder, or condition, 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. In some cases, 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 examples, the target antigens are selected from ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2 / neu (receptor tyrosine kinase erbB2), CD19, CD20, CD22, mesothelin, CEA and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimer, and EGFR. vIII, Folic acid-binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, G protein-coupled receptor 5D (GPCR5D), HMW-MAA, IL-22R-α, kinase insertion domain receptor (kdr), κ light chain, Lewis Y, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, melanoma preferential expression antigen (PRAME), survival protein, TAG72, B7-H6, IL-13 receptor α2 (IL -13Ra2), CA9, GD3, HMW-MAA, CD171, G250 / CAIX, PSCA, folate receptor-α, CD44v6, CD44v7 / 8, avb6 integrin, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, CD44v6, dual antigens, cancer-testis antigen, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncoemulsification antigen (oncofetal) Antigens, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2 / neu, estrogen receptor, progesterone receptor, liver glycoprotein B2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms tumor 1 (WT-1), cyclins, cyclin A2, CCL-1, CD138, pathogen-specific antigens, and antigens associated with universal labels.

[0024] In some examples, the target antigen is selected from receptor tyrosine kinase-like orphan receptor 1 (ROR1), B cell maturation antigen (BCMA), carbonic anhydrase 9 (CA9, also known as CAIX or G250), Her2 / neu (receptor tyrosine kinase erbB2), CD19, CD20, CD22, mesothelin (MSLN), carcinoembryonic antigen (CEA), and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, chondroitin sulfate proteoglycan 4 (CSPG4), EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), liver glycoprotein receptor A2 (EPHa2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, epidermal growth factor receptor type III mutant (EGFR). vIII), folate-binding protein (FBP), Fc receptor-like 5 (FCRL5, also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), ganglioside GD2, ganglioside GD3, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor α (IL-22Rα), kinase insertion domain receptor (kdr), κ light chain, protein family 8 member A containing leucine-rich repeat sequences (LRRC8A), Lewis Y, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, melanoma preferentially expressed antigen (PRAME), survival protein, tumor-associated glycoprotein 72 (TAG72), B7-H3, B7-H6, IL-13 receptor α2 (IL-13Rα2), human high molecular weight melanoma-associated antigen (HMW-M AA), CD171, folate receptor-α, CD44v7 / 8, αvβ6 integrin (avb6 integrin), 8H9, neural cell adhesion molecule (NCAM), vascular endothelial growth factor receptor (VEGF receptor or VEGFR), trophoblast glycoprotein (TPBG, also known as 5T4), NKG2D ligand, CD44v6, dual antigens, cancer-testis antigen, mucin cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), natural killer cell family 2 member D (NKG2D) ligand, cancer / testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), melanin A (MART-1), glycoprotein 100 (gp100), phosphatidylinositol proteoglycan-3 (GPC3), G protein-coupled receptor 5D (GPRC5D), carcinoembryonic antigen, 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 2 (VEGF-R2), carcinoembryonic antigen (CEA), estrogen receptor, progesterone receptor, prostate-specific antigen, liver ligand B2, CD123, CD133, c-Met, O-acetylated GD2 (OGD2), CE7 epitope of L1-CAM, Wilms tumor 1 (WT-1), cyclins, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD138, pathogen-specific antigens or pathogen-expressed antigens, and antigens associated with universal tags, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV, or other pathogens.

[0025] 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 antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igκ, Igλ, CD79a, CD79b, or CD30.

[0026] 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.

[0027] In some embodiments, the recombinant receptor is or contains a functional non-TCR antigen receptor or a TCR or an antigen-binding fragment thereof. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR). In some cases, the chimeric antigen receptor comprises an extracellular domain containing an antigen-binding domain. In some cases, the antigen-binding domain is or contains an antibody or an antibody fragment thereof, said antibody fragment optionally being a single-chain fragment. In some embodiments, said fragment contains an antibody variable region linked by a flexible linker. In some aspects, said fragment contains scFv.

[0028] In some embodiments, the chimeric antigen receptor further comprises a spacer and / or a hinge region. In some embodiments, the chimeric antigen receptor comprises an intracellular signaling region. In some cases, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, 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).

[0029] In some embodiments, the CAR comprises an antigen-specific scFv, a transmembrane domain, a cytoplasmic signaling domain derived from a co-stimulatory molecule (optionally or containing 4-1BB), and a cytoplasmic signaling domain derived from a primary signaling molecule containing ITAM (optionally or containing a CD3ζ signaling domain), and the CAR optionally further comprises a spacer between the transmembrane domain and the scFv; the CAR sequentially comprises an antigen-specific scFv, a transmembrane domain, and a cytoplasmic signaling domain derived from a co-stimulatory molecule. (which may optionally be or include a 4-1BB signaling domain), and a cytoplasmic signaling domain derived from a primary signaling molecule containing ITAM (which may optionally be a CD3ζ signaling domain); or the CAR may sequentially include an antigen-specific scFv, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a co-stimulatory molecule (which may optionally be a 4-1BB signaling domain), and a cytoplasmic signaling domain derived from a primary signaling molecule containing ITAM (which may optionally be or include a CD3ζ signaling domain).

[0030] In some embodiments, the chimeric antigen receptor further includes a transmembrane domain situated between the extracellular domain and the intracellular signaling region. In some embodiments, the intracellular signaling region further includes a co-stimulatory signaling region. In some aspects, the co-stimulatory signaling region contains an intracellular signaling domain or a signaling portion thereof of a T cell co-stimulatory molecule. In some examples, the co-stimulatory signaling region contains an intracellular signaling domain or a signaling portion thereof of CD28, 4-1BB, or ICOS. In some embodiments, the co-stimulatory signaling region is located between the transmembrane domain and the intracellular signaling region.

[0031] In some of these implementations, the subject suffers from a disease or condition, optionally wherein the recombinant receptor specifically recognizes or specifically binds to an antigen associated with or expressed or present on cells of the disease or condition.

[0032] In some embodiments, the method produces an output composition in which the ratio of CD4+ T cells expressing the recombinant receptor to CD8+ T cells expressing the recombinant receptor, optionally the ratio of live cells, varies by no more than 20%, 10%, or 5% compared to the average of such ratios in multiple T cell compositions produced by the method, and / or varies by no more than one standard deviation compared to this average. In some embodiments, the method produces an output composition in which the ratio of CD4+ T cells expressing the recombinant receptor to CD8+ T cells expressing the recombinant receptor, optionally the ratio of live cells, is between about 0.5:1 and 2:1, or between 0.8:1 and 1.6:1, or between 1:1 and 1.5:1, each including end values. In some examples, the ratio of CD4+ T cells expressing the recombinant receptor to CD8+ T cells expressing the recombinant receptor in the output composition, optionally the ratio of live cells, is about 1.2:1, 1.1:1, 1:1, 0.9:1, or 0.8:1. In some cases, the ratio of CD4+ T cells expressing the recombinant receptor to CD8+ T cells expressing the recombinant receptor in the output composition is optionally about 1:1.

[0033] In some embodiments, the live cells contain cells that are negative for apoptosis markers (-), wherein the apoptosis marker is optionally annexin V or active caspase 3.

[0034] In some of these implementations, the method is performed in vitro or ex vivo.

[0035] This document provides output compositions produced by any of the methods described herein. Pharmaceutical compositions containing said output compositions are also provided. In some embodiments, the pharmaceutical composition further comprises a drug carrier.

[0036] This article provides a treatment method comprising administering to a mammalian subject an output composition or any of the pharmaceutical compositions produced by the method. In some embodiments, the cells are derived from the subject to which the cells are administered.

[0037] In some embodiments of the provided method, immature CD4+ cells and / or immature CD8+ cells are surface-positive for CD45RA and CCR7. In some embodiments of the provided method, immature CD4+ cells and / or immature CD8+ cells are surface-positive for CD27 and CCR7. In a particular embodiment of the provided method, immature CD4+ cells and immature CD8+ cells are surface-positive for CD45RA, CD27, and CCR7 and surface-negative for CD45RO.

[0038] In some embodiments of the provided method, immature CD4+ cells and / or immature CD8+ cells are surface-positive for CCR7 and surface-negative for CD62L. In some embodiments of the provided method, the input composition comprises immature CD4+ cells and immature CD8+ cells that are surface-positive for CD45RA and CCR7 at a ratio of about 1.1:1. In a particular embodiment of the provided method, the input composition comprises immature CD4+ cells and immature CD8+ cells that are surface-positive for CD45RA and CD27 at a ratio of about 1.69:1. In some embodiments, the input cell composition comprises immature CD4+ cells and immature CD8+ cells that are surface-positive for CD27 and CCR7 at a ratio of about 1.69:1.

[0039] In some embodiments, this document describes a method for generating a cell composition comprising: combining a first cell composition containing CCR7+CD45RA+CD4+ T cells with a second cell composition containing CCR7+CD45RA+CD8+ T cells to generate an input cell composition, wherein the ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells is between or approximately 0.8:1 and 2.2:1, including end values. In some embodiments of the provided method, the first cell composition is generated by isolating CD4+ T cells from a biological sample obtained from a subject, and / or the second cell composition is generated by isolating CD8+ T cells from a biological sample obtained from said subject.

[0040] In certain embodiments of the provided method, prior to the combination, the method includes determining the number, quantity per volume, quantity per weight, and / or percentage of CCR7+CD45RA+CD4+ T cells in a first cellular composition and / or the number, quantity per volume, quantity per weight, and / or percentage of CCR7+CD45RA+CD8+ T cells in a second composition. In some embodiments of the provided method, prior to the combination, the method includes determining the number, quantity per volume, quantity per weight, and / or percentage of CCR7+CD45RA+CD4+ T cells in a biological sample from a subject and / or the number, quantity per volume, quantity per weight, and / or percentage of CCR7+CD45RA+CD8+ T cells. In some embodiments of the provided method, the ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells in the input composition is adjusted or altered compared to the ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells in the biological sample from the subject.

[0041] In a particular embodiment, this document provides a method for generating a cell composition, the method comprising: determining the number, number per volume, number per weight, and / or percentage of CCR7+CD45RA+CD4+ T cells and CCR7+CD45RA+CD8+ T cells in one or more samples obtained from or derived from a biological sample of a subject; and generating an input composition comprising CD4+ T cells and CD8+ T cells, wherein the ratio of CCR7+CD45RA+CD4+ T cells to naive CD8+ T cells is between or approximately 2.2:1 and 0.8:1, including end values, wherein the ratio in the input composition is adjusted or altered compared to the ratio of CCR7+CD45RA+CD4+ T cells to naive CD8+ T cells in the biological sample of the subject.

[0042] Some embodiments of the provided method further include contacting the input composition with an agent containing a nucleic acid molecule encoding a recombinant receptor under certain conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the input composition.

[0043] This document provides a method for producing a cellular composition, the method comprising: contacting an input composition containing CCR7+CD45RA+CD4+ T cells and CCR7+CD45RA+CD8+ T cells from a biological sample of a subject with an agent containing a nucleic acid molecule encoding a recombinant receptor under certain conditions to introduce the nucleic acid encoding the recombinant receptor into the cells in the composition, wherein the ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells present in the input composition is between or approximately 0.8:1 and 2.2:1, including end values. Some embodiments of the provided method further include stimulating the cells before, during, and / or after the contact, wherein the stimulation comprises incubating the cells in the presence of one or more stimulants, wherein the stimulation leads to activation and / or proliferation of the cells.

[0044] In a particular embodiment, this document provides a method for generating a cell composition, the method comprising: combining a first cell composition comprising CCR7+CD45RA+CD4+ T cells with a second cell composition comprising CCR7+CD45RA+CD8+ T cells to generate an input cell composition, wherein the ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells is between or approximately 0.8:1 and 2.2:1, including end values; contacting the input composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under certain conditions to introduce the nucleic acid encoding the recombinant receptor into the cells in the composition; and stimulating the cells before, during, and / or after said contact, wherein the stimulation comprises incubating the cells in the presence of one or more stimulants, wherein the stimulation results in activation and / or proliferation of the cells.

[0045] In some embodiments of the provided method, the number, number per volume, number per weight, and / or percentage of CCR7+CD45RA+CD4+ T cells and / or the number, number per volume, number per weight, and / or percentage of CCR7+CD45RA+CD8+ T cells are determined by flow cytometry. In some embodiments of the provided method, the ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells has been adjusted compared to the ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells in a biological sample from a subject. In certain embodiments of the provided method, the input composition comprises CCR7+CD45RA+CD4+ cells and CCR7+CD45RA+CD8+ cells in a ratio of or approximately between 0.8:1 and 2.0:1, 0.8:1 and 1.6:1, 0.8:1 and 1.4:1, 0.8:1 and 1.2:1, or 1.0:1 and 1.2:1 (each including end values). In some embodiments of the provided method, the input composition comprises CCR7+CD45RA+CD4+ cells and CCR7+CD45RA+CD8+ cells in a ratio of or approximately 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, or 1.0:1.

[0046] In some embodiments of the provided method, the input composition comprises CCR7+CD45RA+CD4+ cells to CCR7+CD45RA+CD8+ cells at a ratio of 1.1:1 or about 1.1:1. In a particular embodiment, this document provides a method for generating a cell composition comprising: combining a first cell composition comprising CD27+CCR7+CD4+ T cells with a second cell composition comprising CD27+CCR7+CD8+ T cells to generate an input cell composition, wherein the ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells is between 1.2:1 and 2.4:1, including end values.

[0047] In some embodiments of the provided method, the first cell composition is produced by isolating CD4+ T cells from a biological sample obtained from a subject, and / or the second cell composition is produced by isolating CD8+ T cells from a biological sample obtained from said subject. In some embodiments of the provided method, prior to the combination, the method includes determining the number, quantity per volume, quantity per weight, and / or percentage of CD27+CCR7+CD4+ T cells in the first cell composition and / or the number, quantity per volume, quantity per weight, and / or percentage of CD27+CCR7+CD8+ T cells in the second composition. In a particular embodiment of the provided method, prior to the combination, the method includes determining the number, quantity per volume, quantity per weight, and / or percentage of CD27+CCR7+CD4+ T cells in a biological sample from a subject and / or the number, quantity per volume, quantity per weight, and / or percentage of CD27+CCR7+CD8+ T cells.

[0048] In some embodiments of the provided method, the ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells in the input composition is adjusted or altered compared to the ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells in a biological sample from a subject. In some embodiments, this document provides a method for generating a cell composition, the method comprising: determining the number, number per volume, number per weight, and / or percentage of CD27+CCR7+CD4+ T cells and CD27+CCR7+CD8+ T cells in one or more samples obtained from or derived from a biological sample of a subject; and generating an input composition comprising CD4+ T cells and CD8+ T cells, wherein the ratio of CD27+CCR7+CD4+ T cells to naive CD8+ T cells is between or approximately 2.2:1 and 0.8:1, including end values, wherein the ratio in the input composition is adjusted or altered compared to the ratio of CD27+CCR7+CD4+ T cells to naive CD8+ T cells in the biological sample of the subject.

[0049] Some embodiments of the provided method further include contacting the input composition with an agent containing a nucleic acid molecule encoding a recombinant receptor under certain conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the input composition.

[0050] In a particular embodiment, this document provides a method for producing a cellular composition, the method comprising: contacting an input composition comprising CD27+CCR7+CD4+ T cells and CD27+CCR7+CD8+ T cells from a biological sample of a subject with an agent comprising a nucleic acid molecule encoding a recombinant receptor under certain conditions to introduce the nucleic acid encoding the recombinant receptor into the cells in the composition, wherein the ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells present in the input composition is between or approximately 0.8:1 and 2.2:1, including end values. Certain embodiments of the provided method further include stimulating the cells before, during, and / or after the contact, wherein the stimulation comprises incubating the cells in the presence of one or more stimulants, wherein the stimulation results in activation and / or proliferation of the cells.

[0051] In some embodiments, this document provides a method for generating a cell composition, the method comprising: combining a first cell composition comprising CD27+CCR7+CD4+ T cells with a second cell composition comprising CD27+CCR7+CD8+ T cells to generate an input cell composition, wherein the ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells is between or approximately 0.8:1 and 2.2:1, including end values; contacting the input composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under certain conditions to introduce the nucleic acid encoding the recombinant receptor into the cells in the composition; and stimulating the cells before, during, and / or after said contact, wherein the stimulation comprises incubating the cells in the presence of one or more stimulants, wherein the stimulation results in activation and / or proliferation of the cells.

[0052] In certain embodiments of the provided method, the number, number per volume, number per weight, and / or percentage of CD27+CCR7+CD4+ T cells and / or the number, number per volume, number per weight, and / or percentage of CD27+CCR7+CD8+ T cells are determined by flow cytometry. In some embodiments of the provided method, the ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells has been adjusted compared to the ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells in a biological sample from a subject.

[0053] In some embodiments of the provided method, the input composition comprises CD27+CCR7+CD4+ cells and CD27+CCR7+CD8+ cells in a ratio of or approximately between 0.8:1 and 2.0:1, 0.8:1 and 1.6:1, 0.8:1 and 1.4:1, 0.8:1 and 1.2:1, or 1.0:1 and 1.2:1 (each including end values). In specific embodiments of the provided method, the input composition comprises CD27+CCR7+CD4+ cells and CD27+CCR7+CD8+ cells in a ratio of or approximately 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, or 1.0:1. In some embodiments of the provided method, the input composition comprises CD27+CCR7+CD4+ cells and CD27+CCR7+CD8+ cells in a ratio of or approximately 1.1:1. In some embodiments, the input cell composition comprises CD27+CCR7+CD4+ cells and CD27+CCR7+CD8+ cells in a ratio of about 1.69:1.

[0054] In some embodiments, this document provides a method for generating a cell composition, the method comprising: combining a first cell composition comprising CD62L-CCR7+CD4+ T cells with a second cell composition comprising CD62L-CCR7+CD8+ T cells to generate an input cell composition, wherein the ratio of CD62L-CCR7+CD4+ T cells to CD62L-CCR7+CD8+ T cells is between or approximately 0.5:1 and 2:1, including end values.

[0055] In a particular embodiment, this document provides a method for generating a cell composition, the method comprising: determining the number, number per volume, number per weight, and / or percentage of CD62L-CCR7+CD4+ T cells and CD62L-CCR7+CD8+ T cells in a biological sample obtained from or derived from a subject; and generating an input composition comprising CD4+ T cells and CD8+ T cells, wherein the ratio of CD62L-CCR7+CD4+ T cells to naive CD8+ T cells is between or approximately 0.5:1 and 2:1, including end values, wherein the ratio in the input composition is adjusted or altered compared to the ratio of CD62L-CCR7+CD4+ T cells to naive CD8+ T cells in a biological sample from the subject.

[0056] Some embodiments of the provided method further include contacting the input composition with an agent containing a nucleic acid molecule encoding a recombinant receptor under certain conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the input composition. In some embodiments, a method for generating a cell composition is provided herein, the method comprising: combining a first cell composition containing CD62L-CCR7+CD4+ T cells with a second cell composition containing CD62L-CCR7+CD8+ T cells to generate an input cell composition, wherein the ratio of CD62L-CCR7+CD4+ T cells to CD62L-CCR7+CD8+ T cells is between or approximately 0.5:1 and 2:1, including end values; contacting the input composition with an agent containing a nucleic acid molecule encoding a recombinant receptor under certain conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the composition; and stimulating the cells before, during, and / or after said contact, wherein the stimulation comprises incubating the cells in the presence of one or more stimulants, wherein the stimulation results in cell activation and / or proliferation.

[0057] In certain embodiments of the provided method, the number, number per volume, number per weight, and / or percentage of CD62L-CCR7+CD4+ T cells and / or the number, number per volume, number per weight, and / or percentage of CD62L-CCR7+CD8+ T cells are determined by flow cytometry. In some embodiments of the provided method, the ratio of CD62L-CCR7+CD4+ T cells to CD62L-CCR7+CD8+ T cells has been adjusted compared to the ratio of CD62L-CCR7+CD4+ T cells to CD62L-CCR7+CD8+ T cells in a biological sample from a subject. In some embodiments of the provided method, the input composition comprises CD62L-CCR7+CD4+ cells and CD62L-CCR7+CD8+ cells in a ratio of or approximately between 0.5:1 and 1.5:1, 1:1 and 2:1, 0.75:1 and 1.5:1, or 0.8:1 and 1.2:1 (each including end values). In specific embodiments of the provided method, the input composition comprises CD62L-CCR7+CD4+ cells and CD62L-CCR7+CD8+ cells in a ratio of or approximately 1.2:1, 1.1:1, 1.0:1, 0.9:1, or 0.8:1.

[0058] In some embodiments of the provided method, the biological sample is or is obtained from a blood, plasma, or serum sample. In some embodiments of the provided method, the biological sample is or comprises a whole blood sample, erythrocyte sedimentation rate (ESR) amber layer sample, peripheral blood mononuclear cell (PBMC) sample, ungraded T cell sample, lymphocyte sample, leukocyte sample, apheresis product, or leukoablation product. In a specific embodiment of the provided method, the biological sample is or is obtained from an apheresis sample or leukoablation sample. Attached Figure Description

[0059] Figure 1A A graph shows a bivariate fit analysis of the ratio of live CD4+ cells to live CD8+ cells in apheresis samples compared to the ratio of CAR+CD4+ T cells to CAR+CD8+ T cells in the T-cell composition after T-cell activation, transduction with chimeric antigen receptor (CAR) constructs, and expansion (live CD4+ / CD8+ ratio). The curve represents the boundary of the bivariate normal ellipse at p = 0.990. Data points represent the mean ratios from four samples from each subject, including healthy subjects (circles) and subjects with myeloma (plus signs).

[0060] Figure 1B A graph showing a bivariate fit analysis of the CD45RA+ / CCR7+CD4 / CD8 ratio in a starting mixture of selected CD4 and CD8 cells compared to the CAR+CD4+ / CD8+ ratio in the T-cell composition after T-cell activation, transduction with chimeric antigen receptor (CAR) constructs, and expansion. The curve represents the boundary of the bivariate normal ellipse at p = 0.990. Data points represent the mean ratio from four samples from each subject, including healthy subjects (circles) and subjects with myeloma (plus signs).

[0061] Figure 2A-2C A graph showing a bivariate fit analysis of the ratio of different phenotype cells in the starting mixture of selected CD4 and CD8 cells compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. Figure 2A A graph showing a bivariate fit analysis of the ratio of CD45RA+ / CCR7+ / CD4+ to CD45RA+ / CCR7+ / CD8+ cells in the starting mixture of selected CD4 and CD8 cells, compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. Figure 2BA graph showing a bivariate fit analysis of the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells in the starting mixture of selected CD4 and CD8 cells, compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. Figure 2C A graph showing a bivariate fit analysis of the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells in a starting mixture of selected CD4 and CD8 cells, compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. The curve represents the boundary of the bivariate normal ellipse at p = 0.950. Data points represent the mean ratios of multiple compositions from each subject, including healthy donors (circles) and patients with multiple myeloma (plus signs).

