Methods for producing genetically engineered cell compositions and related compositions

Abstract

To provide a method for generating cell compositions for therapeutic use.SOLUTION: Provided herein are methods and compositions for generating engineered cells, such as cells expressing a recombinant receptor, including methods involving stimulation and / or engineering of an input composition 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 a more consistent and / or predictable T cell product and / or a product with lower toxicity compared with other methods.SELECTED DRAWING: Figure 3A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 543,363, filed August 9, 2017, entitled "METHODS FOR PRODUCING GENETICALLY ENGINEERED CELL COMPOSITIONS AND RELATED COMPOSITIONS," and U.S. Provisional Patent 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 by reference in their entireties.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING This application is filed with a Sequence Listing in electronic format, which is provided as a file entitled 735042013140SEQLIST.txt, created on August 9, 2018, which is 35,001 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure relates to methods and compositions for generating engineered cells, such as cells expressing recombinant receptors, including methods involving stimulation and / or manipulation of input compositions with a predetermined 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 engineered receptors, such as engineered antigen receptors, such as engineered (recombinant) TCRs and chimeric antigen receptors (CARs), or other recombinant chimeric receptors. Features of the methods include the production of T cell products that are less variable and / or more predictable than other methods and / or less toxic than other methods. [Background technology]

[0004] background Various methods can be utilized to prepare cells for therapeutic use and administer the cells.For example, methods can be utilized to prepare, manipulate, and perform cell therapy, including T cells, including methods involving depletion or enrichment of certain subpopulations.Improved methods are needed, for example, to reduce the toxicity associated with administering certain adoptive cell therapies, improve manufacturing processes, enable improved administration, and / or reduce costs or other resources.Methods, cells, compositions, kits and systems that meet such needs are provided. Summary of the Invention

[0005] overview Provided herein is a method for generating a cell composition, comprising combining a first cell composition containing naive-like CD4+ T cells with a second cell composition containing naive-like CD8+ T cells to produce an input cell composition having a ratio of naive-like CD4+ T cells to naive-like CD8+ T cells that is 0.8:1 to 2.2:1 or about 0.8:1 to 2.2:1, inclusive. 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 the biological sample obtained from the subject.

[0006] In some embodiments, the method includes determining the number, volume, weight and / or percentage of naive-like CD4+ T cells in the first cell composition and / or the number, volume, weight and / or percentage of naive-like CD8+ T cells in the second composition prior to combining. In some cases, the method includes determining the number, volume, weight and / or percentage of naive-like CD4+ T cells and / or the number, volume, weight and / or percentage of naive-like CD8+ T cells in a biological sample from the subject prior to combining. In some of any such embodiments, the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells in the input composition is adjusted or altered compared to the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells in the biological sample from the subject.

[0007] Provided herein is a method for generating a cell composition comprising: determining the number, number per volume, number per weight and / or percentage of naive-like CD4+ T cells and naive-like CD8+ T cells in a biological sample obtained from a subject or one or more samples derived therefrom; and producing an input composition containing CD4+ T cells and CD8+ T cells and having a ratio of naive-like CD4+ T cells to naive-like CD8+ T cells that is between or about 2.2:1 and 0.8:1, inclusive, wherein the ratio in the input composition is adjusted or altered compared to the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells in the biological sample from the subject.

[0008] In some embodiments, the method further comprises contacting the input composition with an agent containing a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the input composition.

[0009] Provided herein is a method of generating a cell composition, the method comprising contacting an input composition containing naive-like CD4+ T cells and naive-like CD8+ T cells from a biological sample from a subject with an agent containing a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the composition, wherein the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells present in the input composition is at or about 0.8:1 to 2.2:1, inclusive.

[0010] In some embodiments, the method further comprises stimulating the cells before, during and / or after said contacting step, wherein stimulating comprises incubating the cells in the presence of one or more stimuli, wherein the stimulation results in activation and / or proliferation of the cells.

[0011] Provided herein is a method of generating a cell composition comprising: combining a first cell composition containing naive-like CD4+ T cells with a second cell composition containing naive-like CD8+ T cells to produce an input cell composition having a ratio of naive-like CD4+ T cells to naive-like CD8+ T cells that is between or about 0.8:1 and 2.2:1, inclusive; contacting the input composition with an agent containing a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the composition; and stimulating the cells before, during and / or after the contacting step, wherein stimulating comprises incubating the cells in the presence of one or more stimuli, wherein stimulating results in activation and / or proliferation of the cells.

[0012] In some of any such embodiments, the naive-like CD4+ and / or CD8+ cells are surface positive for a marker selected from the group consisting of CD45RA, CD27, CD28, CD62L and CCR7, and / or are surface negative for a marker selected from the group consisting of CD25, CD45RO, CD56, KLRG1, and / or have low expression of CD95, and / or are negative for intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-γ, IL-4, IL-10. In some embodiments, the naive-like CD4+ and / or CD8+ cells are surface positive for a T cell activation marker selected from the group consisting of CD45RA, CD27, CD28 and CCR7, and / or are surface negative for a marker selected from the group consisting of CD45RO, CD56, KLRG1, and / or have low expression of CD95. In some aspects, the naive-like CD4+ cells and / or the naive-like CD8+ cells are surface positive for CD45RA and CCR7. In some embodiments, the naive-like CD4+ cells and the naive-like CD8+ cells are surface positive for CD45RA, CD27 and CCR7, and surface negative for CD45RO.