[0062] Figures 3A-3C A graph showing a bivariate fit analysis of the ratio of different phenotype cells in a starting mixture of selected CD4 and CD8 cells from seven donors with multiple myeloma, compared to the CAR+CD4+ / CD8+ ratio in the resulting engineered CAR+ T cell composition. Figure 3A A graph showing a bivariate fit analysis of the ratio of CD27+ / CCR7+ / CD4+ to CD27+ / CCR7+ / CD8+ cells in the starting mixture of selected CD4 and CD8 cells, compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. Figure 3B A graph showing a bivariate fit analysis of the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells in a starting mixture of selected CD4 and CD8 cells, compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. Figure 3C A graph showing a bivariate fit analysis of the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells in a selected starting mixture of CD4 and CD8 cells, compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. The curve represents the boundary of the bivariate normal ellipse at p = 0.950. Detailed Implementation

[0063] This document provides methods for preparing cell compositions, such as input cell compositions, for genetically engineering cells to express recombinant receptors. In some embodiments, the methods include one or more steps of generating an input cell composition containing naïve CD4+ T cells and naïve CD8+ T cells at a defined, controlled, or desired ratio. In some cases, the input composition provided may be generated by mixing or combining a cell composition containing CD4+ T cells with a known or defined number or percentage of naïve CD4+ T cells and a cell composition containing CD8+ T cells with a known or defined number or percentage of naïve CD8+ T cells, for example, to achieve a selected or desired ratio. Methods for stimulating, expanding, and / or inducing cell proliferation in the input composition are also provided. The provided methods may also include methods related to the genetic engineering of cells, such as transduction methods, including methods for introducing recombinant receptors (e.g., chimeric antigen receptors) into such cells in conjunction with adoptive cell therapy.

[0064] In one embodiment, the resulting input composition includes incubating cells under stimulating conditions, for example, in some aspects, to activate the cells for engineering or transduction or for cell expansion. In some embodiments, the method includes the step of engineering multiple cell types, such as CD4+ cells and CD8+ cells, as those isolated and present in the input composition. In some aspects, engineering is performed to introduce genetically engineered antigen receptors into the cells, such as TCRs, for example, high-affinity TCRs, or functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). In some aspects, the method includes further treatment (e.g., further incubation, for example at or at about 37°C ± 2°C) and / or formulation of the cells and the composition containing said cells. In some embodiments, the treatment produces a resulting output composition containing genetically engineered cells, such as genetically engineered CD4+ cells and CD8+ cells, including cells in which engineered CD4+ and CD8+ cells are present in a desired ratio. In some embodiments, the resulting processed output composition can be used in methods of administering cells and compositions prepared by said method to a patient, for example, in combination with adoptive cell therapy.

[0065] In some respects, output compositions containing engineered cells, such as CAR+ T cells, are advantageous, wherein engineered CD4+ and CD8+ cells are present in a desired ratio, or within a certain permissible margin of error of the desired ratio. For example, this is advantageous for multiple different cell populations or cell types (such as isolated CD4+ cells). +Engineered cells enriched with CD8+ T cell populations and subsets can improve efficacy or reduce or avoid undesirable effects. In some cases, such ratios include those considered optimal for therapeutic use, for example, output ratios considered appropriate or optimal for administration to a patient in combination with adoptive cell therapy. In some embodiments, for administration to a subject, such as in combination with adoptive cell therapy, the desired ratio of CD4+ to CD8+ T cells in the output composition is from or from about 2:1 to 0.5:1, for example, or about 1:1. In some aspects, compositions containing isolated CD8+ and CD4+ T cells (such as those containing a desired ratio of these cells) increase the ability of the cells ultimately administered to the subject to persist, expand, be activated, and / or be transplanted in vivo or after administration to the subject. In some aspects, the composition improves or increases one or more effector functions or activates phenotypes. For example, in some aspects, such advantages can be achieved by administering both CD4+ and CD8+ populations compared to administering only the CD8+ population.

[0066] In some implementations, the method offers one or more advantages compared to other preparation, separation, incubation, and engineering methods, such as cost, time, and / or resource savings. Such advantages, compared to other methods, may include the ability to separate, process (e.g., incubate), and / or engineer multiple cell populations present at or near desired ratios with increased efficiency and / or reduced complexity, time, cost, and / or resource usage.

[0067] In some embodiments, the provided method is based on the observation that in certain cell production processes used for engineered cells, the input ratio of CD4+ to CD8+ cells, or the ratio of their viable cells, may be independent of the output ratio of engineered CD4+ to CD8+ cells, or the ratio of their viable cells. As shown herein, it has been found that the desired ratio of engineered CD4+ to CD8+ T cells in the output composition (e.g., the desired ratio of CAR+CD4 to CAR+CD8+ T cells, or the ratio of their viable cells) is associated with or related to the ratio of naive CD4+ T cells to naive CD8+ T cells, or the ratio of their viable cells, present in the input composition prior to the cell production process, which involves, for example, one or more steps of stimulating, activating, expanding, proliferating, and / or transducing cells. Examples of such naive cells are multiple ratios of CD4+ to CD8+ cells, wherein the CD4+ and CD8+ cells are CD45RA+ and CCR7+, CD62L- / CCR7+, or CD27+ / CCR7+. In some respects, it has been observed that, despite variations in the process used to generate or produce the output composition, there is a correlation between the ratio of naive CD4 / CD8 cells in the input composition and the ratio of engineered CD4+ to CD8+ cells in the output composition (e.g., the ratio of CAR+CD4 to CAR+CD8+ T cells or their viable cell ratio). The sources of this variation can include a variety of different factors, including specific steps or conditions of the process that may introduce variability from one cell composition to the next; or the source may stem from differences in the individuals or samples from which engineered cells are isolated, selected, derived, or obtained.

[0068] In some embodiments, the provided method is based on the observation that the desired ratio of engineered CD4+ to CD8+ T cells in the output composition (e.g., the desired ratio of CAR+CD4 to CAR+CD8+ T cells or their live cell ratio) is associated with or related to the ratio of CD45RA+ / CCR7+CD4+ T cells to CD45RA+ / CCR7+CD8+ T cells or their live cell ratio present in the input composition prior to a manufacturing process involving, for example, one or more steps of stimulating, activating, expanding, proliferating, and / or transducing cells. In some aspects, it has been observed that, despite variations in the donor and / or process used to generate or produce the output composition, this association exists between the ratio of CD45RA+ / CCR7+CD4+ to CD45RA+ / CCR7+ / CD8+ cells in the input composition and the ratio of engineered CD4+ to CD8+ cells in the output composition (e.g., the ratio of CAR+CD4 to CAR+CD8+ T cells or their live cell ratio).

[0069] In a particular embodiment, the provided method is based on the observation that the desired ratio of engineered CD4+ to CD8+ T cells in the output composition (e.g., the desired ratio of CAR+CD4 to CAR+CD8+ T cells or their viable cell ratio) is associated with or related to the ratio of CD62L- / CCR7+CD4+ T cells to CD62L- / CCR7+CD8+ T cells or their viable cell ratio present in the input composition prior to a manufacturing process involving, for example, one or more steps of stimulating, activating, expanding, proliferating, and / or transducing cells. In some aspects, it has been observed that, despite variations in the donor and / or process used to generate or produce the output composition, this association exists between the ratio of CD62L- / CCR7+CD4+ to CD62L- / CCR7+ / CD8+ cells in the input composition and the ratio of engineered CD4+ to CD8+ cells in the output composition (e.g., the ratio of CAR+CD4 to CAR+CD8+ T cells or their viable cell ratio).

[0070] In some embodiments, the provided method is based on the observation that the desired ratio of engineered CD4+ to CD8+ T cells in the output composition (e.g., the desired ratio of CAR+CD4 to CAR+CD8+ T cells or their live cell ratio) is associated with or related to the ratio of CD27+ / CCR7+CD4+ T cells to CD27+ / CCR7+CD8+ T cells or their live cell ratio present in the input composition prior to the cell manufacturing process, which involves, for example, one or more steps of stimulating, activating, expanding, proliferating, and / or transducing cells. In some aspects, it has been observed that, despite variations in the donor and / or process used to generate or produce the output composition, this association exists between the ratio of CD27+ / CCR7+CD4+ to CD27+ / CCR7+ / CD8+ cells in the input composition and the ratio of engineered CD4+ to CD8+ cells in the output composition (e.g., the ratio of CAR+CD4 to CAR+CD8+ T cells or their live cell ratio).

[0071] In some respects, the provided method ensures that the output composition of genetically engineered cells (e.g., CAR+ T cells) produced by the cell production process achieves a relatively consistent and / or controlled ratio of engineered CD4+ to CD8+ T cells or a ratio of their live cells in the composition produced by the process, such ratio exhibiting low or below-acceptable variance or threshold variance, said composition including those derived from samples from multiple different subjects, such as subjects with different characteristics, such as different ages, different numbers and / or types of prior treatment, and different indications and their subtypes or severity or grades. In some respects, such processes produce engineered CD4+ to CD8+ ratios (e.g., the ratio of CAR+CD4 to CAR+CD8+ T cells or their live cell ratios) that vary by no more than 20%, 10%, or 5% compared to the average of such ratios in multiple T cell compositions produced by said process, and / or by no more than one standard deviation compared to this average, or in such different samples and patients, in multiple T cell compositions produced by said process, the variation is no more than 20%, 10%, or 5%.

[0072] In some implementations, such as regarding the ratio of engineered CD4 to CD8 T cells, the use of processes that produce higher consistency in the output composition generated by the cell production process can advantageously ensure consistency in dosing to subjects, which in some respects can optimize the efficacy, potency, and / or safety of the administered composition in the treated subjects. In some aspects, the engineered output composition targets CD4... + Engineered cells enriched with CD8+ T cell populations at a desired output ratio can improve efficacy or reduce or avoid undesirable effects. In some aspects, the isolation or enrichment increases the ability of the cells ultimately given to the subject to persist, expand, be activated, and / or be transplanted in vivo or after administration. In some aspects, the composition improves or increases one or more effector functions or activates phenotypes. Such results can be achieved even in cases where donor variability exists in the starting cell sample used for cell engineering.

[0073] In some embodiments, the methods provided herein allow for the production of therapeutic cell compositions of engineered cells having a desired output ratio in the output composition without separate treatment and / or administration of engineered CD4+ and CD8+ T cells. Therefore, the provided methods offer a more streamlined and / or more controlled approach for preparing compositions of engineered CD4+ T cell populations and CD8+ T cell populations having a desired output ratio or approximately a desired output ratio. In a particular aspect, after producing an input composition containing naive-like CD4+ and CD8+ cells as described, the provided methods can be used in cell production processes where CD4+ and CD8+ T cells are treated together in a single-stream process, e.g., activated, stimulated, expanded, and / or transduced. Therefore, in some aspects, the methods allow for the introduction of genetically engineered antigen receptors for adoptive cell therapy, where cell populations are isolated, incubated, and / or engineered in a combined manner, and compared to methods that isolate, incubate, and / or engineer the populations separately, the methods are associated with increased efficiency and / or reduced complexity, time, cost, and / or resource usage.

[0074] Cells and compositions (including pharmaceutical compositions and formulations) prepared by the method are also provided, as well as kits, systems, and apparatus for performing the method. Methods for using cells and compositions prepared by the method (including therapeutic methods, such as methods for adoptive cell therapy) and pharmaceutical compositions for administration to subjects are also provided.

[0075] 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 set forth in patents, applications, publications, and other publications incorporated herein by reference, the definitions described herein shall prevail over those incorporated herein by reference.

[0076] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0077] I. A method for generating engineered cells at a controlled ratio of CD4+ to CD8+ T cells or specific subtypes thereof.

[0078] This document provides methods for generating cell compositions, such as input cell compositions, for genetically engineering cells to express recombinant receptors. In some embodiments, the input cell composition contains CD4+ T cells and CD8+ T cells. In some embodiments, the input cell composition contains one or more subtypes or populations of CD4+ and / or CD8+ T cells. In some embodiments, the one or more subtypes or populations are naive cells and / or naive-like cells. In a particular embodiment, the input cell composition contains CD4+ T cells, and at least a portion of the CD4+ T cells are naive-like CD4 cells. In some embodiments, the input cell composition contains CD8+ T cells, and at least a portion of the CD8+ T cells are naive cells. In a particular embodiment, the input cell composition contains and / or has naive-like CD4+ T cells and naive-like CD8+ T cells at a fixed ratio, preferred ratio, target ratio, defined ratio, and / or controlled ratio.

[0079] In some embodiments, the method includes one or more steps of mixing or combining cells or cell compositions to generate or produce an input cell composition. In some embodiments, the cells or cell composition have been selected and / or isolated from a sample. In some embodiments, the cells in the input cell composition have been selected and / or isolated from a sample. In some embodiments, the composition of CD4+ T cells and / or CD4+ T cells has been selected or isolated from a sample. In some embodiments, the sample is a biological sample, such as a blood sample, apheresis sample, and / or leukocyte apheresis sample. In some embodiments, the sample is from a subject, such as a human subject. In a particular embodiment, the composition of CD4+ T cells and the composition of CD8+ T cells are isolated and / or selected from the same sample. In some embodiments, the composition of CD4+ T cells and the composition of CD8+ T cells are isolated and / or selected from a sample taken from or obtained from the same subject.

[0080] In some embodiments, the generation or production of the input cell composition includes one or more steps of evaluating, characterizing, and / or identifying cells. In a particular embodiment, cells are evaluated, characterized, and / or identified in a composition of CD4+ T cells. In some embodiments, cells are evaluated, characterized, and / or identified in a composition of CD8+ T cells. In some embodiments, cells are evaluated for cells that are positive for markers indicating an immature state in T cells and / or associated with an immature state in T cells. In some embodiments, cells are evaluated for cells that are negative for markers indicating a non-immature state in T cells and / or associated with a non-immature state in T cells. In a particular embodiment, cells from CD4+ and CD8+ T cell compositions (such as cell compositions isolated from biological samples from a subject) are evaluated, characterized, and / or identified to determine the amount, level, share, and / or percentage of cells that are positive for one or more markers associated with an immature state and / or negative for one or more markers associated with a non-immature state. In some embodiments, cells from CD4+ and CD8+ T cell compositions (such as cell compositions isolated from biological samples from subjects) are evaluated, characterized, and / or identified to determine the quantity, level, share, and / or percentage of cells as naive-like cells.

[0081] In some embodiments, the method includes one or more steps of mixing or combining a cell composition containing CD4+ T cells with a cell composition containing CD8+ T cells to generate or produce a cell composition (e.g., an input cell composition) having a defined ratio of naive-like CD4+ T cells and naive-like CD8+ T cells. In some embodiments, the generation or production of the input cell composition includes one or more of the following steps: (i) isolating or selecting a composition of CD4+ T cells and / or a composition of CD8+ T cells from a sample (e.g., a biological sample); (ii) assessing, characterizing, and / or identifying the amount, level, share, and / or percentage of cells in the composition of CD4+ and / or CD8+ T cells that are positive for one or more markers associated with a naive-like state and / or negative for one or more markers associated with a non-naive-like state; and / or (iii) mixing or combining the composition of CD4+ T cells and the composition of CD8+ T cells in a defined ratio, a fixed ratio, and / or a preferred ratio. In a particular embodiment, the ratio of naive CD4+ T cells to naive CD8+ T cells in the input cell composition is different from the ratio present in the sample.

[0082] In certain embodiments, the content, composition, and / or composition of the input cell composition are associated with the content, composition, and / or composition of the output cell composition, and the content, composition, and / or composition of the output cell composition are controlled to correspond to and / or relate to the content, composition, and / or composition of the output cell composition. In some embodiments, the amount, fraction, percentage, number, quantity per volume, quantity per weight, and / or ratio of immature and / or immature-like cells (e.g., immature-like T cells) in the input cell composition are associated with the content, composition, and / or composition of the output cell composition, and the content, composition, and / or composition of the output cell composition are controlled to correspond to and / or relate to the content, composition, and / or composition of the output cell composition. In certain embodiments, the amount, fraction, quantity, quantity per volume, quantity per weight, and / or ratio of immature-like CD4+ T cells and / or immature-like CD8+ T cells in the input cell composition are associated with the content, composition, and / or composition of the output cell composition, and the content, composition, and / or composition of the output cell composition are controlled to correspond to and / or relate to the content, composition, and / or composition of the output cell composition.

[0083] In some embodiments, the method includes one or more steps of genetically engineering cells of the input cell composition. In some embodiments, genetic engineering includes one or more of the following steps: incubating cells of the input cell composition under conditions of cell activation and / or stimulation; delivering genes (e.g., recombinant and / or heterologous genes) to the cells; expanding the cells by incubating the cells under conditions of activation or stimulation; harvesting the cells; and / or storing the cells by freezing (e.g., cryopreservation). In some embodiments, the one or more steps of genetic engineering produce an output cell composition containing engineered cells. In some embodiments, the engineered cells of the output composition have a fixed ratio, a defined ratio, and / or a target ratio of CD4+ to CD8+ cells.

[0084] In some embodiments, the methods provided herein include one or more steps of generating engineered cells, such as cells expressing a recombinant receptor, said engineered cells having a defined ratio of CD4+ to CD8+ T cells. In some embodiments, the methods provided herein include one or more steps of genetically engineering cells from a starting and / or input cell composition to generate a resulting and / or output cell composition having a defined ratio of genetically engineered CD4+ to CD8+ T cells. In a particular embodiment, genetic engineering is or includes transfecting or transducing cells from the input cell composition to introduce a drug containing nucleic acid into the cells of the input cell composition. In some embodiments, said nucleic acid encodes a recombinant receptor, such as a chimeric antigen receptor (CAR). In some embodiments, generating an output cell composition includes one or more steps of: activating or stimulating cells from the input cell composition; genetically engineering, transducing, or transfecting cells from the input cell composition; and / or amplifying the transfected cells; thereby generating an output cell composition having a defined ratio, such as a 1:1 ratio of genetically engineered CD4+ to CD8+ T cells.

[0085] A. Preparation of cell and imported cell composition

[0086] In some embodiments, the methods provided herein include one or more steps in preparing cells for genetic engineering. In some embodiments, the one or more steps include isolating cells from a biological sample to prepare a composition of cells to be genetically engineered, such as an input cell composition. In some embodiments, the preparation of the input cell composition includes one or more steps of isolating compositions of two or more cell types and / or a specific cell type or cell subtype, and mixing or combining the cell compositions of said cell type and / or said specific cell type into a single input cell composition. In certain embodiments, the cell composition of the specific cell type is evaluated to determine the presence, amount, and / or ratio of other subtypes in the composition. In some embodiments, the cell compositions of the specific cell type are mixed or combined to achieve an input cell composition having a fixed or determined ratio of cell types or subtypes. In some embodiments, the cell type and / or cell subtype are associated with the content, composition, and / or composition of the output cell composition, and the content, composition, and / or composition of the output cell composition are controlled to correspond to and / or relate to the content, composition, and / or composition of the output cell composition.

[0087] In some embodiments, the preparation of the input cell composition includes one or more of the following steps: separating a composition of CD4+ T cells or a composition comprising CD4+ T cells; separating a composition of CD8+ T cells or a composition comprising CD8+ T cells; and mixing or combining CD4+ and CD8+ T cell compositions of specific cell types into a single input cell composition. In some embodiments, the preparation of the input cell composition includes one or more steps of assessing, determining, and / or quantifying the proportion or amount of CD4+ and / or CD8+ T cell subtypes in the cell composition. In specific embodiments, the CD4+ and / or CD8+ T cell subtypes are or include naive and / or naive-like CD4+ and / or CD8+ T cells. In some embodiments, the proportion or amount of naive-like cells in the input cell composition is associated with the content, composition, and / or composition of the output cell composition, controlling the content, composition, and / or composition of the output cell composition to correspond to and / or relate to the content, composition, and / or composition of the output cell composition.

[0088] In some embodiments, the cells isolated from the sample (e.g., CD4+ and / or CD8+ T cells) are eukaryotic cells, such as mammalian cells, and in some embodiments, are human cells. In some embodiments, the cells are derived from the subject's blood, bone marrow, lymph nodes or lymphoid organs, and / or cells of the immune system, such as cells of innate or adaptive immunity. In some embodiments, the cells are lymphocytes. In some embodiments, the lymphocytes are T lymphocytes or T cells. In some embodiments, the cells include CD4+ T cells and CD8+ T cells.

[0089] In some embodiments, the T cell composition (e.g., a CD4+ T cell composition and / or a CD8+ T cell composition) contains cell subtypes further classified according to function, activation status, maturity, differentiation potential, amplification, labeling, or cytokine secretion profile and / or degree of differentiation. For example, in some embodiments, the cells are or comprise naive and / or naive-like CD4+ and / or CD8+ T cells. Regarding the subject to be treated, the cells may be allogeneic and / or autologous. The method includes off-the-shelf methods. In some embodiments, the method includes isolating cells from the subject, preparing, processing, culturing, and / or engineering the cells, and reintroducing the cells into the same subject before or after cryopreservation.

[0090] In some embodiments, the preparation of engineered cells includes one or more culture and / or preparation steps. Cells used to input the cell composition (e.g., a composition of cells to be genetically engineered to express, for example, a recombinant receptor such as CAR) may contain cells already isolated from a sample, such as a biological sample, for example, a 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 or requiring or to whom cell therapy will be administered. In some embodiments, the subject is a person requiring a specific therapeutic intervention (such as adoptive cell therapy, where cells are isolated, processed, and / or engineered).

[0091] In some aspects, the sample from which the cells are derived or isolated (e.g., a biological sample) is blood or a blood-derived sample, or derived from apheresis or leukapheresis products. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, and / or cells derived therefrom. In some embodiments, the cells are derived, isolated, and / or selected from a sample or biological sample, which may include whole blood samples, erythrocyte sedimentation rate (ESR) amber samples, peripheral blood mononuclear cell (PBMC) samples, ungraded T cell samples, lymphocyte samples, leukocyte samples, apheresis products, or leukapheresis products. In the case of cell therapy (e.g., adoptive cell therapy), the sample includes samples from autologous and allogeneic sources.

[0092] 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, and pigs.

[0093] In some embodiments, the isolation of cells or cell compositions (e.g., T lymphocyte or CD4+ T cell compositions and / or CD8+ T cell compositions) includes one or more preparation steps and / or cell isolation steps not based on affinity. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example to remove unwanted components, 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.

[0094] In some instances, cells are obtained from the subject's circulating blood via apheresis or leukoablation. In some aspects, the sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and / or platelets, and in some aspects contains cells other than erythrocytes and platelets.

[0095] In some embodiments, the blood cells collected from the subject are washed to remove plasma fractions, for example, and the cells are placed in a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution is deficient in calcium and / or magnesium and / or many or all divalent cations. In some aspects, the washing step is performed in a semi-automated “flow-through” centrifuge (e.g., a Cobe 2991 cell processor, Baxter) according to the manufacturer’s instructions. In some aspects, the washing step is performed by tangential flow filtration (TFF) according to the manufacturer’s instructions. In some embodiments, the cells are resuspended after washing in a variety of biocompatible buffers (e.g., calcium-free buffers). ++ / Mg ++ In some embodiments, components of the blood cell sample are removed and the cells are directly resuspended in the culture medium.

[0096] 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.

[0097] In some embodiments, the separation method includes separating different cell types based on the expression or presence of one or more specific molecules (such as surface markers (e.g., surface proteins), intracellular markers, or nucleic acids) in the cells. 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 couplers that specifically bind to such markers, followed typically by a washing step and separating cells that have bound the antibodies or binding couplers from those that have not.

[0098] Such separation steps can be based on positive selection (where cells that have already 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.

[0099] 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.

[0100] 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 couplers (each antibody or binding coupler being specific to the marker targeted for negative selection). Similarly, positive selection of multiple cell types can be performed simultaneously by incubating cells with multiple antibodies or binding couplers expressed on various cell types.

[0101] For example, in some aspects, specific subsets or subtypes of T cells, such as CD4+ T cells and / or CD8+ T cells, are isolated using positive or negative selection techniques. In some embodiments, cell compositions of CD4+ T cells and / or CD4+ T cells, or cell compositions comprising CD4+ T cells, are isolated using positive or negative selection techniques. In specific embodiments, cell compositions of CD8+ T cells and / or CD8+ T cells, or cell compositions comprising CD8+ T cells, are isolated using positive or negative selection techniques. In specific embodiments, compositions of CD4+ T cells and / or compositions of CD8+ T cells are isolated using positive or negative selection techniques.

[0102] 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 one or more antibodies or other binding agents being expressed (labeled) on the cells for positive or negative selection, respectively. + It specifically binds to one or more surface markers, either marked with a positive (+) or expressed at a relatively high level (high marking).

[0103] 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... + or CD8 + Select the step for separating CD4 +Helper T cells and CD8 + Cytotoxic T cells. These are CD4 cells. + and CD8 + The composition may contain cells that can be further classified or sorted into subpopulations based on the positive or negative expression of the marker and / or the relative expression level of the marker. Such subtypes may include naive, naive-like, and / or non-naive subtypes or subpopulations.

[0104] In some embodiments, CD4+ T cells (e.g., CD4+ T helper cells) are classified into naive and / or naive-like cells, and immature and / or immature-like cells, by identifying cell populations possessing cell surface antigens. In specific embodiments, CD8+ T cells (e.g., CD8+ T helper cells) are classified into naive and / or naive-like cells, and immature and / or immature-like cells, by identifying cell populations possessing cell surface antigens.

[0105] In some embodiments, immunomagnetic (or affinity magnetic) separation techniques are used to separate or isolate T lymphocytes. In specific embodiments, immunomagnetic separation techniques are used to separate or isolate CD4+ T cells and / or combinations of CD4+ T cells. In some embodiments, immunomagnetic separation techniques are used to separate or isolate CD8+ T cells and / or combinations of CD8+ T cells. Separation and isolation using immunomagnetic (or affinity magnetic) separation techniques are reviewed in the following literature: Methods in Molecular Medicine, Vol. 58; Metastasis Research Protocols, Vol. 2; Cell Behavior In Vitro and In Vivo, pp. 17-25 (edited by S.A. Brooks and U. Schumacher). HumanaPress Inc., Totowa, New Jersey.

[0106] In some respects, a sample or composition of cells to be separated is combined with small magnetizable or magnetically responsive materials (such as magnetically responsive particles or microparticles, such as paramagnetic beads, e.g., like... or The materials are incubated together with the beads. Magnetic responsive materials (e.g., particles) are typically attached directly or indirectly to binding partners (e.g., antibodies) that specifically bind to molecules (e.g., surface markers) present on a single cell, multiple cells, or cell population that need to be separated (e.g., for negative or positive selection).

[0107] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material that binds to a specific binding member, such as an antibody or other binding partner. Many well-known magnetically responsive materials exist for use in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Patent No. 4,452,773, and European Patent Specification EP 452342B (which is hereby incorporated by reference). Colloidal-sized particles, such as those described in Owen, U.S. Patent No. 4,795,698, and Liberti et al., U.S. Patent No. 5,200,084, are other examples.

[0108] Incubation is typically carried out under conditions in which antibodies or binding couplers, or molecules (such as secondary antibodies or other reagents) specifically bound to such antibodies or binding couplers attached to magnetic particles or beads, specifically bind to cell surface molecules (if present on the cells within the sample).

[0109] 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 to 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 further separation steps.

[0110] In some embodiments, the magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In some embodiments, the magnetic particles are attached to cells by coating with a primary antibody specific to one or more labels. In some embodiments, the cells are labeled with a primary antibody or binding partner instead of beads, and then magnetic particles coated with a cell type-specific secondary antibody or other binding partner (e.g., streptavidin) are added. In some embodiments, streptavidin-coated magnetic particles are used in combination with biotinylated primary or secondary antibodies.

[0111] In some embodiments, the magnetically responsive particles remain attached to the cells, which are subsequently incubated, cultured, and / or engineered; in some aspects, the particles remain attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competitive unlabeled antibodies and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0112] In some embodiments, affinity-based selection is performed using magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). MACS systems enable high-purity selection of cells attached to magnetized particles. In some embodiments, MACS operates in a manner where non-target and target species are eluted sequentially after an external magnetic field is applied. That is, cells attached to the magnetized particles are held in place, while unattached species are eluted. Then, after the first elution step is completed, species trapped in the magnetic field and prevented from elution are released in a manner that allows them to be eluted and recovered. In some embodiments, non-target cells are labeled and depleted from a heterogeneous cell population.

[0113] In some embodiments, a system, apparatus, or device is used for separation or splitting, performing one or more of the separation, cell preparation, splitting, processing, incubation, culture, and / or preparation 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 Patent Application Publication No. WO2009 / 072003 or US 20110003380A1.

[0114] In some implementations, 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, and / or adjust various aspects of the processing, separation, engineering, and formulation steps.

[0115] In some aspects, the separation and / or other steps are performed using, for example, the CliniMACS system (Miltenyi Biotec) for automating cell separation at a clinical-scale level 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.

[0116] In some aspects, the CliniMACS system uses magnetizable particles of conjugated antibodies 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 is in turn connected 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 are retained within the column, while unlabeled cells are removed through a series of washing steps. In some embodiments, the cell population used for use with the methods described herein is unlabeled and not retained in the column. In some embodiments, the cell population used for use with the methods described herein is labeled and retained in the column. In some embodiments, the cell population used for use with the methods described herein is eluted from the column after removal of the magnetic field and collected in a cell collection bag.

[0117] 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 for automated washing and grading of cells by 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 erythrocytes, leukocytes, 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.

[0118] In some embodiments, the cell populations described herein are collected and enriched (or depleted) by flow cytometry, in which cells stained against 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 microelectromechanical systems (MEMS) chip in combination with a FACS-based detection system (see, for example, WO 2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5): 355-376). In both cases, cells can be labeled with multiple markers, thereby allowing for the separation of well-defined T cell subgroups with high purity.

[0119] In some implementations, the antibody or binding partner is labeled with one or more detectable markers to facilitate separation for positive and / or negative selection. For example, separation can be based on binding to a fluorescently labeled antibody. In some examples, cell separation based on binding to an antibody or other binding partner specific to one or more cell surface markers is carried in a fluid stream, such as by fluorescence-activated cell sorting (FACS), including fabrication-scale (FACS) and / or microelectromechanical systems (MEMS) chips, for example, in combination with a flow cytometry detection system. Such methods allow for simultaneous positive and negative selection based on multiple markers.

[0120] Injection cell composition

[0121] In some embodiments, this document provides methods for generating and / or preparing input cell compositions. In some embodiments, the input cell composition is a composition for cells used in genetic engineering, said cells being, for example, cells that will be genetically engineered with nucleic acids encoding recombinant proteins (such as recombinant receptors, e.g., CAR), and / or cells that will undergo a process to produce genetically engineered cells expressing recombinant proteins (such as recombinant receptors, e.g., CAR). In some embodiments, the cells of the input composition are treated, contacted, and / or incubated with nucleic acids encoding recombinant receptors. In some embodiments, the input cell composition contains CD4+ T cells and CD8+ T cells. In a particular embodiment, the input cell composition contains CD4+ T cells and CD8+ T cells that comprise specific desired ratios, fixed ratios, and / or controlled ratios of CD4+ and CD8+ T cells as naive and / or naive-like T cells.

[0122] In some embodiments, the desired, fixed, and / or controlled ratio of naive and / or naive-like T cells to CD4+ CD8+ T cells is a ratio or number of cells at which the two types of cells or isolated cell populations are included in the input cell composition, which is designed to produce an output cell composition having a desired, defined, and / or controlled ratio of engineered CD4+ to CD8+ T cells, or within its permissible error rate or variation, upon completion of the incubation and / or engineering or other processing steps and / or after thawing and / or just before administration to a subject.

[0123] In some embodiments, the input cell composition contains a ratio (e.g., a defined ratio, a controlled ratio, and / or a fixed ratio) of CD4+ immature T cells to CD8+ immature T cells. In specific embodiments, the ratio of CD4+ immature T cells to CD8+ immature T cells is between 10:1 and 0.05:1, between 8:1 and 0.1:1, between 5:1 and 0.2:1, between 2.5:1 and 0.25:1, between 2.2:1 and 0.8:1, between 2:1 and 0.5:1, or between 1.5:1 and 1:1, including end values. In certain embodiments, the ratio of CD4+ immature T cells to CD8+ immature T cells is between 2:1 and 0.8:1, between 1.6:1 and 0.8:1, between 1.4:1 and 0.8:1, between 1.2:1 and 0.8:1, or between 1.2:1 and 0.8:1, including end values. In some embodiments, the ratio is between 2.2:1 and 0.8:1, including end values. In some embodiments, the ratio of CD4+ immature T cells to CD8+ immature T cells is or is about 2.2:1, 2.1:1, 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, or 0.8:1. In some implementations, the ratio is or is about 1.1:1.

[0124] In a particular embodiment, the input cell composition has a density of 1 x 10⁻⁶ cells / cm². 6 5 x 10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1 x 10 8 1.2 x 10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6 x 108 1.7 x 10 8 1.8x10 8 1.9 x 10 8 2.0x 10 8 2.1 x 10 8 2.2 x 10 8 2.3 x 10 8 2.4 x 10 8 2.5 x 10 8 2.6 x 10 8 2.7x10 8 2.8 x 10 8 2.9 x 10 8 3.0 x 10 8 3.5 x 10 8 4.0 x 10 8 4.5 x 10 8 5 x 10 8 5 x 10 8 Or 1x 10 9 The total number of cells or total viable cells. In some embodiments, the input cell composition has a quantity of 1 x 10⁻⁶ cells / units or approximately 1 x 10⁻⁶ cells / units. 6 5x10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1 x 10 8 1.2 x 10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6x10 8 1.7 x 10 8 1.8 x 10 8 1.9 x 10 8 2.0x 10 8 2.1 x 10 8 2.2 x 10 8 2.3 x 10 8 2.4 x 10 8 2.5x10 8 2.6 x 10 8 2.7 x 10 8 2.8 x 10 8 2.9 x 10 8 3.0 x 10 8 3.5 x 10 8 4.0 x 108 4.5 x 10 8 5x10 8 5 x 10 8 Or 1x 10 9 The amount of cells expressing CD4 or CD8. In some embodiments, the input cell composition has a concentration of 1 x 10⁻⁶ cells / mL or approximately 1 x 10⁻⁶ cells / mL. 6 5 x 10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1 x 10 8 1.2 x 10 8 1.3 x 10 8 1.4x10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8 x 10 8 1.9 x 10 8 2.0x 10 8 2.1 x 10 8 2.2 x 10 8 2.3x10 8 2.4 x 10 8 2.5 x 10 8 2.6 x 10 8 2.7 x 10 8 2.8 x 10 8 2.9 x 10 8 3.0 x 10 8 3.5 x 10 8 4.0x10 8 4.5 x 10 8 5 x 10 8 5 x 10 8 Or 1x 10 9 The amount of immature CD4+ and immature CD8+ T cells.

[0125] In a particular embodiment, the input cell composition has a density of 1 x 102 6 One and 1x 10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3x10 8The total number of cells or total viable cells is between [number] and [number] units. In some embodiments, the input cell composition has a [number] or approximately [number] units of [cells] at 1 x 10 [units]. 6 One and 1x 10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3 x 10 8 The cell composition expresses either CD4 or CD8 in amounts between [number] and [number] cells. In some embodiments, the input cell composition has a concentration of [number] or approximately [number] cells per 1 x 10[number] cells. 6 One and 1x10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3 x 10 8 Between the number of immature CD4+ and immature CD8+ T cells.

[0126] In some embodiments, the input cell composition has or contains at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 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% immature cells. In a particular embodiment, the input cell composition contains or includes no more than 100%, no more than 99%, no more than 98%, no more than 97%, no more than 96%, no more than 95%, no more than 90%, or no more than 85% immature cells.

[0127] In certain embodiments, the methods provided herein include one or more steps of generating, producing, and / or preparing an input cell composition. In some embodiments, generating, producing, and / or preparing an input cell composition includes one or more steps of mixing or combining a composition of CD4+ T cells with a composition of CD8+ T cells.

[0128] In some embodiments, the cells in the input composition (e.g., CD4+ T cells and CD8+ T cells) have been isolated and / or selected from a sample (e.g., a biological sample). In some embodiments, the source of the cells in the input composition is a composition of cells that have been isolated and / or selected from a sample, such as a composition of CD4+ T cells and a composition of CD8+ T cells. In specific embodiments, the composition of CD4+ T cells and the composition of CD8+ T cells are isolated and / or selected from a sample (e.g., a biological sample), such as a composition of cells isolated and / or selected separately. In some embodiments, the composition of CD4+ T cells and the composition of CD8+ T cells are isolated and / or selected from the same sample. In some embodiments, the composition of CD4+ T cells and the composition of CD8+ T cells are isolated and / or selected from a sample taken from or obtained from the same subject.

[0129] In certain embodiments, the CD4+ T cell composition contains or includes at least 60%, 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 CD4+ T cells. In some embodiments, the CD4+ T cell composition contains or includes no more than 100%, no more than 99%, no more than 98%, no more than 97%, no more than 96%, no more than 95%, no more than 90%, or no more than 85% of CD4+ T cells.

[0130] In some embodiments, the CD8+ T cell composition contains or includes at least 60%, 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 CD8+ T cells. In a particular embodiment, the CD8+ T cell composition contains or includes no more than 100%, no more than 99%, no more than 98%, no more than 97%, no more than 96%, no more than 95%, no more than 90%, or no more than 85% of CD8+ T cells.

[0131] In some embodiments, generating, producing, and / or preparing the input cell composition includes one or more of the following steps: for example, measuring, determining, and / or quantifying the amount, fraction, number, quantity per volume, quantity per weight, and / or percentage of live CD4+ T cells and / or live CD8+ T cells present in the CD4+ T cell composition and / or CD8+ T cell composition before combining or mixing the cells of the cell composition. In a particular embodiment, generating, producing, and / or preparing the input cell composition includes one or more of the following steps: measuring, determining, and / or quantifying the amount, fraction, number, quantity per volume, quantity per weight, and / or percentage of immature CD4+ T cells and / or immature CD8+ T cells present in the CD4+ T cell composition and / or CD8+ T cell composition. In some embodiments, the immature CD4+ and / or immature CD8+ T cells are live immature cells.

[0132] In some embodiments, generating, producing, and / or preparing the input cell composition includes one or more steps of measuring, determining, and / or quantifying the amount, fraction, number, quantity per volume, quantity per weight, and / or percentage of live CD4+ T cells and / or live CD8+ T cells present in a sample (e.g., a biological sample). In specific embodiments, generating, producing, and / or preparing the input cell composition includes one or more steps of measuring, determining, and / or quantifying the amount, fraction, number, quantity per volume, quantity per weight, and / or percentage of immature CD4+ T cells and / or immature CD8+ T cells present in a sample. In some embodiments, the immature CD4+ and / or immature CD8+ T cells are live immature cells.

[0133] In some embodiments, the cells in the input composition are isolated and / or selected from a sample (e.g., a biological sample). In specific embodiments, the proportion of immature cells in the sample (e.g., the proportion of immature CD4+ and CD8+ T cells) is known or has been determined, measured, or evaluated. In some embodiments, cells from the sample are isolated and / or selected to directly produce a cell composition (e.g., the input cell composition) having a defined ratio, a fixed ratio, or a controlled ratio of immature CD4+ T cells to immature CD8+ T cells. In some embodiments, cells are isolated and / or selected using immunoaffinity beads. In some embodiments, cells are isolated and / or selected using affinity columns. In specific embodiments, cells from the sample are isolated or selected according to any method described in WO 2015 / 164675 to produce a cell composition having a defined ratio, a controlled ratio, and / or a fixed ratio of immature CD4+ cells to immature CD8+ cells.

[0134] In some embodiments, the input cell composition contains cells directly isolated and / or selected from a sample by first and second separation or selection. In some embodiments, the input composition is produced by performing first and second selection to separate an amount, number, or concentration of CD4+ T cells and CD8+ T cells sufficient to produce a defined ratio, a fixed ratio, and / or a controlled ratio of naïve CD4+ T cells and naïve CD8+ T cells.

[0135] In some embodiments, cells from a sample are directly isolated, selected, and / or enriched to produce an input cell composition enriched with CD4+ and CD8+ cells. In some embodiments, the amount, quantity, percentage, number per volume, and / or number per weight of immature CD4+ and immature CD8+ cells in the sample have been measured, evaluated, and / or determined, and CD4+ and CD8+ cells are isolated, selected, and / or enriched in an amount sufficient to achieve the input cell composition having a defined, fixed, or controlled ratio of immature CD4+ T cells to immature CD8+ T cells. In some embodiments, cells directly isolated, selected, and / or enriched from the sample are the input cell composition and used in subsequent processing steps, such as subsequent processing steps involving the incubation, stimulation, activation, engineering, and / or formulation of the enriched cells.

[0136] In some embodiments, cells isolated, selected, and / or enriched from a sample, such as an input cell composition, contain a defined ratio of CD4+ cells to CD8+ cells at a predetermined, fixed, or controlled ratio of naive CD4+ cells to naive CD8+ cells. In embodiments of the methods provided herein, first and / or second selection of the sample, or selection of its subpopulations, can be performed in a manner that produces an input cell composition having the desired ratio of naive CD4+ T cells to naive CD8+ cells.

[0137] In some embodiments, the ratio of CD4+ to CD8+ T cells in a sample (e.g., a biological sample) is determined before performing first and / or second selection from the sample. In some embodiments, the ratio of naive CD4+ T cells to naive CD8+ T cells in the sample is determined before performing first and / or second selection. Based on a specific ratio of CD4+ to CD8+ T cells in the sample and / or a specific ratio of naive CD4+ to CD8+ T cells (which can vary between samples), specific selection methods can be personalized for a sample, for example, by sizing the column or selecting the amount or concentration of the immunoaffinity reagent to achieve a desired ratio, a fixed ratio, or a controlled ratio. The relative levels or frequencies of various cell populations in a subject can be determined based on assessing the surface expression of one or more markers present on such populations or subpopulations. A variety of well-known methods for assessing the expression levels of surface markers or proteins can be used, such as detection by affinity-based methods, such as immunoaffinity-based methods, for example, in the case of cell surface proteins, such as detection by flow cytometry.

[0138] In some contexts, the appropriate ratio of naive CD4+ to CD8+ T cells can vary depending on factors such as: the specific disease, condition, or prior treatment of the subject in which the derived cells are located, and / or the specific antigen specificity of the cells, the relative representation between specific cell types (e.g., CD4+ cells) of various subsets (e.g., effector cells versus memory cells versus naive cells), and / or one or more conditions to be used to incubate the cells, such as culture medium, stimulants, culture time, buffer, oxygen content, carbon dioxide content, antigens, cytokines, antibodies, and other components. Therefore, a cell type that is typically or generally known to proliferate or expand faster than another cell type may not always exhibit this characteristic in every situation. Thus, in some respects, the ratio of naive CD4+ to naive CD8+ T cells is determined based on the known capacity of the cell type under normal or typical conditions, combined with an assessment of the phenotype or condition of the subject in which the cells or derived cells are located, and / or empirical evidence.

[0139] In some embodiments, the separation and / or steps are performed using immunomagnetic beads. In some embodiments, a cell sample containing CD4+ and CD8+ cells is contacted with magnetic beads containing a first immunoaffinity reagent bound to CD4 or CD8 and a second immunoaffinity reagent bound to the other of CD4 or CD8. The separation and / or steps may be performed simultaneously and / or sequentially.

[0140] In some embodiments, the first and / or second immunoaffinity reagents are present in the incubation composition at a suboptimal yield concentration, thereby enriching the composition to contain less than all (e.g., 70%) of the total CD4+ cells and / or less than all (e.g., 70%) of the CD8+ cells in the incubation composition, thereby producing a composition enriched with CD4+ and CD8+ T cells.

[0141] In some embodiments, the suboptimal yield concentration of the affinity reagent is a concentration lower than that used or required to achieve the optimal or maximum yield of bound cells in a given selection or enrichment, which involves incubating cells with the reagent and recovering or separating cells already bound to the reagent (e.g., "yield" is the number of cells thus recovered or selected compared to the total number of cells incubated that are targeted by the reagent, or that the reagent is specific to, or that have a label (the reagent is specific to and able to bind the label)). The suboptimal yield concentration is typically the concentration or amount of reagent used to achieve less than (e.g., no more than 70%) of the yield of bound cells (e.g., CD4+ and / or CD8+ T cells) after the recovery of cells already bound to the reagent in this process or step. In some embodiments, yields of no more than or equal to about 50%, 45%, 40%, 30%, or 25% are achieved by the suboptimal concentration of the affinity reagent. The concentration can be expressed as the number or mass of particles or surface area per cell and / or the mass number or number of molecules of the agent (e.g., antibody, such as antibody fragment) per cell. In a particular embodiment, the suboptimal yield concentration is sufficient to drive or achieve a fixed, controlled, and / or defined ratio of naive CD4+ T cells to naive CD8+ T cells.

[0142] In some implementations, for example, when operating with suboptimal yield concentrations of one or more of two or more selectable reagents having affinity for CD4+ and / or CD8+ T cells, one or more of such reagents are used at higher concentrations than one or more of the other such reagents to favor the cell types identified by that reagent, as compared to the cell types identified by the other one or more reagents. For example, depending on how much the ratio is expected to increase, the reagent that binds specifically to the marker desired to favor the ratio may be included at a concentration (e.g., agent or mass per cell) that is half, 1, 2, 3, 4, 5, 10, or more than that of the other one or more reagents.

[0143] In some embodiments, when operating within suboptimal ranges and / or with cell manipulation sufficient to achieve reagent saturation, the amount of immunoaffinity reagent is proportional to the approximate yield of enriched cells. In some embodiments, the appropriate amount or concentration of immunoaffinity reagent can be determined in a conventional manner, depending on the desired ratio of the resulting composition containing enriched or selected CD4+ and CD8+ T cells.

[0144] In some embodiments, magnetic beads are used for the separation and / or isolation steps, in which immunoaffinity reagents are reversibly bound, such as through interaction with peptide ligands of streptavidin mutant proteins, as described in WO 2015 / 164675. Examples of such magnetic beads are... In some implementations, the separation and / or steps are performed using magnetic beads, such as those commercially available from Miltenyi Biotec.

[0145] In some embodiments, the first selection or enrichment of CD4+ and CD8+ cells from a sample is performed using an immunoaffinity-based reagent comprising at least first and second affinity chromatographic matrices, on which antibodies are immobilized. In some embodiments, one or both of the first and / or second selections may employ multiple affinity chromatographic matrices and / or antibodies, thereby tandemly linking the multiple matrices and / or antibodies used for the same selection (i.e., the first or second selection). In some embodiments, one or more affinity chromatographic matrices employed in the first and / or second selections adsorb or are capable of selecting or enriching at least about 50 x 10⁻⁶ cells. 6 cells / mL, 100 x 10 6 cells / mL, 200 x 10 6 1 cell / mL or 400 x 10 6 Cells / mL. In some embodiments, the adsorption capacity can be adjusted based on the column diameter and / or length. In some embodiments, the culture initiation rate of the selected or enriched composition is achieved by assuming, for example, based on the adsorption capacity of one or more columns used for cell selection, an amount of substrate sufficient to achieve the culture initiation rate and / or a relative amount sufficient to achieve the culture initiation rate.

[0146] In one exemplary embodiment, CD4+ T cells and CD8+ T cells have an equal or similar proportion of immature-like cells, and the adsorption capacity of one or more substrates is the same between the first and second selections, for example, approximately 1 x 10⁻⁶ for both. 8Cells / mL, thereby enriching or selecting cells in a first and second selection to produce a composition containing CD4+ and CD8+ cells, said composition having an immature CD4+ to CD8+ T cell ratio of 1:1 or about 1:1. In a particular embodiment, the appropriate volume, diameter, or number of affinity matrix columns for the first and / or second selection can be selected or determined in a conventional manner, depending on the fraction of immature cells and the desired ratio of the resulting input cell composition.

[0147] In some implementations, the adsorption capacity of one or more column matrices is modulated to compensate for the difference in frequency of naive cells (e.g., naive CD4+ or CD8+ cells) compared to the frequency of cells from the corresponding CD4+ or CD8+ parental population in the subject's initial sample. The relative levels or frequencies of various cell populations in the subject can be determined based on assessing the surface expression of one or more markers present on such populations or subpopulations. A variety of well-known methods for assessing the expression levels of surface markers or proteins can be used, such as assays performed via affinity-based methods, such as immunoaffinity-based methods, for example, in the case of cell surface proteins, such as detection by flow cytometry.

[0148] In some embodiments, naïve-like cells (e.g., naïve CD4+ and / or CD8+ T cell compositions) are evaluated, measured, and / or detected in a cell composition (e.g., a CD4+ and / or CD8+ T cell composition) or in a sample (e.g., a biological sample). In some embodiments, naïve-like T cells are T cells that are positive for the expression of one or more markers indicating that the cell is a naïve cell and / or a naïve-like cell. In some embodiments, naïve-like T cells are cells that are positive for the expression of a marker associated with a naïve or naïve-like state in the T cell. In a particular embodiment, naïve-like T cells are T cells that are negative for the expression of one or more markers indicating that the cell is not a naïve cell and / or not a naïve-like cell. In some embodiments, naïve-like T cells are cells that are negative for the expression of a marker associated with a non-naïve or non-naïve-like state in the T cell. In some implementations, the non-naive or non-naive-like state in T cells includes, for example, but not limited to, effector T (TEFF) cells, memory T cells, central memory T cells (TCM), effector memory T (TEM) cells, and combinations thereof.

[0149] In some embodiments, naive-like T cells are positive for expression of at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten markers, said markers indicating that the cells are naive cells and / or naive-like cells, and / or said markers are associated with an naive or naive-like state in T cells. In some embodiments, said markers are expressed on the cell surface. In some embodiments, naive-like T cells are negative for expression of at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten markers, said markers indicating that the cells are non-naive cells and / or non-naive-like cells, and / or said markers are associated with a non-naive or non-naive-like state in T cells.

[0150] Indicators that T cells are naive cells and / or naive-like T cells, and / or markers associated with naive or naive-like states in T cells, include, but are not limited to, CD27, CD28, CD45RA, CD62L, and / or CCR7. In some embodiments, naive-like T cells (e.g., naive-like CD4+ and / or CD8+ T cells) are positive for expression of CD27, CD28, CD45RA, and / or CCR7. In some embodiments, naive-like T cells are positive for surface expression of one or more of CD27, CD28, CD45RA, and / or CCR7. In some embodiments, naive-like T cells (e.g., naive-like CD4+ and / or CD8+ T cells) are negative for expression of CD62L.

[0151] Indicator cells are non-immature cells and / or non-immature-like T cells, and / or markers associated with the non-immature or non-immature-like state of T cells, including but not limited to CD25, CD45RO, CD56, KLRG1, and / or CD95. In some embodiments, immature-like T cells (e.g., immature-like CD4+ and / or CD8+ T cells) are negative for expression of CD25, CD45RO, CD56, and / or KLRG1. In certain embodiments, immature-like T cells (e.g., immature-like CD4+ and / or CD8+ T cells) have low expression of markers associated with non-immature cells or non-immature-like cells. In certain embodiments, immature-like T cells have low expression of CD95. In some embodiments, immature-like T cells are negative for surface expression of one or more of CD25, CD45RO, CD56, and / or KLRG1.

[0152] In some embodiments, low expression of markers associated with non-immature cells or non-immature-like cells is or includes expression that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% less than the expression of the marker in cells that are non-immature-like cells and / or for cells positive for one or more markers, the markers indicating that the cells are non-immature T cells and / or non-immature-like T cells, and / or the markers are associated with a non-immature or non-immature-like state in T cells. In some embodiments, low expression of markers associated with non-immature cells or non-immature-like cells is or includes expression that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% less than the expression of the markers in effector T cells. EFF ) cells, memory T cells, central memory T cells (T cells) CM ) and / or effect memory T(T EM The expression in cells is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

[0153] In some embodiments, the markers indicating whether cells are non-immature T cells and / or non-immature-like T cells, and / or associated with the non-immature or non-immature-like state in T cells, include one or more cytokines. For example, in some embodiments, non-immature T cells or non-immature-like T cells are negative for the expression and / or production of one or more of IL-2, IFN-γ, IL-4, and IL-10. In some embodiments, the one or more cytokines are secreted. In specific embodiments, for example, during or after treatment with an agent that prevents, inhibits, or reduces secretion, non-immature-like T cells internally express the one or more cytokines.

[0154] In some embodiments, naïve T cells are positive for the expression (e.g., surface expression) of CD45RA and CCR7. In specific embodiments, naïve CD4+ T cells are positive for the expression (e.g., surface expression) of CD45RA and CCR7. In some embodiments, naïve CD8+ T cells are positive for the expression (e.g., surface expression) of CD45RA, CD27, and CCR7, and negative for the expression (e.g., surface expression) of CD45RO. In specific embodiments, naïve CD4+ T cells are positive for the expression (e.g., surface expression) of CD45RA, CD27, and CCR7, and negative for the expression (e.g., surface expression) of CD45RO. In some implementations, naive CD8+ T cells are positive for expression (e.g., surface expression) of CD45RA, CD27, and CCR7, and negative for expression (e.g., surface expression) of CD45RO.

[0155] In some embodiments, CD4+ and / or CD8+ T cells are live cells. In some embodiments, CD4+ and / or CD8+ T cells are live naive-like cells. The live cells are positive for the expression of a marker indicating that the cells have undergone normal functional cellular processes and / or have not yet undergone necrosis or programmed cell death, or are not in the process of undergoing necrosis or programmed cell death. In some embodiments, viability can be assessed by the cell's redox potential, cell membrane integrity, or mitochondrial activity or function. In some embodiments, viability is the absence of specific molecules associated with cell death, or the absence of indicators of cell death, in the assay.

[0156] In some embodiments, cell viability is assessed by assays, which may include, but are not limited to, dye uptake assays (e.g., calcein AM assay), XTT cell viability assays, and dye exclusion assays (e.g., trypan blue, eosin, or propidium dye exclusion assays). In certain embodiments, the live cells have negative expression of one or more apoptosis markers (e.g., annexin V or active caspase 3). In some embodiments, the live cells are negative for the expression or activation of one or more apoptosis markers, which may include, but are not limited to, caspases (e.g., caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, and caspase 10), Bcl-2 family members (e.g., Bax, Bad, and Bid), annexin V, and / or TUNEL staining.

[0157] In some embodiments, expression is or includes the amount, level, concentration, and / or presence of a label. In a particular embodiment, the label is a polypeptide. In some embodiments, the label is mRNA. In some embodiments, expression is or includes the amount, level, concentration, and / or presence of a polypeptide (e.g., a labeled polypeptide). In some embodiments, the amount, level, concentration, and / or presence of a polynucleotide encoding the label (e.g., mRNA or cDNA derived from mRNA). In some embodiments, expression is or includes the amount, level, concentration, and / or presence of a label located on or exposed on the cell surface or within the cell membrane. In some embodiments, expression is or includes the amount, level, concentration, and / or presence of a label located on or exposed on the cell surface or within the cell membrane. In a particular embodiment, expression is or includes internal expression, such as the amount, level, concentration, and / or presence of a label within the cell, such as in the cytosol, nucleus, endoplasmic reticulum, and / or Golgi apparatus.

[0158] In some embodiments, the marker is measured, evaluated, and / or quantified by in vitro assays. In some examples, the in vitro assay is an immunoassay, an aptamer-based assay, a histological or cytological assay, or an mRNA expression level assay. In some cases, the in vitro assay used may be an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry, surface plasmon resonance (SPR), chemiluminescence assay, lateral flow immunoassay, inhibition assay, and affinity assay. In some embodiments, the marker expression is measured, evaluated, and / or quantified by RNA-seq. In certain embodiments, the marker expression is measured, evaluated, and / or quantified by immunostaining techniques. In certain embodiments, the marker expression is measured, evaluated, and / or quantified by flow cytometry analysis. In some embodiments, the marker expression is measured, evaluated, and / or quantified by internal cytokine staining.

[0159] In some embodiments, the markers are measured, evaluated, and / or quantified in the cells of the CD4+ T cell composition. In specific embodiments, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or at least 99% of the CD4+ T cells are naive CD4+ T cells. In some embodiments, CD4+ T cells with a percentage between 10% and 50%, between 20% and 60%, between 25% and 75%, between 30% and 80%, between 40% and 90%, between 50% and 100%, between 30% and 50%, between 40% and 60%, between 50% and 70%, between 60% and 80%, between 70% and 90%, between 80% and 100%, between 5% and 25%, between 25% and 50%, between 50% and 75%, or between 75% and 99% are naive CD4+ T cells. In some embodiments, naive CD4+ T cells are live naive CD4+ T cells.

[0160] In some embodiments, the markers are measured, evaluated, and / or quantified in the cells of the CD8+ T cell composition. In specific embodiments, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or at least 99% of the CD8+ T cells are naive CD8+ T cells. In some embodiments, CD8+ T cells at percentages between 10% and 50%, between 20% and 60%, between 25% and 75%, between 30% and 80%, between 40% and 90%, between 50% and 100%, between 30% and 50%, between 40% and 60%, between 50% and 70%, between 60% and 80%, between 70% and 90%, between 80% and 100%, between 5% and 25%, between 25% and 50%, between 50% and 75%, or between 75% and 99% are naive CD4+ T cells. In some embodiments, naive CD8+ T cells are live naive CD8+ T cells.

[0161] In some embodiments, cells from a composition of CD4+ T cells are mixed or combined with cells from a composition of CD8+ T cells in an amount and / or ratio sufficient to produce an input cell composition having a CD4+ naive T cell to CD8+ naive T cell ratio between 10:1 and 0.05:1, between 8:1 and 0.1:1, between 5:1 and 0.2:1, between 2.5:1 and 0.25:1, between 2.2:1 and 0.8:1, between 2:1 and 0.5:1, or between 1.5:1 and 1:1, including end values. In some embodiments, cells are mixed in an amount and / or ratio sufficient to achieve a CD4+ naive T cell to CD8+ naive T cell ratio between 2.2:1 and 0.8:1 (including end values). In some embodiments, the ratio of CD4+ immature T cells to CD8+ immature T cells is mixed or combined at a ratio of approximately 2.2:1, 2.1:1, 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, or 0.8:1. In some embodiments, the ratio of cells is mixed or combined at a ratio of approximately 1.1:1.

[0162] In some implementations, it will be or approximately 5 x 10 5 1x10 6 5 x 10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1 x 10 8 1.2 x 10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8 x 10 8 1.9x10 8 2.0x 10 8 2.1 x 10 8 2.2 x 10 8 2.3 x 10 8 2.4 x 10 8 2.5 x 10 8 2.6 x 10 8 2.7 x 10 8 2.8x10 8 2.9 x 10 83.0 x 10 8 3.5 x 10 8 4.0 x 10 8 4.5 x 10 8 5 x 10 8 5 x 10 8 Or 1x 10 9 The total number of CD4+ T cells or total live CD4+ T cells is approximately 5 x 10^6. 5 1x10 6 5 x 10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1x10 8 1.2 x 10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8 x 10 8 1.9 x 10 8 2.0x 10 8 2.1 x 10 8 2.2 x 10 8 2.3 x 10 8 2.4 x 10 8 2.5 x 10 8 2.6 x 10 8 2.7 x 10 8 2.8x10 8 2.9 x 10 8 3.0 x 10 8 3.5 x 10 8 4.0 x 10 8 4.5 x 10 8 5 x 10 8 5.5 x 10 8 Or 1x 10 9 A mixture or combination of total CD8+ T cells or total live CD8+ T cells is used to produce an input cell composition having a defined ratio of CD4+ naive T cells to CD8+ naive T cells. In some embodiments, 1 x 10 6 One and 1x 10 10 Between 1x10 7 One and 1x 10 9 Between 5x107 5 x 10 8 Between 1x10 8 3 x 10 8 The total CD4+ T cells or total active CD4+ T cells are approximately 1 x 10^6 T cells. 6 One and 1x 10 10 Between 1x10 7 One and 1x10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3 x 10 8 A mixture or combination of total CD8+ T cells or total live CD8+ T cells of varying amounts is used to produce an input cell composition having a defined ratio of CD4+ immature T cells to CD8+ immature T cells.

[0163] In some implementations, it will be or approximately 5 x 10 5 1x10 6 5 x 10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1 x 10 8 1.2 x 10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8 x 10 8 1.9x10 8 2.0x 10 8 2.1 x 10 8 2.2 x 10 8 2.3 x 10 8 2.4 x 10 8 2.5 x 10 8 2.6 x 10 8 2.7 x 10 8 2.8x10 8 2.9 x 10 8 3.0 x 10 8 3.5 x 10 8 4.0 x 10 8 4.5 x 10 8 5 x 10 85 x 10 8 Or 1x 10 9 The number of immature CD4+ T cells was approximately 5 x 10^12. 5 1x10 6 5 x 10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1 x 10 8 1.2x10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8 x 10 8 1.9 x 10 8 2.0x 10 8 2.1 x 10 8 2.2 x 10 8 2.3 x 10 8 2.4 x 10 8 2.5 x 10 8 2.6 x 10 8 2.7 x 10 8 2.8 x 10 8 2.9x10 8 3.0 x 10 8 3.5 x 10 8 4.0 x 10 8 4.5 x 10 8 5 x 10 8 5.5 x 10 8 Or 1x 10 9 A certain amount of immature CD8+ T cells are mixed or combined to produce an input cell composition of CD4+ immature T cells and CD8+ immature T cells in a defined ratio. In some embodiments, 5 x 10 5 One and 1x 10 10 Between 1 and 10 6 One and 1x 10 10 Between, in 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3 x 10 8The number of immature CD4+ T cells was approximately 5 x 10^6. 5 One and 1x 10 10 Between 1 and 10 6 One and 1x 10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3 x 10 8 A mixture or combination of immature CD8+ T cells of varying amounts is used to produce an input cell composition of CD4+ immature T cells and CD8+ immature T cells in a defined ratio.

[0164] In certain embodiments, the ratio of naïve CD4+ T cells to naïve CD8+ T cells in the input cell composition has been adjusted, varied, and / or altered compared to the ratio of naïve CD4+ T cells to naïve CD8+ T cells in a sample (e.g., a biological sample). In certain embodiments, the adjustment, variation, or alteration of the naïve CD4+ T cell to naïve CD8+ T cell ratio compared to the biological sample is or is about or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1x, 1.5x, 2x, 2.5x, 3x, 4x, 5x, 10x, 20x, 50x, or 100x. In some embodiments, the sample is a sample of cells from which the input cell composition is derived, isolated, selected, and / or obtained.

[0165] In some embodiments, the input cell composition contains a ratio (e.g., a defined ratio, a controlled ratio, and / or a fixed ratio) of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells. In a specific embodiment, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells is between 10:1 and 0.05:1, between 8:1 and 0.1:1, between 5:1 and 0.2:1, between 2.5:1 and 0.25:1, between 2.2:1 and 0.8:1, between 2:1 and 0.5:1, or between 1.5:1 and 1:1, including end values. In certain embodiments, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells is between 2:1 and 0.8:1, between 1.6:1 and 0.8:1, between 1.4:1 and 0.8:1, between 1.2:1 and 0.8:1, or between 1.2:1 and 0.8:1, including end values. In some embodiments, the ratio is between 2.2:1 and 0.8:1, including end values. In some embodiments, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells is or is about 2.2:1, 2.1:1, 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, or 0.8:1. In some embodiments, the ratio is or is about 1.1:1.

[0166] In a particular embodiment, the input cell composition has a density of 1 x 10⁻⁶ cells / cm². 6 5 x 10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1 x 10 8 1.2 x 10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8x10 8 1.9 x 10 8 2.0x 10 8 2.1 x 10 8 2.2 x 10 8 2.3 x 108 2.4 x 10 8 2.5 x 10 8 2.6 x 10 8 2.7x10 8 2.8 x 10 8 2.9 x 10 8 3.0 x 10 8 3.5 x 10 8 4.0 x 10 8 4.5 x 10 8 5 x 10 8 5 x 10 8 Or 1x 10 9 The amount of total cells or total viable cells. In some embodiments, the input cell composition has a quantity of 5 x 10⁻⁶ cells / units or approximately 10⁻⁶ cells / units. 5 1x10 6 5 x 10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1 x 10 8 1.2 x 10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8 x 10 8 1.9 x 10 8 2.0x 10 8 2.1 x 10 8 2.2 x 10 8 2.3 x 10 8 2.4x10 8 2.5 x 10 8 2.6 x 10 8 2.7 x 10 8 2.8 x 10 8 2.9 x 10 8 3.0 x 10 8 3.5 x 10 8 4.0 x 10 8 4.5x10 8 5 x 10 8 5 x 10 8 Or 1x 10 9The input cell composition contains a quantity of cells expressing CD4 or CD8. In some embodiments, the input cell composition has a quantity of 5 x 10⁻⁶ cells or approximately 10⁻⁶ cells. 5 1x10 6 5 x 10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1 x 10 8 1.2 x 10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8 x 10 8 1.9 x 10 8 2.0x 10 8 2.1x10 8 2.2 x 10 8 2.3 x 10 8 2.4 x 10 8 2.5 x 10 8 2.6 x 10 8 2.7 x 10 8 2.8 x 10 8 2.9 x 10 8 3.0x10 8 3.5 x 10 8 4.0 x 10 8 4.5 x 10 8 5 x 10 8 5 x 10 8 Or 1x 10 9 The number of CD45RA+ / CCR7+ / CD4+ and CD45RA+ / CCR7+ / CD8+ T cells.

[0167] In a particular embodiment, the input cell composition has a density of 1 x 102 6 One and 1x 10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3x10 8 The total number of cells or total viable cells is between [number] and [number] units. In some embodiments, the input cell composition has a [number] or approximately [number] units of [cells] cells. 5One and 1x 10 10 Between, 1x10 6 One and 1x 10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3 x 10 8 The cell composition expresses either CD4 or CD8 in amounts between [number] and [number] cells. In some embodiments, the input cell composition has a concentration of [number] or approximately [number] cells per 5 x 10[ ... 5 One and 1x 10 10 Between 1 and 10 6 With 1x 10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3 x 10 8 The number of CD45RA+ / CCR7+ / CD4+ and CD45RA+ / CCR7+ / CD8+ T cells varies between individuals.

[0168] In some embodiments, the input cell composition has or contains at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 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 CD45RA+ / CCR7+ cells. In a particular embodiment, the input cell composition contains or includes no more than 100%, no more than 99%, no more than 98%, no more than 97%, no more than 96%, no more than 95%, no more than 90%, or no more than 85% of CD45RA+ / CCR7+ cells.

[0169] In some implementations, it will be or approximately 5 x 10 5 1x10 6 5 x 10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1 x 10 8 1.2 x 10 8 1.3 x 10 81.4 x 10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8 x 10 8 1.9x10 8 2.0x 10 8 2.1 x 10 8 2.2 x 10 8 2.3 x 10 8 2.4 x 10 8 2.5 x 10 8 2.6 x 10 8 2.7 x 10 8 2.8x10 8 2.9 x 10 8 3.0 x 10 8 3.5 x 10 8 4.0 x 10 8 4.5 x 10 8 5 x 10 8 5 x 10 8 Or 1x 10 9 The total number of CD4+ T cells or total live CD4+ T cells is approximately 5 x 10^6. 5 1x10 6 5 x 10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1x10 8 1.2 x 10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8 x 10 8 1.9 x 10 8 2.0x 10 8 2.1 x 10 8 2.2 x 10 8 2.3 x 10 8 2.4 x 10 8 2.5 x 10 8 2.6 x 10 8 2.7 x 10 8 2.8x10 82.9 x 10 8 3.0 x 10 8 3.5 x 10 8 4.0 x 10 8 4.5 x 10 8 5 x 10 8 5.5 x 10 8 Or 1x 10 9 A mixture or combination of total CD8+ T cells or total live CD8+ T cells is used to produce an input cell composition having a defined ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells. In some embodiments, 5 x 10 5 One and 1x 10 10 Between 1 and 10 6 One and 1x 10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5x10 8 Between 1x10 8 3 x 10 8 The total CD4+ T cells or total active CD4+ T cells were approximately 5 x 10⁻⁶. 5 One and 1x 10 10 Between 1 and 10 6 One and 1x 10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3 x 10 8 Mixing or combining amounts of total CD8+ T cells or total live CD8+ T cells to produce an input cell composition of CD45RA+ / CCR7+ / CD4+ T cells with a defined ratio of CD45RA+ / CCR7+ / CD8+ T cells.

[0170] In certain embodiments, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells in the input cell composition has been adjusted, varied, and / or altered compared to the ratio of CD45RA+ / CCR7+ / CD8+ T cells in a sample (e.g., a biological sample). In certain embodiments, the adjustment, variation, or alteration of the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells compared to the biological sample is or is about or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1x, 1.5x, 2x, 2.5x, 3x, 4x, 5x, 10x, 20x, 50x, or 100x. In some embodiments, the sample is a sample of cells from which the input cell composition is derived, isolated, selected, and / or obtained.

[0171] In some embodiments, the input cell composition contains CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells in a predetermined ratio (e.g., a defined ratio, a controlled ratio, and / or a fixed ratio). In a specific embodiment, the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells is between 10:1 and 0.05:1, between 8:1 and 0.1:1, between 5:1 and 0.2:1, between 2.5:1 and 0.25:1, between 2.2:1 and 0.8:1, between 2:1 and 0.5:1, or between 2:1 and 1:1, including end values. In certain embodiments, the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells is between 2:1 and 0.8:1, between 1.8:1 and 1:1, between 1.8:1 and 1.2:1, between 1.2:1 and 1.4:1, or between 1.8:1 and 1.6:1, including end values. In some embodiments, the ratio is between 1.8:1 and 1.6:1, including end values. In some embodiments, the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells is or is about 2.2:1, 2.1:1, 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.69:1, 1.6:1, 1.5:1, 1.4:1, or 1.3:1. In some implementations, the ratio is or is about 1.69:1.

[0172] In a particular embodiment, the input cell composition has a density of 1 x 10⁻⁶ cells / cm². 65 x 10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1 x 10 8 1.2 x 10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8x10 8 1.9 x 10 8 2.0x 10 8 2.1 x 10 8 2.2 x 10 8 2.3 x 10 8 2.4 x 10 8 2.5 x 10 8 2.6 x 10 8 2.7x10 8 2.8 x 10 8 2.9 x 10 8 3.0 x 10 8 3.5 x 10 8 4.0 x 10 8 4.5 x 10 8 5 x 10 8 5 x 10 8 Or 1x 10 9 The amount of total cells or total viable cells. In some embodiments, the input cell composition has a quantity of 5 x 10⁻⁶ cells / units or approximately 10⁻⁶ cells / units. 5 1x10 6 5 x 10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1 x 10 8 1.2 x 10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8 x 10 8 1.9 x 10 8 2.0x 10 8 2.1 x 10 82.2 x 10 8 2.3 x 10 8 2.4x10 8 2.5 x 10 8 2.6 x 10 8 2.7 x 10 8 2.8 x 10 8 2.9 x 10 8 3.0 x 10 8 3.5 x 10 8 4.0 x 10 8 4.5x10 8 5 x 10 8 5 x 10 8 Or 1x 10 9 The input cell composition contains a quantity of cells expressing CD4 or CD8. In some embodiments, the input cell composition has a quantity of 5 x 10⁻⁶ cells or approximately 10⁻⁶ cells. 5 1x10 6 5 x 10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1 x 10 8 1.2 x 10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8 x 10 8 1.9 x 10 8 2.0x 10 8 2.1x10 8 2.2 x 10 8 2.3 x 10 8 2.4 x 10 8 2.5 x 10 8 2.6 x 10 8 2.7 x 10 8 2.8 x 10 8 2.9 x 10 8 3.0x10 8 3.5 x 10 8 4.0 x 10 8 4.5 x 10 8 5 x 10 8 5 x 10 8 Or 1x 10 9The number of CD27+ / CCR7+ / CD4+ and CD27+ / CCR7+ / CD8+ T cells.

[0173] In a particular embodiment, the input cell composition has a density of 1 x 102 6 One and 1x 10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3x10 8 The total number of cells or total viable cells is between [number] and [number] units. In some embodiments, the input cell composition has a [number] or approximately [number] units of [cells] cells. 5 One and 1x 10 10 Between, 1x10 6 With 1x 10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3 x 10 8 The cell composition expresses either CD4 or CD8 in amounts between [number] and [number] cells. In some embodiments, the input cell composition has a concentration of [number] or approximately [number] cells per 5 x 10[ ... 5 One and 1x 10 10 Between 1 and 10 6 One and 1x 10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3 x 10 8 The number of CD27+ / CCR7+ / CD4+ and CD27+ / CCR7+ / CD8+ T cells varies between individuals.

[0174] In some embodiments, the input cell composition has or contains at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 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 CD27+ / CCR7+ cells. In a particular embodiment, the input cell composition contains or includes no more than 100%, no more than 99%, no more than 98%, no more than 97%, no more than 96%, no more than 95%, no more than 90%, or no more than 85% of CD27+ / CCR7+ cells.

[0175] In some implementations, it will be or approximately 5 x 10 5 1x10 6 5 x 10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1 x 10 8 1.2 x 10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8 x 10 8 1.9x10 8 2.0x 10 8 2.1 x 10 8 2.2 x 10 8 2.3 x 10 8 2.4 x 10 8 2.5 x 10 8 2.6 x 10 8 2.7 x 10 8 2.8x10 8 2.9 x 10 8 3.0 x 10 8 3.5 x 10 8 4.0 x 10 8 4.5 x 10 8 5 x 10 8 5x10 8 Or 1x 10 9 The total number of CD4+ T cells or total active CD4+ T cells is approximately 5 x 10^6. 51x10 6 5 x 10 6 1x10 7 5 x 10 7 1.0x10 8 1.1 x 10 8 1.2 x 10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8 x 10 8 1.9x10 8 2.0x 10 8 2.1 x 10 8 2.2 x 10 8 2.3 x 10 8 2.4 x 10 8 2.5 x 10 8 2.6 x 10 8 2.7 x 10 8 2.8x10 8 2.9 x 10 8 3.0 x 10 8 3.5 x 10 8 4.0 x 10 8 4.5 x 10 8 5 x 10 8 5.5 x 10 8 Or 1x 10 9 A certain amount of total CD8+ T cells or total live CD8+ T cells are mixed or combined to produce an input cell composition having a defined ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells. In some embodiments, 5 x 10 5 One and 1x10 10 Between 1 and 10 6 One and 1x 10 10 Between, in 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3x10 8 The total CD4+ T cells or total active CD4+ T cells are approximately 5 x 10^6. 5 One and 1x 1010 Between 1 and 10 6 One and 1x 10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3 x 10 8 Mixing or combining amounts of total CD8+ T cells or total live CD8+ T cells to produce an input cell composition of CD27+ / CCR7+ / CD4+ T cells with a defined ratio of CD27+ / CCR7+ / CD8+ T cells.

[0176] In certain embodiments, the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells in the input cell composition has been adjusted, varied, and / or altered compared to the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells in a sample (e.g., a biological sample). In certain embodiments, the adjustment, variation, or alteration of the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells compared to the biological sample is or is about or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1x, 1.5x, 2x, 2.5x, 3x, 4x, 5x, 10x, 20x, 50x, or 100x. In some embodiments, the sample is a sample of cells from which the input cell composition is derived, isolated, selected, and / or obtained.

[0177] In some embodiments, the input cell composition contains a ratio (e.g., a defined ratio, a controlled ratio, and / or a fixed ratio) of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells. In a specific embodiment, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells is between 10:1 and 0.05:1, between 8:1 and 0.1:1, between 5:1 and 0.2:1, between 2.5:1 and 0.25:1, between 2.2:1 and 0.8:1, between 2:1 and 0.5:1, or between 1.5:1 and 1:1, including end values. In certain embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells is between 2:1 and 0.8:1, between 1.6:1 and 0.8:1, between 1.4:1 and 0.8:1, between 1.2:1 and 0.8:1, or between 1.2:1 and 0.8:1, including end values. In some embodiments, the ratio is between 2.2:1 and 0.8:1, including end values. In some embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells is or is about 2.2:1, 2.1:1, 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, or 0.8:1. In some embodiments, the ratio is or is about 1.1:1.

[0178] In a particular embodiment, the input cell composition has a density of 1 x 102 6 One and 1x 10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3x10 8 The total number of cells or total viable cells is between [number] and [number] units. In some embodiments, the input cell composition has a [number] or approximately [number] units of [cells] at 1 x 10 [units]. 6 One and 1x 10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3 x 10 8The cell composition expresses either CD4 or CD8 in amounts between [number] and [number] cells. In some embodiments, the input cell composition has a concentration of [number] or approximately [number] cells per 1 x 10[number] cells. 6 One and 1x10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3 x 10 8 The number of CD62L- / CCR7+ / CD4+ and CD62L- / CCR7+ / CD8+ T cells varies between individuals.

[0179] In some embodiments, the input cell composition has or contains at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 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 CD62L- / CCR7+ cells. In a particular embodiment, the input cell composition contains or includes no more than 100%, no more than 99%, no more than 98%, no more than 97%, no more than 96%, no more than 95%, no more than 90%, or no more than 85% of CD62L- / CCR7+ cells.

[0180] In some implementations, it will be or approximately 1 x 10 6 5 x 10 6 1x10 7 5 x 10 7 1.0x10 8 1.1x10 8 1.2 x 10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8 x 10 8 1.9 x 10 8 2.0x10 8 2.1 x 10 8 2.2 x 10 8 2.3 x 10 8 2.4 x 10 8 2.5 x 10 8 2.6 x 108 2.7 x 10 8 2.8 x 10 8 2.9x10 8 3.0 x 10 8 3.5 x 10 8 4.0 x 10 8 4.5 x 10 8 5 x 10 8 5 x 10 8 Or 1x 10 9 The total number of CD4+ T cells or total active CD4+ T cells is approximately 1 x 10^6. 6 5 x 10 6 1x10 7 5 x 10 7 1.0x 10 8 1.1 x 10 8 1.2x10 8 1.3 x 10 8 1.4 x 10 8 1.5 x 10 8 1.6 x 10 8 1.7 x 10 8 1.8 x 10 8 1.9 x 10 8 2.0x 10 8 2.1x10 8 2.2 x 10 8 2.3 x 10 8 2.4 x 10 8 2.5 x 10 8 2.6 x 10 8 2.7 x 10 8 2.8 x 10 8 2.9 x 10 8 3.0x10 8 3.5 x 10 8 4.0 x 10 8 4.5 x 10 8 5 x 10 8 5.5x10 8 Or 1x 10 9 A mixture or combination of total CD8+ T cells or total live CD8+ T cells is used to produce an input cell composition having a defined ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells. In some embodiments, 1 x 10 6One and 1x 10 10 Between, in 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between or in 1x 10 8 3 x 10 8 The total CD4+ T cells or total active CD4+ T cells are approximately 1 x 10^6 T cells. 6 One and 1x 10 10 Between 1x10 7 One and 1x 10 9 Between 5x10 7 5 x 10 8 Between 1x10 8 3 x 10 8 A mixture or combination of total CD8+ T cells or total live CD8+ T cells of varying amounts is used to produce an input cell composition of CD62L- / CCR7+ / CD4+ T cells with a defined ratio of CD62L- / CCR7+ / CD8+ T cells.

[0181] In certain embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells in the input cell composition has been adjusted, varied, and / or altered compared to the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells in a sample (e.g., a biological sample). In certain embodiments, the adjustment, variation, or alteration of the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells compared to the biological sample is or is about or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1x, 1.5x, 2x, 2.5x, 3x, 4x, 5x, 10x, 20x, 50x, or 100x. In some embodiments, the sample is a sample of cells from which the input cell composition is derived, isolated, selected, and / or obtained.

[0182] In some embodiments, generating, producing, and / or preparing the input cell composition includes one or more of the following steps: mixing or combining cells from a CD4+ T cell composition with cells from a CD8+ T cell composition to produce an input cell composition having a ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells between 2.2:1 and 0.8:1. In certain embodiments, prior to mixing or combining, the number, quantity per volume, quantity per weight, and / or amount, level, or percentage of CD45RA+ / CCR7+ cells in the CD4+ T cell composition and the CD8+ T cell composition are measured, evaluated, and / or quantified. In some embodiments, the quantity, level, number, quantity per volume, quantity per weight, and / or percentage of CD45RA+ / CCR7+ T cells are measured, evaluated, and / or quantified by detecting CD45RA+;CCR7+ T cells. In certain embodiments, the input cell composition has a CD45RA+ / CCR7+ / CD4+ T cell to CD45RA+ / CCR7+ / CD8+ T cell ratio between 2.2:1 and 0.8:1. In some embodiments, the input cell composition has a CD45RA+ / CCR7+ / CD4+ T cell to CD45RA+ / CCR7+ / CD8+ T cell ratio of 1.1:1 or about.

[0183] In some embodiments, generating, producing, and / or preparing the input cell composition includes one or more of the following steps: mixing or combining cells from a CD4+ T cell composition with cells from a CD8+ T cell composition to produce an input cell composition having a ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells between 2.4:1 and 1:1. In certain embodiments, prior to mixing or combining, the number, quantity per volume, quantity per weight, and / or amount, level, or percentage of CD27+ / CCR7+ cells in the CD4+ T cell composition and the CD8+ T cell composition are measured, evaluated, and / or quantified. In some embodiments, the quantity, level, number, quantity per volume, quantity per weight, and / or percentage of CD27+ / CCR7+ cells are measured, evaluated, and / or quantified by detecting CD45RA+;CCR7+ T cells. In certain embodiments, the introduced cell composition has a CD27+ / CCR7+ / CD4+ T cell to CD27+ / CCR7+ / CD8+ T cell ratio between 2.4:1 and 1:1. In some embodiments, the introduced cell composition has a CD27+ / CCR7+ / CD4+ T cell to CD27+ / CCR7+ / CD8+ T cell ratio of 1.69:1 or more.

[0184] In some embodiments, generating, producing, and / or preparing the input cell composition includes one or more of the following steps: mixing or combining cells from a CD4+ T cell composition with cells from a CD8+ T cell composition to produce an input cell composition having a ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells between 2.2:1 and 0.8:1. In certain embodiments, prior to mixing or combining, the number, quantity per volume, quantity per weight, and / or amount, level, or percentage of CD62L- / CCR7+ cells in the CD4+ T cell composition and the CD8+ T cell composition are measured, evaluated, and / or quantified. In some embodiments, the quantity, level, number, quantity per volume, quantity per weight, and / or percentage of CD62L- / CCR7+ T cells are measured, evaluated, and / or quantified by detecting CD62L- / CCR7+ T cells. In certain embodiments, the input cell composition has a CD62L- / CCR7+ / CD4+ T cell to CD62L- / CCR7+ / CD8+ T cell ratio between 2.2:1 and 0.8:1. In some embodiments, the input cell composition has a CD62L- / CCR7 / CD4+ T cell to CD62L- / CCR7 / CD8+ T cell ratio of 1.1:1 or more.

[0185] In some embodiments, generating, producing, and / or preparing the input cell composition includes one or more of the following steps: mixing or combining cells from a CD4+ T cell composition with cells from a CD8+ T cell composition to produce an input cell composition having an immature CD4+ T cell to immature CD8+ T cell ratio between 2.2:1 and 0.8:1. In a particular embodiment, prior to mixing or combining, the number, number per volume, number and / or amount, level, or percentage of immature cells in the CD4+ T cell composition and CD8+ T cell composition are measured, evaluated, and / or quantified. In some embodiments, the amount, level, number, number per volume, number and / or percentage of immature cells are measured, evaluated, and / or quantified by detecting CD45RA+;CCR7+ T cells. In a particular embodiment, the input cell composition has a CD45RA+ / CCR7+ / CD4+ T cell to CD45RA+ / CCR7+ / CD8+ T cell ratio between 2.2:1 and 0.8:1. In some embodiments, the input cell composition has a ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells of about 1.1:1.

[0186] B. Cell activation or stimulation

[0187] In some embodiments, cells are incubated and / or cultured before, in conjunction with, and / or after the genetic engineering process. In some embodiments, cells are incubated and / or cultured immediately before, during, or after one or more steps associated with the genetic engineering protocol or process. Incubation steps may include culturing, nurturing, stimulating, activating, and / or proliferating. Incubation and / or engineering can be performed in culture vessels, such as units, chambers, wells, columns, tubes, tube assemblies, valves, vials, petri dishes, bags, or other containers used for culturing or nurturing 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).

[0188] In some embodiments, the methods provided herein include one or more steps of incubating cells under stimulation or activation conditions. In some embodiments, cell incubation is performed for approximately 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days. In some embodiments, cell incubation is performed for between 5 minutes and 90 minutes, between 1 hour and 4 hours, between 2 hours and 8 hours, between 6 hours and 24 hours, between 12 hours and 48 hours, between 16 hours and 32 hours, between 18 hours and 30 hours, between 1 day and 4 days, or between 2 days and 7 days. In some embodiments, cell incubation lasts between 2 and 15 days, 2 and 12 days, 2 and 10 days, 2 and 8 days, 2 and 6 days, 2 and 4 days, 4 and 12 days, 4 and 10 days, 4 and 8 days, 4 and 6 days, 6 and 12 days, 6 and 10 days, 6 and 8 days, 8 and 12 days, 8 and 10 days, or 10 and 12 days. In some embodiments, cell incubation lasts at least 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.

[0189] In some embodiments, cells are genetically engineered by methods or processes including one or more steps for gene transfer. In some embodiments, gene transfer is performed by first stimulating the cells, such as by incubating the cells under stimulating conditions, for example, incubating the cells with a stimulus that induces responses such as proliferation, survival, and / or activation, followed by gene transfer. In some embodiments, cells are incubated under stimulating conditions prior to gene transfer for the purpose of activating the cells. In some embodiments, gene transfer is or includes the transduction of activated cells. In certain embodiments, the stimulant or condition induces and / or is capable of inducing primary signaling, signal transduction, stimulation, activation, and / or amplification of the cells.

[0190] In some embodiments, cells (e.g., cells into which the cell composition is introduced) are incubated under stimulating or activating conditions prior to gene transfer. In some embodiments, the cells are incubated under stimulating or activating conditions for approximately 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days. In some embodiments, cells into which the cell composition is introduced are incubated under stimulating or activating conditions prior to gene transfer for between 5 minutes and 90 minutes, between 1 hour and 4 hours, between 2 hours and 8 hours, between 6 hours and 24 hours, between 12 hours and 48 hours, between 16 hours and 32 hours, between 18 hours and 30 hours, between 1 day and 4 days, or between 2 days and 7 days. In certain embodiments, cells to which the cell composition is introduced are incubated under stimulating or activating conditions for a duration between 2 and 15 days, 2 and 12 days, 2 and 12 days, 2 and 8 days, 2 and 6 days, 2 and 4 days, 4 and 12 days, 4 and 10 days, 4 and 8 days, 4 and 6 days, 6 and 12 days, 6 and 10 days, 6 and 8 days, 8 and 12 days, 8 and 10 days, or 10 and 12 days. In some embodiments, cells are incubated for a duration of at least 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.

[0191] In some embodiments, cells are incubated under stimulating or activating conditions during some or all of the gene transfer process. For example, in some embodiments, cells are incubated under stimulating conditions during all or part of the process of transducing or transfecting cells to introduce heterologous nucleic acids. In some embodiments, cells are incubated under stimulating or activating conditions in the presence of a viral vector, such as a retroviral vector, like a gamma retroviral vector or a lentiviral vector.

[0192] In certain embodiments, for example, cells are incubated under stimulating or activating conditions immediately after gene transfer. In some embodiments, cells are incubated under stimulating or activating conditions after gene transfer to expand the cells, for example, to expand the cells to a quantity sufficient for clinical application. In some embodiments, cells are incubated under stimulating or activating conditions for a duration of approximately 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days after gene transfer. In some embodiments, before and after gene transfer, the cell composition is incubated under stimulating or activating conditions for a duration between 5 and 90 minutes, 1 and 4 hours, 2 and 8 hours, 6 and 24 hours, 12 and 48 hours, 16 and 32 hours, 18 and 30 hours, 1 and 4 days, or 2 and 7 days. In some embodiments, after gene transfer, the cells of the composition are incubated under stimulating or activating conditions for a duration between 2 and 15 days, 2 and 12 days, 2 and 12 days, 2 and 8 days, 2 and 6 days, 2 and 4 days, 4 and 12 days, 4 and 10 days, 4 and 8 days, 4 and 6 days, 6 and 12 days, 6 and 10 days, 6 and 8 days, 8 and 12 days, 8 and 10 days, or 10 and 12 days. In some implementations, 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 some implementations, the cells are expanded for a sufficient number of cells to produce a quantity suitable for clinical use.

[0193] 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)).

[0194] In some implementations, the stimulating conditions or agents induce and / or are capable of inducing primary signals, signal transduction, stimulation, activation, and / or amplification of cells.

[0195] In some embodiments, the stimulating condition or agent includes one or more agents (e.g., ligands) capable of activating the intracellular signaling domains of the TCR complex. In some aspects, the agent turns on 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 a co-stimulatory receptor, such as 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 (e.g., at a concentration of at least about 0.5 ng / ml) to the culture medium. In some embodiments, the stimulant includes IL-2, IL-15, and / or IL-7. In a particular embodiment, the agent is an antibody that specifically binds to the recombinant receptor. In a particular embodiment, the recombinant receptor is an antigen-binding receptor, such as a CAR, and the agent is an anti-idiotype antibody that specifically binds to the antigen-binding receptor.

[0196] In some embodiments, the agent and / or ligand are attached to magnetic beads, such as paramagnetic beads (e.g., Dynal beads or MACS beads). In some embodiments, the beads can be removed from the cells by placing the cells in a magnetic field, thereby removing and / or separating the beads from the cells, e.g., debeading the cells. In some embodiments, the beads are removed immediately after incubation to activate the cells. In some embodiments, the beads are removed before gene delivery, for example, by transduction or transfection. In some embodiments, the beads are removed during gene delivery or immediately after gene delivery. In certain embodiments, the beads are removed before or during incubation to expand the cells. In some embodiments, the beads are removed before harvesting and / or cryoprotecting the cells.

[0197] 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.

[0198] In some embodiments, T cells are expanded by: adding feeder cells (such as non-dividing peripheral blood mononuclear cells (PBMCs)) to the culture starting composition (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 incubating the culture (e.g., for a duration sufficient to expand the number of said T cells). In some aspects, the non-dividing feeder cells may comprise γ-irradiated PBMC feeder cells. In some embodiments, 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 before the T cell population is added.

[0199] In some embodiments, the stimulation conditions include a temperature suitable for the growth of human T lymphocytes, for example, 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).

[0200] In the implementation scheme, antigen-specific T cells, such as antigen-specific CD4+ and / or CD8+ T cells, are obtained by stimulating naive T lymphocytes 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.

[0201] C. Genetic engineering

[0202] In some embodiments, the provided methods involve generating or producing compositions of engineered cells, such as cell compositions containing cells expressing recombinant antigen receptors. Various methods for introducing genetically engineered components, such as recombinant receptors (e.g., CARs or TCRs), are well known and can be used in conjunction with the provided methods and compositions. Exemplary methods include those for transferring nucleic acids encoding receptors, including via viruses (e.g., retroviruses or lentiviruses), transduction, transposons, and electroporation.

[0203] In some embodiments, cells of an input cell composition are contacted with one or more agents containing nucleic acids (e.g., encoding recombinant receptors such as CARs), and / or said one or more agents are introduced into the cells of the input cell composition. In some embodiments, the agent is a vector, such as a viral vector, plasmid, or transposon. In some embodiments, the cells are CD4+ T cells. In some embodiments, the cells are CD8+ T cells. In certain embodiments, the introduction of nucleic acids into cells and / or contacting the cells with nucleic acids produces engineered cells having a defined ratio, a desired ratio, or a fixed ratio of engineered CD4+ T cells to engineered CD8+ T cells. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in cells or samples obtained from cells, such as nucleic acids obtained from another organism or cell, for example, nucleic acids that are not normally found in the engineered cells and / or the organisms from which such cells are 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.

[0204] In some embodiments, the preparation method includes the step of freezing (e.g., cryopreserving) cells before (e.g., immediately before), during, or after (e.g., immediately after) any step or stage for genetic engineering and / or producing an output cell composition containing genetically engineered cells. In some embodiments, cells are cryopreserved before, during, or after steps such as isolating or selecting cells, mixing or combining cells into an input cell composition, incubating, activating, transferring genes, transducing, transfecting, amplifying, and / or harvesting. In some embodiments, all or a portion of the cells are collected for freezing. In some embodiments, a portion of the cells is collected before, during, or after a stage or step in the process of genetically engineering the cells for subsequent or subsequent analysis, such as analysis after the composition of engineered cells is administered to a subject.

[0205] In some embodiments, freezing and subsequent thawing steps remove granulocytes from the cell population and, to some extent, monocytes. In some embodiments, for example, after a washing step to remove plasma and platelets, the cells are suspended in a freezing solution. In some aspects, any of a variety of known freezing solutions and parameters can be used. 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 a final concentration of 10% DMSO and 4% HSA, respectively. The cells are then typically frozen to -80°C at a rate of 1° / min and stored in the gas phase of a liquid nitrogen tank.

[0206] 1. Vectors and methods for genetic engineering

[0207] In some embodiments, recombinant nucleic acids are transferred to cells using recombinant infectious viral particles (e.g., vectors derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV)). In some embodiments, recombinant nucleic acids are transferred to T cells using recombinant lentiviral vectors or retroviral vectors (e.g., gamma-retroviral vectors) (see, for example, Koste et al. (2014) Gene Therapy 2014 Apr 3. doi:10.1038 / gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10):1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2,e93; Park et al., Trends Biotechnol. 2011 Nov 29(11):550–557. In some embodiments, the virus is adeno-associated virus (AAV).

[0208] 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. The retroviruses are generally amphiphilic, meaning they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the gag, pol, and / or env sequences of the retrovirus. Numerous illustrative retroviral systems have been described (e.g., U.S. Patent Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, AD (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

[0209] Methods of lentiviral transduction are known. Exemplary methods are described in, for example, the following literature: Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101: 1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.

[0210] In some embodiments, the viral vector particle contains a genome derived from a retroviral genome-based vector (such as a lentiviral genome-based vector or a gamma retrovirus-based vector). In some aspects of the provided viral vector, a heterologous nucleic acid encoding a recombinant receptor (e.g., an antigen receptor, such as a CAR) is contained and / or located between the 5' LTR and 3' LTR sequences of the vector genome.

[0211] In some embodiments, the viral vector genome is a lentiviral genome, such as the HIV-1 genome or the SIV genome. For example, lentiviral vectors have been generated by repeatedly attenuating virulence genes, such as by deleting the genes env, vif, vpu, and nef, making the vector safer for therapeutic purposes. Lentiviral vectors are known. See Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Patent Nos. 6,013,516 and 5,994,136. In some embodiments, these viral vectors are plasmid-based or virus-based and are configured to carry a basic sequence for incorporating foreign nucleic acids, for selection, and for transferring said nucleic acids into host cells. Known lentiviruses can be readily obtained from depository institutions or collections such as the American Type Culture Collection (“ATCC”; 10801 University Blvd., Manassas, Va. 20110-2209) or isolated from known sources using common techniques.

[0212] Non-limiting examples of lentiviral vectors include those derived from lentiviruses, such as human immunodeficiency virus 1 (HIV-1), HIV-2, simian immunodeficiency virus (SIV), human T-lymphotropic virus 1 (HTLV-1), HTLV-2, or equine infection anemia virus (E1AV). Lentiviral vectors have been produced, for example, by repeatedly attenuating HIV virulence genes, such as by deleting the genes env, vif, vpr, vpu, and nef, making the vectors safer for therapeutic purposes. Lentiviral vectors are known in the art; see Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Patent Nos. 6,013,516 and 5,994,136. In some embodiments, these viral vectors are plasmid-based or virus-based and are configured to carry basic sequences for incorporating foreign nucleic acids, for selection, and for transferring said nucleic acids into host cells. Known lentiviruses can be readily obtained from depository institutions or collections such as the American Type Culture Collection (“ATCC”; 10801 University Blvd., Manassas, Va. 20110-2209) or isolated from known sources using common techniques.

[0213] In some implementations, the viral genome vector may contain sequences of the 5' and 3' LTRs of a retrovirus (e.g., lentivirus). In some aspects, the viral genome construct may contain sequences of the 5' and 3' LTRs from a lentivirus, and specifically may contain the R and U5 sequences of the 5' LTR from the lentivirus and an inactivated or self-inactivated 3' LTR from the lentivirus. The LTR sequence can be an LTR sequence from any lentivirus of any species. For example, they can be LTR sequences from HIV, SIV, FIV, or BIV. Typically, the LTR sequence is an HIV LTR sequence.

[0214] In some embodiments, the nucleic acid of the viral vector (e.g., the HIV viral vector) lacks an additional transcription unit. The vector genome may contain an inactivated or self-inactivated 3'LTR (Zufferey et al., J Virol 72: 9873, 1998; Miyoshi et al., J Virol 72: 8150, 1998). For example, a deletion in the U3 region of the 3'LTR of the nucleic acid used to generate viral vector RNA can be used to generate a self-inactivated (SIN) vector. This deletion can then be transferred to the 5'LTR of the proviral DNA during reverse transcription. Self-inactivated vectors typically have deletions of enhancer and promoter sequences from the 3' long terminal repeat (LTR), which are copied into the 5'LTR during vector integration. In some embodiments, sufficient sequences, including removal of the TATA box, can be eliminated to eliminate the transcriptional activity of the LTR. This prevents the generation of full-length vector RNA in transduced cells. In some aspects, the U3 element of the 3'LTR contains deletions of its enhancer sequence, TATA box, Sp1, and NF-κB sites. Due to the self-inactivation of the 3'LTR, the provirus generated after entry and reverse transcription contains an inactivated 5'LTR. This can improve safety by reducing the risk of mobilizing the vector genome and the effect of the LTR on nearby cellular promoters. The self-inactivating 3'LTR can be constructed using any method known in the art. In some embodiments, this does not affect the vector titer or the in vitro or in vivo properties of the vector.

[0215] Optionally, the U3 sequence from the lentiviral 5'LTR can be replaced in the viral construct with a promoter sequence (e.g., a heterologous promoter sequence). This can increase the titer of the virus recovered from the packaging cell line. Enhancer sequences may also be included. Any enhancer / promoter combination that increases viral RNA genome expression in the packaging cell line can be used. In one example, a CMV enhancer / promoter sequence (US Patent Nos. 5,385,839 and 5,168,062) is used.

[0216] In some embodiments, the risk of insertional mutagenesis can be minimized by constructing the retroviral vector genome (e.g., a lentiviral vector genome) as integration-deficient. Various approaches can be used to generate non-integrating vector genomes. In some embodiments, one or more mutations can be engineered into the integrase component of the pol gene, such that it encodes a protein with an inactive integrase. In some embodiments, the vector genome itself can be modified to prevent integration by, for example, mutating or deleting one or two attachment sites, or by deleting or modifying the 3'LTR proximal polypurine bundle (PPT) to render it non-functional. In some embodiments, non-genetic approaches can be used; these approaches include pharmacological agents that inhibit one or more functions of the integrase. These approaches are not mutually exclusive; that is, more than one of the aforementioned approaches can be used at a time. For example, neither the integrase nor the attachment site may be functional, or neither the integrase nor the PPT site may be functional, or neither the attachment site nor the PPT site may be functional, or both may be non-functional. Such methods and viral vector genomes are known and available (see Philpott and Thrasher, Human Gene Therapy 18:483, 2007; Engelman et al. J Virol 69:2729, 1995; Brown et al. J Virol 73:9011 (1999); WO 2009 / 076524; McWilliams et al., J Virol 77:11150, 2003; Powell and Levin J Virol 70:5288, 1996).

[0217] In some embodiments, the provided method relates to transducing cells by contacting (e.g., incubating) a cell composition containing multiple cells with viral particles. In some embodiments, the cells to be transfected or transduced are or contain primary cells obtained from a subject, such as cells enriched and / or selected from the subject.

[0218] In some embodiments, the concentration of cells to be transduced in the composition is from or from about 1.0 x 10⁻⁶. 5 cells / mL to 1.0 x 10⁻⁶ 8 Cells / mL, for example, at least or about 1.0 x 10⁻⁶. 5 cells / mL, 5 x 10 5 cells / mL, 1x10 6 cells / mL, 5 x 10 6 cells / mL, 1 x 10 7 cells / mL, 5 x 10 7 1 cell / mL or 1 x 10 8 Cells / mL.

[0219] In some embodiments, the viral particles are provided at a ratio (IU / cell) of viral vector particle copies or infectious units (IU) to the total number of cells to be transduced. For example, in some embodiments, the viral particles are present during contact at approximately or at least 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or 60 IU of viral vector particles per cell.

[0220] In some implementations, the titer of the viral vector particles is at or around 1 x 10⁻⁶. 6 IU / mL and 1x10 8 Between IU / mL, for example at or around 5 x 10⁻⁶. 6 IU / mL and 5x 10 7 Between IU / mL, for example at least 6 x 10⁻⁶ 6 IU / mL, 7 x 10 6 IU / mL, 8 x 10 6 IU / mL, 9 x 10 6 IU / mL, 1x 10 7 IU / mL, 2 x 10 7 IU / mL, 3 x 10 7 IU / mL, 4 x 10 7 IU / mL or 5x10 7 IU / mL.

[0221] In some implementations, transduction can be achieved at a multiplicity of infection (MOI) of less than 100, for example typically less than 60, 50, 40, 30, 20, 10, 5 or smaller.

[0222] In some embodiments, the method involves contacting or incubating cells with viral particles. In some embodiments, the contact may last from 30 minutes to 72 hours, such as 30 minutes to 48 hours, 30 minutes to 24 hours, or 1 hour to 24 hours, such as at least or about 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, or longer.

[0223] In some embodiments, contact can be achieved by centrifugation, such as rotational inoculation (e.g., centrifugal inoculation). In some embodiments, the composition containing cells, virus particles, and reagents can be rotated, typically at a relatively low force or speed, for example, a speed lower than that used for cell precipitation, such as about 600 rpm to 1700 rpm (e.g., about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm). In some embodiments, rotation is performed with a force (e.g., relative centrifugal force) of about 100 g to 3200 g (e.g., about or at least about 100 g, 200 g, 300 g, 400 g, 500 g, 1000 g, 1500 g, 2000 g, 2500 g, 3000 g, or 3200 g), as measured, for example, at the inner or outer wall of a chamber or cavity. The term "relative centrifugal force" or RCF is generally understood as the effective force exerted on an object or substance (e.g., a cell, sample, or granule, and / or a point in a rotating chamber or other container) at a specific point in space, relative to the Earth's gravity, such as relative to the axis of rotation. This value can be determined using well-known formulas that take into account gravity, the speed of rotation, and the radius of rotation (distance from the axis of rotation and the object, substance, or particle on which the RCF is measured).

[0224] In some implementations, the input cells are treated, incubated, or contacted with particles containing binding molecules that bind to or recognize recombinant receptors encoded by viral DNA.

[0225] In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, for example, Chicaybam et al., (2013) PLoS ONE 8(3):e60298; and Van Tedeloo et al., (2000) Gene Therapy 7(16):1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, for example, Manuri et al., (2010) Hum Gene Ther 21(4):427-437; Sharma et al., (2013) Molec Ther Nucl Acids 2,e74; and Huang et al., (2009) Methods Mol Biol 506:115-126). Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-promoted microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)).

[0226] 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.

[0227] In some embodiments, recombinant nucleic acids are transferred to T cells via transposons. Examples of transposons suitable for use with mammalian cells (e.g., human primary leukocytes) include, but are not limited to, Sleeping Beauty and Piggybac. Transposon-based transfection is a two-component system consisting of a transposase and a transposon. In some embodiments, the transposon-containing system is engineered to contain foreign DNA (also referred to herein as cargo DNA) (e.g., a gene encoding a recombinant receptor) flanked by inverted / reverse repeat (IR / DR) sequences recognized by the accompanying transposon. In some embodiments, a nonviral plasmid encodes a transposon under promoter control. In some embodiments, transfection of the plasmid into a host cell results in transient expression of the transposon at a level sufficient to integrate the transposon into the genomic DNA.

[0228] Sleeping Beauty (SB) is a synthetic member of the Tc / 1-Sailor superfamily of transposons, reconstructed from a dormant element present in the salmonid genome. SB transposon transfection is a two-component system consisting of a transposon containing an inverted / forward repeat (IR / DR) sequence that leads to precise integration into a TA dinucleotide. The transposon is engineered with a target expression cassette flanked by IR / DR sequences. The SB transposon binds to a specific binding site located on the IR of the Sleeping Beauty transposon. The SB transposon mediates the integration of the transposon, a mobile element encoding a cargo sequence flanked by inverted terminal repeat sequences with catalytic enzyme (SB) binding sites. Stable expression is achieved when the SB inserts the gene sequence into the vertebrate chromosome at the TA target dinucleotide via a cut-and-paste mechanism. This system has been used to engineer various vertebrate cell types, including human primary peripheral blood leukocytes. In some embodiments, cells are contacted with, incubated with, and / or treated with SB transposons containing a cargo gene (e.g., a gene encoding a recombinant receptor or CAR) flanked by an SB IR sequence. In certain embodiments, cells to be transfected are contacted with, incubated with, and / or treated with a plasmid containing an SB transposon containing a cargo gene (e.g., a gene encoding a CAR) flanked by an SB IR sequence. In some embodiments, the plasmid also contains a gene encoding an SB transposase without an SB IR sequence flanking it.

[0229] PiggyBac (PB) is another transposon system that can be used to integrate cargo DNA into the genomic DNA of a host (e.g., human). PB transposases recognize PB transposon-specific inverted terminal repeats (ITRs) located at both ends of the transposon and efficiently remove the contents from the original site and efficiently integrate the contents into the TTAA chromosomal site. The PB transposon system enables the movement of a target gene between the two ITRs in a PB vector into the target genome. The PB system has been used to engineer various vertebrate cell types, including primary human cells. In some embodiments, cells to be transfected are contacted with, incubated with, and / or treated with PB transposons containing a cargo gene (e.g., a gene encoding a CAR) flanked by PB IR sequences. In certain embodiments, cells to be transfected are contacted with a plasmid containing a PB transposon, incubated with a plasmid containing a PB transposon, and / or treated with a plasmid containing a PB transposon that contains a cargo gene (e.g., a gene encoding a CAR) flanked by a PB IR sequence. In some embodiments, the plasmid also contains a gene encoding an SB transposase that is not flanked by a PB IR sequence.

[0230] In some embodiments, transduction with transposons is performed using a plasmid containing a transposase gene and a plasmid containing a transposon, the transposon containing a cargo DNA sequence flanked by an inverted / forward repeat (IR / DR) sequence recognized by the transposase. In some embodiments, the cargo DNA sequence encodes a heterologous protein, such as a recombinant T-cell receptor or CAR. In some embodiments, the plasmid contains both a transposase and a transposon. In some embodiments, the transposase is under the control of a pervasive promoter or any promoter suitable for driving the expression of the transposase in target cells. Pervasive promoters include, but are not limited to, EF1a, CMB, SV40, PGK1, Ubc, human β-actin, CAG, TRE, UAS, Ac5, CaMKIIa, and U6. In some embodiments, the cargo DNA contains a selection cassette that allows selection of cells that stably integrate the cargo DNA into the genomic DNA. Suitable selection boxes include, but are not limited to, those encoding the following selection boxes: kanamycin resistance gene, spectinomycin resistance gene, streptomycin resistance gene, ampicillin resistance gene, carbenicillin resistance gene, hygromycin resistance gene, bleomycin resistance gene, erythromycin resistance gene, and polymyxin B resistance gene.

[0231] In some implementations, the components for transposon transduction (e.g., plasmids containing SB transposase and SB transposons) are introduced into the target cells. Any convenient protocol can be used, depending on the location of the target cells, to introduce the system components into the target cells in vitro or in vivo. For example, when the target cells are isolated cells, the system can be introduced directly into the cells, for example, using standard transformation techniques under cell culture conditions that allow for target cell viability. Such techniques include, but are not limited to, viral infection, transformation, conjugation, protoplast fusion, electroporation, particle gun techniques, calcium phosphate precipitation, direct microinjection, viral vector delivery, etc. The choice of method generally depends on the type of cells to be transformed and the environment in which the transformation occurs (i.e., in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

[0232] In some embodiments, the SB transposon and SB transposase source are introduced into target cells of a multicellular organism (e.g., a mammal or a human) under conditions sufficient to cleave the inverted repeat flanking nucleic acid from the vector carrying the transposon and subsequently integrate the cleaved nucleic acid into the genome of the target cell. Some embodiments also include the step of ensuring that the necessary transposase activity is present in the target cell along with the introduced transposon. Depending on the structure of the transposon vector itself, i.e., whether the vector includes a region encoding a product having transposase activity, the method may also include introducing a second vector into the target cell encoding the necessary transposase activity.

[0233] In some implementations, the amount of vector nucleic acid containing the transposon and the amount of vector nucleic acid encoding the transposase introduced into the cell are sufficient to provide the necessary cleavage of the transposon nucleic acid and insertion into the target cell genome. Thus, the amount of vector nucleic acid introduced should provide sufficient transposase activity and a sufficient copy number of the desired insertion into the target cell. The amount of vector nucleic acid introduced into the target cell varies depending on the efficiency of the specific introduction protocol employed (e.g., the specific in vitro administration protocol).

[0234] Once the vector DNA, combined with the necessary transposase, enters the target cell, the vector nucleic acid region flanked by inverted repeat sequences (i.e., the vector nucleic acid located between inverted repeat sequences recognized by the Sleeping Beauty transposase) is cleaved from the vector via the provided transposase and inserted into the genome of the target cell. Thus, the vector DNA is introduced into the target cell and subsequently subjected to transposase-mediated excision, and the foreign nucleic acid carried by the vector is inserted into the genome of the target cell. In certain embodiments, the vector integrates into at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, or at least 20% of the genome of cells transfected with SB transposons and / or SB transposase. In some embodiments, the integration of the nucleic acid into the target cell genome is stable; that is, the vector nucleic acid remains present in the target cell genome for more than a transient period, and a portion of the chromosomal genetic material is transferred to the offspring of the target cell.

[0235] In some embodiments, transposons are used to integrate nucleic acids (i.e., polynucleotides) of various sizes into the target cell genome. In some embodiments, the size of the DNA inserted into the target cell genome using a subject-matter approach ranges from about 0.1 kb to 200 kb, from about 0.5 kb to 100 kb, from about 1.0 kb to about 8.0 kb, from about 1.0 kb to about 200 kb, from about 1.0 kb to about 10 kb, from about 10 kb to about 50 kb, from about 50 kb to about 100 kb, or from about 100 kb to about 200 kb. In some embodiments, the size of the DNA inserted into the target cell genome using a subject-matter approach ranges from about 1.0 kb to about 8.0 kb. In some embodiments, the size of the DNA inserted into the target cell genome using a subject-matter approach ranges from about 1.0 kb to about 200 kb. In a particular embodiment, the size of the DNA inserted into the target cell genome using a subject-matter approach ranges from about 1.0 kb to about 8.0 kb.

[0236] 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, to introduce the gene for the desired receptor, can be performed using any suitable retroviral vector. The genetically modified cell population can then be evacuated from the initial stimulus (e.g., an anti-CD3 / anti-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 antigenic stimuli in the form of peptide / MHC molecules, homologous (crosslinked) ligands of the genetically introduced receptor (e.g., natural ligands 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., “Chimeric antigen receptors for T-cell based therapy” Methods Mol Biol. 2012; 907:645-66; or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine, Vol. 65:333-347 (2014).

[0237] 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.

[0238] Other 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 sensitive 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 selective fusion genes derived by fusing a dominant positive-selective marker with a negative-selective marker. See, for example, Riddell et al., U.S. Patent No. 6,040,177, columns 14-17.

[0239] 2. Recombinant receptor

[0240] In some embodiments, the cells used in or in combination with the provided methods contain or are engineered to contain engineered receptors, such as engineered antigen receptors (e.g., chimeric antigen receptors (CARs)) or T-cell receptors (TCRs). Populations of such cells, compositions containing such cells and / or enriched with such cells are also provided, such as those enriched with or selected for a particular cell type, such as T cells or CD8+ or CD4+ T cells. The compositions include pharmaceutical compositions and formulations for administration (e.g., for adoptive cell therapy). Methods for administering cells and compositions to a subject (e.g., a patient) according to the provided methods and / or with the provided products or compositions are also provided.

[0241] In some embodiments, the cells include one or more nucleic acids introduced through genetic engineering, thereby expressing recombinant or genetically engineered products of such nucleic acids. In some embodiments, gene transfer is accomplished by first stimulating the cells, such as by combining the cells with a stimulus that induces a response (e.g., proliferation, survival, and / or activation, as measured, for example, by the expression of cytokines or activation markers), then transducing the activated cells, and expanding them in culture to a quantity sufficient for clinical application.

[0242] The cells typically express recombinant receptors, such as antigen receptors (including functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs)) and other antigen-binding receptors (such as transgenic T-cell receptors (TCRs)). Other chimeric receptors are also included.

[0243] a. Chimeric antigen receptor (CAR)

[0244] In some embodiments of the provided methods and uses, the chimeric receptor (such as a chimeric antigen receptor) contains one or more domains that combine a ligand-binding domain (e.g., an antibody or antibody fragment) specific for a desired antigen (e.g., a tumor antigen) with an intracellular signaling domain. In some embodiments, the intracellular signaling domain is an activating intracellular domain portion, such as a T-cell activation domain, thereby providing a primary activation signal. In some embodiments, the intracellular signaling domain contains or additionally contains a co-stimulatory signaling domain to promote effector function. In some embodiments, when the chimeric receptor is genetically engineered into immune cells, it can modulate T-cell activity and, in some cases, T-cell differentiation or homeostasis, thereby producing genetically engineered cells with improved lifespan, survival, and / or persistence in vivo, such as for adoptive cell therapy.

[0245] 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, the antigen 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.

[0246] In a particular embodiment, 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-ζ (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).

[0247] In some embodiments, the chimeric receptor further comprises 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.

[0248] Exemplary antigen receptors (including CARs) and methods for engineering such receptors and introducing them into cells include, for example, International Patent Application Publication Nos. WO 200014257, WO 2013126726, WO2012 / 129514, WO 2014031687, WO2013 / 166321, WO 2013 / 071154, WO2013 / 123061; and U.S. Patent Application Publication No. US 2002131960, US2013287748, US20130149337; 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 those described in European Patent Application No. EP 2537416, and / or 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.

[0249] In some embodiments, the CAR is constructed to be specific for 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).

[0250] 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). The antigen receptor includes 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, an 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 signaling via a natural antigen receptor (such as a TCR), and optionally via a combination of such receptors and co-stimulatory receptors.

[0251] In some embodiments, the recombinant receptor (such as a chimeric receptor (e.g., CAR)) includes a ligand-binding domain that binds (e.g., specifically to) an antigen (or ligand). The antigens targeted by the chimeric receptor include antigens expressed in the context of a disease, condition, or cell type targeted via adoptive cell therapy. The 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.

[0252] In some embodiments, the antigen (or ligand) is a polypeptide. In some embodiments, the antigen is a carbohydrate or other molecule. In some embodiments, the antigen (or ligand) is selectively expressed or overexpressed on cells of the disease or condition (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.

[0253] In some embodiments, the CAR contains an antibody or antigen-binding fragment (e.g., scFv) that specifically recognizes an antigen (e.g., an intact antigen) expressed on the cell surface.

[0254] In some embodiments, the antigen (or ligand) is a tumor antigen or cancer marker. In some embodiments, the antigen (or ligand) is or includes orphan tyrosine kinase receptor (ROR1), B-cell maturation antigen (BCMA), carbonic anhydrase 9 (CA9, also known as CAIX or G250), Her2 / neu (receptor tyrosine kinase erbB2), CD19, CD20, CD22, mesothelin (MSLN), carcinoembryonic antigen (CEA), and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, chondroitin sulfate proteoglycan 4 (CSPG4), EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), liver glycoprotein receptor A2 (EPHa2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, epidermal growth factor receptor type III mutant (EGFR). vIII), folate-binding protein (FBP), Fc receptor-like 5 (FCRL5, also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), ganglioside GD2, ganglioside GD3, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor α (IL-22Rα), kinase insertion domain receptor (kdr), κ light chain, protein family 8 member A containing leucine-rich repeat sequences (LRRC8A), Lewis Y, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, melanoma preferentially expressed antigen (PRAME), survival protein, tumor-associated glycoprotein 72 (TAG) 72), B7-H3, B7-H6, IL-13 receptor α2 (IL-13Rα2), human high molecular weight melanoma-associated antigen (HMW-MAA), CD171, folate receptor-α, CD44v7 / 8, αvβ6 integrin (avb6 integrin), 8H9, neural cell adhesion molecule (NCAM), vascular endothelial growth factor receptor (VEGF receptor or VEGFR), trophoblast glycoprotein (TPBG, also known as 5T4), NKG2D ligand, CD44v6, dual antigens, cancer-testis antigen, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), natural killer cell family 2 member D (NKG2D) ligand, cancer / testis antigen 1B (CTAG),Also known as NY-ESO-1 and LAGE-2), melanin A (MART-1), glycoprotein 100 (gp100), phosphatidylinositol proteoglycan-3 (GPC3), G protein-coupled receptor 5D (GPRC5D), carcinoembryonic antigen, 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 2 (VEGF-R2), carcinoembryonic antigen (CEA) The receptor-targeted antigens include estrogen receptors, progesterone receptors, prostate-specific antigens, hepatin B2, CD123, CD133, c-Met, O-acetylated GD2 (OGD2), the CE7 epitope of L1-CAM, Wilms tumor 1 (WT-1), cyclins, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD138, pathogen-specific antigens or pathogen-expressed antigens, and 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 receptor-targeted antigens are CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igκ, Igλ, CD79a, CD79b, or CD30.

[0255] In some embodiments, the antigen is a pathogen-specific antigen. 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.

[0256] 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, for example, as disclosed in U.S. Patent Publication No. US 2016 / 0152723.

[0257] 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 contains CDRH1 and H2 as shown in SEQ ID NO:38 and 39, respectively, and CDRH3 as shown in SEQ ID NO:40 or 54, and CDRL1 as shown in SEQ ID NO:35, and CDR L2 36 or 55, and CDR L3 sequence 37 or 34. 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 shown in SEQ ID NO:56. In some embodiments, the scFv sequentially comprises V H , connectors and V L In some implementations, scFv sequentially includes V L , connectors and V H 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% identity with the sequence of 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% identity with the sequence of SEQ ID NO:43.

[0258] In some implementations, scFv is derived from SJ25C1. SJ25C1 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 SJ25C1 antibody contains the CDRH1, H2, and H3 sequences shown in SEQ ID NO:47-49, and the 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 shown in SEQ ID NO:52. In some embodiments, the scFv sequentially comprises V H , connectors and V L In some implementations, scFv sequentially includes V L , connectors and V H In some embodiments, scFv comprises the amino acid sequence shown in SEQ ID NO:53 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:53.

[0259] 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 the antibody or antibody fragment shown in International Patent Application Publication Nos. WO 2016 / 090327 and WO 2016 / 090320.

[0260] 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 the antibody or antibody fragment shown in International Patent Application Publications WO 2016 / 090329 and WO 2016 / 090312.

[0261] 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). The antigen receptor includes 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.

[0262] The term "major histocompatibility complex" (MHC) refers to a protein, typically a glycoprotein, containing a polymorphic peptide-binding site or binding groove. In some cases, this protein can complex with peptide antigens of polypeptides, including peptide antigens processed by cellular structures. In some cases, MHC molecules can be displayed or expressed on cell surfaces, including as peptide-peptide complexes, i.e., MHC-peptide complexes, for presenting antigens with conformations that can be recognized 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 the effective portion of the MHC containing antigen-binding sites or sites for binding peptides, as well as the sequence required for recognition by appropriate antigen receptors. In some embodiments, MHC class I molecules deliver cytosol-derived peptides to the cell surface, where the MHC-peptide complex is recognized by T cells (typically CD8+ T cells, but in some cases CD4+ T cells). In some embodiments, MHC class II molecules deliver peptides derived from the vesicle system to the cell surface, where the peptides are typically recognized by CD4+ T cells. Generally, MHC molecules are encoded by a set of linked loci collectively referred to as H-2 in mice and as human leukocyte antigens (HLA) in humans. Therefore, human MHC can also be referred to as human leukocyte antigens (HLA).

[0263] 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 a 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).

[0264] 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 a fragment of a longer biomolecule (such as a polypeptide or protein). 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 background 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 a T cell, inducing a T cell response, such as T cell proliferation, cytokine production, cytotoxic T cell response, or other responses.

[0265] In some embodiments, the TCR-like antibody or antigen-binding moiety is known or can be generated by known methods (see, for example, U.S. 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. WO03 / 068201).

[0266] 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, such as 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 elicit an immune response, wherein the immunogen remains in its three-dimensional form for a period of time sufficient to elicit an immune response against the three-dimensional presentation 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 presentation of the peptide in the binding groove of the MHC molecule has been produced. In some embodiments, the produced antibody can be evaluated to confirm that the antibody can distinguish the MHC-peptide complex from a single MHC molecule, a single target peptide, and a complex of MHC with an unrelated peptide. The desired antibody can then be isolated.

[0267] 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, where 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.

[0268] 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 antigen-binding fragments (Fab), F(ab')2, Fab', Fv, recombinant IgG (rIgG) fragments, and variable heavy chains (V) capable of specifically binding antigens. HThis 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).

[0269] In some embodiments, the antigen-binding protein, antibody, and its antigen-binding fragment 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.

[0270] 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.

[0271] 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 VH 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).

[0272] A single-domain antibody is an antibody fragment containing all or part of the variable heavy chain domain or all or part of the variable light chain domain of an antibody. 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.

[0273] Antibody fragments can be prepared using a variety of techniques, including but not limited to the 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.

[0274] 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 a 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.

[0275] Therefore, in some embodiments, the chimeric antigen receptor (including TCR-like CARs) includes an extracellular portion containing an antibody or antibody fragment. In some embodiments, the antibody or fragment includes scFv. In some aspects, the chimeric antigen receptor includes 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).

[0276] 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 Fc region. In some embodiments, the recombinant receptor further comprises a spacer and / or hinge region. In some embodiments, the constant region or portion is of human IgG (such as IgG4 or IgG1). In some aspects, the portion of the constant region serves as a spacer between an antigen recognition component (e.g., scFv) and a transmembrane domain. The length of the spacer can provide enhanced cellular reactivity after antigen binding compared to the absence of a spacer. In some examples, the length of the spacer is 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 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.

[0277] 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:23-31.

[0278] In some embodiments, the antigen receptor includes an intracellular domain directly or indirectly connected to an extracellular domain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain connecting an extracellular domain to an intracellular signaling domain. In some embodiments, the intracellular signaling domain includes an ITAM. For example, in some aspects, an antigen recognition domain (e.g., an extracellular domain) is connected to one or more intracellular signaling components, such as signaling components that mimic activation via an antigen receptor complex (e.g., a TCR complex) and / or signal transduction via another cell surface receptor domain in the case of CAR. In some embodiments, the chimeric receptor includes a transmembrane domain connected or fused between an extracellular domain (e.g., scFv) and an intracellular signaling domain. Thus, in some embodiments, the antigen-binding component (e.g., an antibody) is connected to one or more transmembrane and intracellular signaling regions. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain naturally associated with one of the domains of the receptor (e.g., CAR) is used. In some cases, the transmembrane domains are selected or modified by amino acid substitution to prevent such domains from binding to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0279] 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 one or more of the following): 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 comprises hydrophobic residues such as leucine and valine. In some respects, a triplet of phenylalanine, tryptophan, and valine is 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. In some respects, the transmembrane domain contains a transmembrane portion of CD28.

[0280] In some embodiments, 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 receptor contains an extracellular portion of a molecule from which the transmembrane domain is derived, such as the extracellular portion of CD28.

[0281] The intracellular signal transduction domains include those that mimic or approximate signals via natural antigen receptors, via a combination of such receptors and co-stimulatory receptors, and / or via co-stimulatory receptors alone. In some embodiments, short oligopeptide or polypeptide linkers (e.g., linkers with a length between 2 and 10 amino acids, such as linkers containing glycine and serine, e.g., glycine-serine duplexes) are present and form a connection between the transmembrane domains of the CAR and the cytoplasmic signal transduction domains.

[0282] The receptor (e.g., a CAR) typically includes 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., a 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-ζ (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

[0283] In some embodiments, upon CAR conjugation, the cytoplasmic domain or intracellular signaling domain of the receptor activates 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 cell lysis activity or T helper activity, such as the secretion of cytokines or other factors. In some embodiments, a truncated portion (e.g., if it transduces effector function signals) of the intracellular signaling region of an antigen receptor component or co-stimulatory molecule is used instead of the complete immune stimulation chain. In some embodiments, one or more intracellular signaling domains comprise a cytoplasmic sequence of a T cell receptor (TCR) and, in some respects, also include co-receptors (which, in their natural context, synergize with such receptors 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.

[0284] In the context of a natural TCR, full activation typically requires not only signal transduction via the TCR but also co-stimulatory signals. Therefore, in some embodiments, the CAR includes components for generating secondary or co-stimulatory signals to promote full 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.

[0285] 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, the CAR includes one or both of these signaling components.

[0286] In some aspects, the 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 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.

[0287] In some embodiments, the CAR includes a signaling 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 a signaling region and a co-stimulatory component. In some embodiments, the chimeric antigen receptor contains an intracellular domain derived from a T-cell co-stimulatory molecule or a functional variant thereof, such as one located between the transmembrane domain and the intracellular signaling domain. In some aspects, the T-cell co-stimulatory molecule is CD28 or 41BB.

[0288] In some embodiments, the signal transduction region and / or activation domain are included within a CAR, while the co-stimulatory component is provided by another CAR that recognizes a different antigen. In some embodiments, the CAR comprises an activating or stimulating CAR and a co-stimulating CAR expressed on the same cell (see WO 2014 / 055668). In some aspects, the cell comprises one or more stimulating or activating CARs and / or co-stimulating CARs. In some embodiments, the cell also includes an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5(215)(December 2013)), such as a CAR that recognizes antigens other than those associated with and / or specific to the disease or condition, thereby reducing or inhibiting the activation signal delivered by the disease-targeting CAR through binding to its ligand, for example, to reduce off-target effects.

[0289] In some embodiments, the intracellular signaling domain includes a CD28 transmembrane and signaling domain connected to the CD3 (e.g., CD3-ζ) intracellular domain. In some embodiments, the intracellular signaling domain includes a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domain connected to the CD3ζ intracellular domain.

[0290] In some embodiments, 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.

[0291] In some embodiments, the antigen receptor further includes a label, and / or cells expressing CAR or other antigen receptors further include alternative labels such as cell surface labels, which can be used to confirm that the cells have been transduced or engineered to express the receptor. In some aspects, the label includes all or part (e.g., truncated forms) of CD34, NGFR, or epidermal growth factor receptors, such as truncated forms of cell surface receptors (e.g., tEGFR). Exemplary alternative labels may include truncated forms of cell surface peptides, such as non-functional and non-transducing or unable to transduce signals or signals normally transduced by the full-length form of the cell surface peptide, and / or non-internalized or unable to internalize truncated forms. 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 cetuximab. Or other therapeutic anti-EGFR antibodies or binding molecules that recognize epitopes that can be used to identify or select cells engineered with tEGFR constructs and the encoded exogenous protein, and / or to eliminate or isolate cells expressing the encoded exogenous protein. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. April 2016; 34(4):430-434. In some aspects, the marker (e.g., alternative markers) includes all or part (e.g., truncated forms) of CD34, NGFR, CD19, or truncated CD19 (e.g., truncated non-human CD19) or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the marker is or comprises a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP, such as superfolded GFP (sfGFP)), red fluorescent protein (RFP, such as 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 (such as luciferase), the lacZ gene from *E. coli*, alkaline phosphatase, secreted 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.

[0292] 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.

[0293] 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.

[0294] 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., an MMP cleavable adapter sequence). For example, the tag and optionally the adapter sequence can be any of those disclosed in published patent application number WO 2014031687. For example, the tag can be a truncated EGFR (tEGFR) optionally linked to an adapter sequence, such as a T2A cleavable adapter sequence.

[0295] In some embodiments, the marker is a molecule (e.g., a cell surface protein) or a portion thereof that is not naturally found on or on the surface of T cells. In some embodiments, the molecule is a non-self molecule, such as a non-self protein, i.e., a molecule that is not recognized as "self" by the host's immune system, and the cell will be adopted into the host.

[0296] In some embodiments, the marker does not have any therapeutic effect and / or produces no effect other than serving as a marker for genetic engineering (e.g., for selecting successfully engineered cells). In other embodiments, the marker may be a therapeutic molecule or a molecule that otherwise performs some desired function, such as a ligand for cells to be encountered in vivo, such as a co-stimulatory or immune checkpoint molecule, to enhance and / or attenuate the cell's response upon adoptive transfer and encounter with the ligand.

[0297] 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.

[0298] In some embodiments, the chimeric antigen receptor includes an extracellular portion containing an antibody or antibody fragment as described herein. In some aspects, the chimeric antigen receptor includes an extracellular portion containing an antibody or fragment as described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment includes scFv or a single-domain V. H The antibody, and the intracellular domain contains 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.

[0299] In some aspects, the transmembrane domain contains a transmembrane portion of CD28. The extracellular domain and the transmembrane may be directly or indirectly connected. In some embodiments, the extracellular domain and the transmembrane are connected by a spacer (as described herein). In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T-cell costimulatory molecule, such as between the transmembrane domain and the intracellular signal transduction domain. In some aspects, the T-cell costimulatory molecule is CD28 or 4-1BB.

[0300] 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 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 4-1BB or a functional variant thereof and a signaling portion of CD3ζ or a functional variant thereof. In some such embodiments, the receptor further includes a spacer containing a portion of an Ig molecule (such as a human Ig molecule) (such as an Ig hinge, such as an IgG4 hinge), such as a hinge-only spacer.

[0301] In some embodiments, the transmembrane domain of the receptor (e.g., CAR) is a 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 comprising the amino acid sequence shown in SEQ ID NO:8 or exhibiting amino acid sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of SEQ ID NO:8; in some embodiments, the transmembrane domain containing a portion of the recombinant receptor comprises 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 of SEQ ID NO:9.

[0302] In some embodiments, 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.

[0303] In some embodiments, the intracellular signaling region comprises an intracellular co-stimulatory 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.

[0304] 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 AAs 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.

[0305] 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.

[0306] For example, in some embodiments, a CAR includes an antibody (such as an antibody fragment, including scFv), a spacer (such as a spacer containing a portion of an immunoglobulin molecule (such as a hinge region and / or one or more constant regions of a heavy chain molecule), such as a spacer containing an Ig hinge), a transmembrane domain containing all or part of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3ζ signaling domain. In some embodiments, a CAR includes an antibody or fragment (such as scFv), a spacer (such as any spacer containing an Ig hinge), a CD28-derived transmembrane domain, a 4-1BB-derived intracellular signaling domain, and a CD3ζ-derived signaling domain.

[0307] In some embodiments, the nucleic acid molecule encoding such a CAR construct further includes, for example, a sequence encoding a T2A ribosomal jumping element and / or a tEGFR sequence downstream of the CAR-encoding sequence. In some embodiments, the sequence encodes the T2A ribosomal jumping element shown in SEQ ID NO:6 or 17, 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:6 or 17. In some embodiments, T cells expressing an antigen receptor (e.g., CAR) can also be generated to express a truncated EGFR (EGFRt) as a non-immunogenic selective epitope (e.g., by introducing a CAR and EGFRt encoded by a T2A ribosomal switch to express two proteins from the same construct), and the non-immunogenic selective epitope can then be used as a marker for detecting such cells (see, for example, U.S. Patent No. 8,802,374). In some embodiments, the sequence encodes the tEGFR sequence shown in SEQ ID NO:7 or 16, or exhibits an amino acid sequence that is 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, the peptide (such as T2A) can lead to the synthesis of a ribosome-skipped (ribosome-jumping) 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)). Many 2A elements are known. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein include, but are not limited to, 2A sequences from the following viruses: foot-and-mouth disease virus (F2A, e.g., SEQ ID NO:22), equine rhinitis A virus (E2A, e.g., SEQ ID NO:21), thosea asigna virus (T2A, e.g., SEQ ID NO:6 or 17), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO:19 or 20), as described in U.S. Patent Publication No. 20070116690.

[0308] Recombinant receptors (such as CARs) expressed by cells administered to a subject typically recognize or specifically bind to molecules expressed in, associated with, or specific to the disease or condition being treated or its cells. Upon specific binding to a molecule, such as an antigen, the receptor typically delivers an immune-stimulating signal (such as an ITAM-transduced signal) into the cell, thereby promoting an immune response targeting the disease or condition. For example, in some embodiments, cells express CARs that specifically bind to antigens expressed by or associated with cells or tissues of the disease or condition.

[0309] b. Chimeric autoantibody receptor (CAAR)

[0310] 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, rather than 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 said 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.

[0311] 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 (secondary intracellular signaling region).

[0312] 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.

[0313] c. Multi-target

[0314] 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. WO2014055668A1 (describing a combination of activating and co-stimulatory CARs, e.g., targeting two different antigens that are present alone on off-target (e.g., normal cells) but co-present only on cells of the disease or condition to be treated) and Fedorov et al., Sci. Transl. Medicine, 5(215)(2013) (describing cells expressing activating and repressive CARs, such as those in which the activating CAR binds to an antigen expressed on both 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 do not require treatment).

[0315] 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 further 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.

[0316] In some embodiments, the 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 ITAM-like motifs. 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 immune synapses 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.

[0317] 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.

[0318] In some implementations, the first and / or second receptors include an intracellular signal transduction domain containing an ITAM or ITAM-like motif and an intracellular signal transduction domain of a co-stimulatory receptor.

[0319] In some embodiments, the first receptor contains an intracellular signaling domain comprising an ITAM or 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).

[0320] In some embodiments, neither the connection of a first receptor alone nor the connection of a 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 is 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 first and second antigens, the desired response, such as full immune activation or stimulation, is achieved, for example, as indicated by the secretion, proliferation, persistence, and / or performance of immune effector functions (such as cytotoxic killing of target cells) of one or more cytokines.

[0321] 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 signals that inhibit or attenuate that response. An example is a combination of an activating CAR and an inhibitory CAR or iCAR. For instance, this 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.

[0322] In some implementations, a multi-targeting strategy is used where antigens associated with a specific disease or condition are expressed transiently (e.g., after a genetically engineered stimulus) or permanently on disease-free cells and / or on the engineered cells themselves. In such cases, specificity, selectivity, and / or efficacy can be improved due to the need to connect two separate and individually specific antigen receptors.

[0323] In some embodiments, 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 because multiple receptors need to be linked to achieve a cellular response.

[0324] dT cell receptor (TCR)

[0325] 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.

[0326] 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 that bind to MHC molecules. In some embodiments, said TCR is in αβ form. Typically, TCRs present in αβ and γδ forms are structurally similar, 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 typically responsible for recognizing antigens that bind to major histocompatibility complex (MHC) molecules.

[0327] Unless otherwise stated, the term "TCR" should be understood to encompass the complete TCR and its antigen-binding moiety or antigen-binding 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 that binds to an MHC molecule (e.g., to 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 that bind to the complete TCR (e.g., to an MHC-peptide complex). In some cases, the antigen-binding moiety contains variable domains of the TCR (e.g., 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.

[0328] 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 regions 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 the most important of the three CDRs on a given TCR variable region for antigen recognition of the processed peptide moiety of the peptide-MHC complex and / or for interaction with the processed peptide moiety of the peptide-MHC complex. In some contexts, CDR1 of the α chain may interact with the N-terminal portion of some antigenic peptides. In some contexts, CDR1 of the β chain may interact with the C-terminal portion of the peptide. In some contexts, CDR2 has the strongest role in interaction with or recognition of the MHC moiety of the MHC-peptide complex, or is the major responsible CDR. In some embodiments, the variable region of the β chain may contain additional hypervariable regions (CDR4 or HVR4), which are typically involved in superantigen binding rather than antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

[0329] 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.

[0330] 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 based on Kabat numbered amino acids 1 to 116, 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 based on Kabat numbered positions 117 to 259; or the β chain constant domain or Cα). β Typically based on the position of the Kabat chain (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.

[0331] 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 device or complex. The intracellular tail of the CD3 signaling subunit (e.g., the CD3γ, CD3δ, CD3ε, and CD3ζ chains) contains one or more tyrosine-based activation motifs or ITAMs involved in the signaling ability of the TCR complex.

[0332] 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, for example, one or more disulfide bonds.

[0333] 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 full-length TCR sequences (including V chain sequences) from cellular sources are well known. 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 TCR-encoding nucleic acids from one or more given cells or isolated from said one or more given cells, or by synthesis of publicly available TCR DNA sequences.

[0334] In some embodiments, the TCR is obtained from a biological source, such as from cells (e.g., from T cells, such as 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 synthetically based on knowledge of the TCR sequence.

[0335] In some embodiments, the TCR is generated from TCRs identified or selected by screening a library of candidate TCRs 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, the T cells can be amplified from tumor-infiltrating lymphocytes (TILs). In some embodiments, the TCR library can be generated from CD4+ or CD8+ T 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 native Vα and Vβ libraries, wherein the amplified products are cloned or assembled to be separated by adapters. Depending on the source of the subject and cells, the library may be HLA allele-specific. Alternatively, in some embodiments, the TCR library may 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 may be modified by affinity maturation. In some embodiments, antigen-specific T cells may be selected, such as by screening to assess CTL activity against the peptide. In some aspects, TCRs present on antigen-specific T cells may be selected, for example, by binding activity (e.g., specific affinity or affinity for the antigen).

[0336] In some embodiments, the TCR or its antigen-binding portion is modified or engineered. In some embodiments, directed evolution methods are used to generate TCRs with altered properties, such as higher affinity for specific MHC-peptide complexes. In some embodiments, directed evolution is achieved 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 pathway 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.

[0337] 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.

[0338] HLA-A0201 binding motifs and proteasome and immunoproteasome cleavage sites are known using computer prediction models. For predicting MHC class I binding sites, such models include, but are not limited to, ProPred1 (described in more detail in the following literature: Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17(12):1236-12372001) and SYFPEITHI (see Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction. Immunoinformatics Methods in Molecular Biology, Vol. 409(1):75-932007).

[0339] In some embodiments, the TCR or its antigen-binding portion may be a recombinant 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 various 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 method, the TCR is in a cell-bound form expressed on the cell surface.

[0340] 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.

[0341] 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.

[0342] 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, the 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.

[0343] In some embodiments, dTCR comprises a TCRα chain containing 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 containing 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.

[0344] In some implementations, the TCR is a scTCR. Typically, scTCRs can be generated using known methods, 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. WO96 / 13593, WO96 / 18105, WO99 / 60120, WO99 / 18129, WO03 / 020763, WO2011 / 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. WO03 / 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. WO99 / 18129).

[0345] 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 connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0346] In some embodiments, the scTCR includes a first segment consisting of an α-chain variable region sequence fused to the N-terminus of the α-chain extracellular constant domain sequence, a second segment consisting of a β-chain variable region sequence fused to the N-terminus of the β-chain extracellular constant and transmembrane sequence, and optionally a connector sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0347] In some embodiments, the scTCR comprises a first segment consisting of a TCR β chain variable region sequence fused to the N-terminus of the β chain extracellular constant domain sequence, a second segment consisting of an α chain variable region sequence fused to the N-terminus of the α chain extracellular constant and transmembrane sequence, and optionally a connector sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0348] In some embodiments, the linker connecting the first and second TCR segments in 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-(SGGG G)5-P-, where P is proline, G is glycine, and S is serine (SEQ ID NO:32). In some embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO:33).

[0349] 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 native TCRs. For example, in some embodiments, one or more cysteine ​​residues may be incorporated into the extracellular sequences of the constant regions of the first and second segments of the scTCR polypeptide. In some cases, both natural and non-natural disulfide bonds may be required.

[0350] In some embodiments of dTCRs or scTCRs containing introduced interchain disulfide bonds, native disulfide bonds are absent. In some embodiments, the one or more native cysteine ​​residues forming the 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. WO2006 / 000830.

[0351] In some embodiments, the TCR or its antigen-binding fragment exhibits affinity for the target antigen at an equilibrium binding constant, which is in 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.

[0352] 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 methods 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.

[0353] In some embodiments, the vector may be one of the following series: pUC series (Fermentas LifeSciences), pBluescript series (Stratagene, La Jolla, California), pET series (Novagen, Madison, Wisconsin), pGEX series (Pharmacia Biotech, Uppsala, Sweden), or pEX series (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 implementations, viral vectors, such as retroviral vectors, are used.

[0354] In some embodiments, standard recombinant DNA techniques can be used to prepare recombinant expression vectors. 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 operatively linked to a nucleotide sequence encoding the 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.

[0355] In some embodiments, to generate a vector encoding a TCR, total cDNA isolated from a T cell clone 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 generated α and β chains are incorporated into a retroviral (e.g., lentiviral) vector.

[0356] D. Characteristics of the exported cell composition

[0357] In certain embodiments, the methods provided herein produce or generate cell compositions containing genetically engineered cells, for example, export cell compositions. In some embodiments, the export cell composition is a cell composition obtained through some or all of the steps used for genetically engineered cells. In some embodiments, the export cell composition is obtained from 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, such as cells obtained from the input cell composition. In some embodiments, the export cell composition contains cells that have already been genetically engineered. In certain embodiments, the cells of the export cell composition have undergone all steps of the genetic engineering process.

[0358] In some embodiments, the output cell composition contains cells comprising one or more nucleic acids introduced through genetic engineering, thereby expressing a recombinant or genetically engineered product 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, nucleic acids not typically found in the engineered cells and / or the organisms 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 a chimeric combination of nucleic acids encoding various domains from multiple different cell types.

[0359] In some embodiments, the exported cell 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 cell 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 cell 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.

[0360] In some embodiments, the exported cell composition has an engineered CD4+ T cell to CD8+ T cell ratio between 5:1 and 0.2:1, between 4:1 and 0.25:1, between 3:1 and 0.33:1, between 2:1 and 0.5:1, between 1.5:1 and 0.66:1, or between 1.25:1 and 0.8:1. In a particular embodiment, the exported cell composition has an engineered CD4+ T cell to CD8+ T cell ratio between 2:1 and 0.5:1. In some embodiments, the exported cell composition has an engineered CD4+ T cell to CD8+ T cell ratio of 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, or 0.5:1. In a particular embodiment, the exported cell composition has an engineered CD4+ T cell to CD8+ T cell ratio of 1:1. In a particular embodiment, the engineered T cells express a recombinant receptor. In a particular embodiment, the exported cell composition has a ratio of CD4+ T cells expressing the recombinant receptor to CD8+ T cells expressing the recombinant receptor of 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, or 0.5:1. In a particular embodiment, the exported cell composition has a ratio of CD4+ T cells expressing the recombinant receptor to CD8+ T cells expressing the recombinant receptor of 1:1. In some embodiments, the recombinant receptor is a TCR or a CAR. In a particular embodiment, the recombinant receptor is a CAR.

[0361] In some embodiments, the method produces an output cell composition having an engineered CD4+ T cell to engineered CD8+ T cell ratio, a recombinant receptor-expressing CD4+ T cell to recombinant receptor-expressing CD8+ T cell ratio, and / or a CAR-expressing CD4+ T cell to CAR-expressing CD8+ T cell ratio, within a certain tolerance range or error range of such defined ratio, desired ratio, or fixed ratio, and / or the method produces this ratio at a certain percentage of the time the method is performed, such as at least or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% of the time. In some embodiments, the tolerance for differences is within approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 60%, approximately 70%, or approximately 75% of the ratio. In some aspects, the ratio is within 10%, 20%, or 30% of the desired ratio, and / or within that ratio for at least 70%, 80%, or 90% of the time the method is performed. In some embodiments, the ratio is within 50% of the 1:1 ratio for at least 90% of the time the method is performed.

[0362] In some implementations, the tolerance difference and / or limiting ratio of engineered CD4+ to CD8+ T cells expressing recombinant receptors and / or CARs is or has been determined by administering different cell types, such as CD4+ and CD8+ T cells, to one or more subjects at multiple test ratios or quantities, and evaluating one or more parameters. In some aspects, determining the limiting ratio or fixed ratio or tolerance difference includes evaluating one or more outcomes after administration to the subject. In some aspects, said outcomes include those selected from: improvement in disease symptoms, and results indicating safety and / or low or no toxicity.

[0363] In some embodiments, the output cell composition produced by the methods provided herein has an engineered CD4+ T cell to engineered CD8+ T cell ratio between 2:1 and 0.5:1. In some embodiments, the output composition contains an engineered CD4+ T cell to engineered CD8+ T cell ratio of 1:1 or about. In some embodiments, the output cell composition is produced from an input cell composition described herein (e.g., the input cell composition described in Section IA) and has an engineered CD4+ T cell to engineered CD8+ T cell ratio between 2:1 and 0.5:1. In a particular embodiment, the output cell composition produced from the input cell composition has a 1:1 engineered CD4+ T cell to engineered CD8+ T cell ratio, with tolerances of 50%, 25%, 10%, or less. In some embodiments, the engineered T cells express a recombinant receptor. In some embodiments, the engineered T cells express CAR.

[0364] II. Compositions and Formulations

[0365] 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 obtain export cell compositions having a defined ratio of CD4+ to CD8+ T cells expressing the recombinant receptor, such as for use as therapeutic cell compositions. Export cell compositions produced by any of the methods described herein are also provided herein.

[0366] In some embodiments, cells generated using any of the methods described herein (e.g., cells from an export cell composition) are provided as a composition, including pharmaceutical compositions and formulations, such as unit dosage form compositions comprising a number of cells to be administered at a given dose or fraction thereof. In certain embodiments, the cells from the export cell composition are genetically engineered with a recombinant receptor (e.g., CAR-T cells). In some embodiments, the 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.

[0367] In some embodiments, the cell composition (e.g., the output cell composition) is generated or manufactured for cell therapy purposes. In some embodiments, the cell composition is a pharmaceutical composition or formulation. Such compositions may 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 of detection, diagnosis, and prognosis.

[0368] The term "pharmaceutical formulation" refers to a preparation in a form that renders the biological activity of the active ingredient contained therein effective, and which contains no additional components that would cause unacceptable toxicity to a subject 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.

[0369] "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.

[0370] 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, mixtures of two or more preservatives are used. Preservatives or mixtures thereof are typically present in an amount from 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 esters of p-hydroxybenzoate, 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).

[0371] 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).

[0372] 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 activity is complementary to that of the cells and / or whose respective activities do not adversely affect each other. Such active ingredients are present in a suitable combination in an amount 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.

[0373] In some embodiments, the pharmaceutical composition contains cells, such as cells that efflux a cell composition, in an amount effective in treating or preventing a 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 subject. For repeated administration over several days or longer, treatment is repeated as appropriate until the desired suppression of disease symptoms is achieved. 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.

[0374] Cells (e.g., cells from which cell compositions are exported) can be formulated for administration using standard administration techniques, formulations, and / or devices. Formulations and devices (such as syringes and vials) for storing and administering compositions are provided. Regarding cells, administration can be autologous or allogeneic. For example, immune-response 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-response 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-response cells), they are typically formulated into injectable, unit-dose forms (solutions, suspensions, emulsions).

[0375] 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.

[0376] In some embodiments, the composition is provided as a sterile liquid formulation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which may 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 may 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.

[0377] 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 lyophi...

Claims

1. A method for producing a cell composition, the method comprising: An input cell composition comprising CCR7+CD45RA+CD4+ T cells and CCR7+CD45RA+CD8+ T cells from a biological sample of a healthy subject is contacted under certain conditions with an agent containing a nucleic acid molecule encoding a recombinant receptor to introduce the nucleic acid encoding the recombinant receptor into the cells in the composition. The ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells in the input cell composition is between 0.8:1 and 2.2:1, including end values. The ratios are determined based on the number, number per volume, number per weight, or percentage of CCR7+CD45RA+CD4+ T cells and the number, number per volume, number per weight, or percentage of CCR7+CD45RA+CD8+ T cells obtained from the subject's biological sample.

2. The method of claim 1, further comprising stimulating the cells before, during, and / or after the contact, wherein the stimulation comprises incubating the cells in the presence of one or more stimulants, wherein the stimulation results in activation and / or proliferation of the cells.

3. A method for producing a cell composition, the method comprising: (a) By incubating an input cell composition comprising naïve CD4+ T cells and naïve CD8+ T cells from a biological sample obtained from a healthy subject in the presence of one or more stimulants, wherein the naïve CD4+ T cells and naïve CD8+ T cells are surface-positive for CCR7 and CD45RA, wherein the ratio of naïve CCR7+CD45RA+CD4+ T cells to naïve CCR7+CD45RA+CD8+ T cells present in the input cell composition is between 0.8:1 and 2.2:1, including the end values, wherein the ratio is determined based on the number, number per volume, number per weight, or percentage of CCR7+CD45RA+CD4+ T cells and the number, number per volume, number per weight, or percentage of CCR7+CD45RA+CD8+ T cells obtained from the biological sample obtained from the subject; and (b) The input cell composition is contacted under certain conditions with an agent containing a nucleic acid molecule encoding a recombinant receptor to introduce the nucleic acid encoding the recombinant receptor into the cells in the composition.

4. The method according to claim 1 or 3, wherein the input cell composition comprises CCR7+CD45RA+CD4+ cells and CCR7+CD45RA+CD8+ cells in a ratio between 0.8:1 and 1.6:1, including end values.

5. The method according to claim 1 or 3, wherein the input cell composition comprises CCR7+CD45RA+CD4+ cells and CCR7+CD45RA+CD8+ cells in a ratio between 0.8:1 and 1.2:1, including end values.

6. The method according to claim 1 or 3, wherein the input cell composition comprises CCR7+CD45RA+CD4+ cells and CCR7+CD45RA+CD8+ cells in a ratio of 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1 or 1.0:

1.

7. The method according to claim 1 or 3, wherein the input cell composition comprises CCR7+CD45RA+CD4+ cells and CCR7+CD45RA+CD8+ cells in a ratio of 1.1:

1.

8. A method for producing a cell composition, the method comprising: An input cell composition comprising CD27+CCR7+CD4+ T cells and CD27+CCR7+CD8+ T cells from a biological sample derived from a healthy or patient subject is contacted under certain conditions with an agent comprising a nucleic acid molecule encoding a recombinant receptor to introduce the nucleic acid encoding the recombinant receptor into the cells of the composition. The ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells in the input cell composition is between 0.8:1 and 2.2:1, including end values. The ratios are determined based on the number, number per volume, number per weight, or percentage of CD27+CCR7+CD4+ T cells and CD27+CCR7+CD8+ T cells obtained from the subject's biological sample.

9. The method of claim 8, further comprising stimulating the cells before, during, and / or after the contact, wherein the stimulation comprises incubating the cells in the presence of one or more stimulants, wherein the stimulation results in activation and / or proliferation of the cells.

10. A method for producing a cell composition, the method comprising: (a) By incubating an input cell composition comprising naïve CD4+ T cells and naïve CD8+ T cells from a biological sample obtained from a healthy or patient subject in the presence of one or more stimulants, wherein the naïve CD4+ T cells and naïve CD8+ T cells are surface-positive for CD27 and CCR7, wherein the ratio of naïve CD27+CCR7+CD4+ T cells to naïve CD27+CCR7+CD8+ T cells present in the input cell composition is between 0.8:1 and 2.2:1, including end values, wherein the ratio is determined based on the number, number per volume, number per weight, or percentage of CD27+CCR7+CD8+ T cells and the number, number per volume, number per weight, or percentage of CD27+CCR7+CD8+ T cells obtained from the biological sample obtained from the subject; and (b) The input cell composition is contacted under certain conditions with an agent containing a nucleic acid molecule encoding a recombinant receptor to introduce the nucleic acid encoding the recombinant receptor into the cells in the composition.

11. The method of claim 8 or 10, wherein the input cell composition comprises CD27+CCR7+CD4+ cells and CD27+CCR7+CD8+ cells in a ratio between 0.8:1 and 2.0:1, including end values.

12. The method of claim 8 or 10, wherein the input cell composition comprises CD27+CCR7+CD4+ cells and CD27+CCR7+CD8+ cells in a ratio between 0.8:1 and 1.6:1, including end values.

13. The method according to claim 8 or 10, wherein the input cell composition comprises CD27+CCR7+CD4+ cells and CD27+CCR7+CD8+ cells in a ratio of 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1 or 1.0:

1.

14. The method according to claim 8 or 10, wherein the input cell composition comprises CD27+CCR7+CD4+ cells and CD27+CCR7+CD8+ cells in a ratio of 1.1:

1.

15. The method of claim 8 or 10, wherein the input cell composition comprises CD27+CCR7+CD4+ cells and CD27+CCR7+CD8+ cells in a ratio of 1.69:

1.

16. A method for producing a cell composition, the method comprising: An input cell composition comprising CD62L-CCR7+CD4+ T cells and CD62L-CCR7+CD8+ T cells from a biological sample derived from a healthy or patient subject is contacted under certain conditions with an agent comprising a nucleic acid molecule encoding a recombinant receptor to introduce the nucleic acid encoding the recombinant receptor into the cells of the composition. The ratio of CD62L-CCR7+CD4+ T cells to CD62L-CCR7+CD8+ T cells in the input cell composition is between 0.5:1 and 2:1, including end values, and The ratios are determined based on the number, number per volume, number per weight, or percentage of CD62L-CCR7+CD4+ T cells and CD62L-CCR7+CD8+ T cells obtained from the subject's biological sample.

17. The method of claim 16, further comprising stimulating the cells before, during, and / or after the contact, wherein the stimulation comprises incubating the cells in the presence of one or more stimulants, wherein the stimulation results in activation and / or proliferation of the cells.

18. A method for producing a cell composition, the method comprising: (a) By incubating an input cell composition comprising naïve CD4+ T cells and naïve CD8+ T cells from a biological sample obtained from a healthy or patient subject in the presence of one or more stimulants, wherein the naïve CD4+ T cells and naïve CD8+ T cells are surface-negative for CD62L and surface-positive for CCR7, wherein the ratio of naïve CD4+ T cells to naïve CD8+ T cells present in the input cell composition is between 0.5:1 and 2:1, including end values, wherein the ratio is determined based on the number, number per volume, number per weight, or percentage of CD62L-CCR7+CD4+ T cells and the number, number per volume, number per weight, or percentage of CD62L-CCR7+CD8+ T cells obtained from the biological sample obtained from the subject; and (b) The input cell composition is contacted under certain conditions with an agent containing a nucleic acid molecule encoding a recombinant receptor to introduce the nucleic acid encoding the recombinant receptor into the cells in the composition.

19. The method of claim 16 or 18, wherein the input cell composition comprises CD62L-CCR7+CD4+ cells and CD62L-CCR7+CD8+ cells in a ratio between 0.5:1 and 1.5:1, including end values.

20. The method of claim 16 or 18, wherein the input cell composition comprises CD62L-CCR7+CD4+ cells and CD62L-CCR7+CD8+ cells in a ratio between 0.8:1 and 1.2:1, including end values.

21. The method according to claim 16 or 18, wherein the input cell composition comprises CD62L-CCR7+CD4+ cells and CD62L-CCR7+CD8+ cells in a ratio of 0.8:1, 0.9:1, 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1 or 1.5:

1.

22. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the biological sample is obtained from a blood sample.

23. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the biological sample is obtained from a plasma sample.

24. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the biological sample is obtained from a serum sample.

25. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the biological sample is a blood sample.

26. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the biological sample is a plasma sample.

27. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the biological sample is a serum sample.

28. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the biological sample is or comprises a whole blood sample.

29. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the biological sample is or comprises an erythrocyte sedimentation rate (ESR) brown-yellow layer sample.

30. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the biological sample is or comprises a peripheral blood mononuclear cell (PBMC) sample.

31. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the biological sample is or comprises an ungraded T cell sample.

32. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the biological sample is or comprises a lymphocyte sample.

33. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the biological sample is or comprises a leukocyte sample.

34. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the biological sample is or comprises agarose product.

35. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the biological sample is or contains leukapheresis products.

36. The method according to any one of claims 1-3, 8-10, and 16-18, wherein the input cell composition comprises from 1 x 10 7 Up to 5 x 10 9 Total cells, from 5 x 10 7 Up to 1 x 10 9 Total cells, from 1 x 10 8 Up to 5 x 10 8 Total cells, or from 2 x 10 8 Up to 5 x 10 8 A total of cells, or a living population of any of the aforementioned.

37. The method according to any one of claims 1-3, 8-10, and 16-18, wherein the input cell composition comprises from 1 x 10 7 Up to 5 x 10 9 Total T cells, from 5 x 10 7 Up to 1 x 10 9 Total T cells, from 1 x 10 8 Up to 5 x 10 8 One total T cell, or from 2 x 10 8 Up to 5 x 10 8 Total T cells, or any of the aforementioned live populations.

38. The method according to any one of claims 1-3, 8-10, and 16-18, wherein the input cell composition comprises at least 1 x 10 8 2 x 10 8 3 x 10 8 4 x 10 8 Or 5 x 10 8 A total of cells, or a living population of any of the aforementioned.

39. The method according to any one of claims 1-3, 8-10, and 16-18, wherein the input cell composition comprises at least 1 x 10 8 2 x 10 8 3 x 10 8 4 x 10 8 Or 5 x 10 8 Total T cells, or any of the aforementioned live populations.

40. The method according to any one of claims 2-3, 9-10 and 17-18, wherein the one or more stimulants are capable of activating T cells; capable of inducing signaling via the TCR complex; or capable of inducing T cell proliferation.

41. The method according to any one of claims 2-3, 9-10 and 17-18, wherein the one or more stimulants are capable of activating CD4+ T cells and / or CD8+ T cells; capable of inducing signaling via the TCR complex; or capable of inducing the proliferation of CD4+ T cells and / or CD8+ T cells.

42. The method according to claims 2-3, 9-10 and 17-18, wherein one or more stimulants comprise anti-CD3 antibodies and anti-CD28 antibodies.

43. The method according to any one of claims 2-3, 9-10 and 17-18, wherein the incubation is performed for 2 to 15 days.

44. The method according to any one of claims 2-3, 9-10 and 17-18, wherein the incubation is performed for at least 4 days.

45. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the agent comprising the nucleic acid molecule is a viral vector or a transposon.

46. ​​The method according to any one of claims 1-3, 8-10, and 16-18, wherein the recombinant receptor is a chimeric antigen receptor (CAR), wherein the CAR comprises: an extracellular domain containing an antigen-binding domain that specifically binds to an antigen, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain is or contains a CD3 chain. The intracellular signal transduction domain further includes a co-stimulatory signal transduction region; and The co-stimulatory signal transduction region includes the intracellular signal transduction domain of 4-1BB or ICOS or its signal transduction portion.

47. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the method produces an output composition wherein the ratio of CD4+ T cells expressing the recombinant receptor to CD8+ T cells expressing the recombinant receptor is between 0.5:1 and 2:1, including end values.

48. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the method produces an output composition wherein the ratio of CD4+ T cells expressing the recombinant receptor to CD8+ T cells expressing the recombinant receptor is between 0.5:1 and 2:1, including end values.

49. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the ratio of CD4+ T cells expressing recombinant receptor to CD8+ T cells expressing recombinant receptor in the output composition is 0.8:1, 0.9:1, 1.0:1, 1.1:1 or 1.2:

1.

50. The method according to any one of claims 1-3, 8-10 and 16-18, wherein the ratio of CD4+ T cells expressing recombinant receptor to CD8+ T cells expressing recombinant receptor in the output composition is 1:

1.

51. Use of the output composition produced by the method according to any one of claims 1-3, 8-10 and 16-18 in the preparation of a medicament for treating cancer in a subject.

52. Use of the output composition produced by the method according to any one of claims 1-3, 8-10 and 16-18 in the preparation of a medicament for treating an autoimmune disease in a subject.

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