[0013] In some of any such embodiments, the number, volume, weight and / or percentage of naive-like CD4+ T cells and / or the number, volume, weight and / or percentage of naive-like CD8+ T cells are determined by flow cytometry. In some aspects, the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells is adjusted compared to the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells in a biological sample from the subject.

[0014] In some embodiments, the biological sample is or is obtained from a blood, plasma or serum sample. In some aspects, the biological sample is or includes a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product or a leukapheresis product. In some cases, the biological sample is or is obtained from an apheresis sample or a leukapheresis sample. In some embodiments, the subject is a human subject.

[0015] In some of any such embodiments, the input composition contains a ratio of naive-like CD4+ cells to naive-like CD8+ cells of at or about 0.8:1 to 2.0:1, 0.8:1 to 1.6:1, 0.8:1 to 1.4:1, 0.8:1 to 1.2:1, or 1.0:1 to 1.2:1, inclusive, at or about 0.8:1 to 2.0:1, about 0.8:1 to 1.6:1, about 0.8:1 to 1.4:1, about 0.8:1 to 1.2:1, or about 1.0:1 to 1.2:1, inclusive. In some embodiments, the input composition comprises a ratio of naive-like CD4+ cells to naive-like CD8+ cells of or about 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 a ratio of naive-like CD4+ cells to naive-like CD8+ cells of or about 1.1:1.

[0016] In some of any such embodiments, the input composition is 1×10 7 ~5×10 9 pcs or approx. 1×10 7 ~5×10 9 Total cells or total T cells, 5 x 10 7 ~1×10 9 Pieces or about 5 x 10 7 ~1×10 9 Total cells or total T cells, 1 x 10 8 ~5×10 8pcs or approx. 1×10 8 ~5×10 8 total cells or total T cells, or 2 x 10 8 ~5×10 8 pcs or approx. 2×10 8 ~5×10 8 In some cases, the input composition contains at least 1×10 total cells or total T cells, or a viable population of any of the foregoing. 8 , 2×10 8 , 3×10 8 , 4×10 8 Or 5 x 10 8 or at least about 1 x 10 8 , about 2×10 8 , about 3×10 8 , about 4×10 8 Or about 5 x 10 8 The subject invention relates to a method for producing a culture of a subject comprising administering to a subject the invention comprising administering to a subject a total population of cells or total T cells, or a viable population of any of the foregoing.

[0017] In some embodiments, the one or more stimulatory substances can activate T cells, CD4+ T cells, and / or CD8+ T cells, induce signals through the TCR complex, and / or induce proliferation of T cells, CD4+ T cells, and / or CD8+ T cells. In some aspects, the one or more stimulatory substances contain a primary agent that binds to a member of the TCR complex and optionally specifically binds to CD3. In some cases, the one or more stimulatory substances further contain a secondary agent that specifically binds to a T cell costimulatory molecule. In some examples, the costimulatory molecule is selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS.

[0018] In some embodiments, the primary and secondary agents contain antibodies, and optionally, the one or more stimulatory agents include incubation with anti-CD3 and anti-CD28 antibodies. In some embodiments, the one or more stimulatory agents are present on the surface of a solid support, optionally a bead. In some embodiments, the one or more stimulatory agents are present on the surface of a bead, and the bead is a paramagnetic bead. In some aspects, the one or more stimulatory agents are selected from the group consisting of a CD3 binding molecule, a CD28 binding molecule, recombinant IL-2, recombinant IL-15, and recombinant IL-7, a vaccine containing an antigen that is specifically recognized by an antigen receptor, and an anti-idiotypic antibody that specifically binds to an antigen receptor, or a combination thereof.

[0019] In some of any such embodiments, incubation is carried out for 2-15 days, 2-12 days, 2-12 days, 2-8 days, 2-6 days, 2-4 days, 4-12 days, 4-10 days, 4-8 days, 4-6 days, 6-12 days, 6-10 days, 6-8 days, 8-12 days, 8-10 days, or 10-12 days. In some cases, incubation is carried out for at least 4, 6, 8, 10, or 12 days or for at least about 4, 6, 8, 10, or 12 days or for 4, 6, 8, 10, or 12 days.

[0020] In some of any of such embodiments, the agent containing the nucleic acid molecule is a viral vector or a transposon.In some cases, the agent containing the nucleic acid molecule is a viral vector, and the viral vector is a retroviral vector.In some examples, the viral vector is a lentiviral vector or a gamma retroviral vector.

[0021] In some embodiments, the recombinant receptor can bind to the target antigen associated with, specific to, and / or expressed on the cell or tissue of a 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 antigen is 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, anti-folate 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, EGFR vIII, folate binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, G protein-coupled receptor 5D (GPCR5D), HMW-MAA, IL-22R-α, kinase insert domain receptor (kdr), kappa light chain, Lewis Y, L1 cell adhesion molecule, (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, preferentially expressed antigen in melanoma (PRAME), survivin, TAG72, B7-H6, IL-13 receptor α2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250 / CAIX, PSCA, folate receptor-a, CD44v6, CD44v7 / 8, avb6 integrin, 8H9 , NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, CD44v6, dual antigen, cancer-testis antigen, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms' tumor 1 (WT-1), cyclin, cyclin A2, CCL-1, CD138, pathogen-specific antigen and universal tag associated antigen.

[0022] In some examples, the target antigen is selected from the group consisting of 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, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, chondroitin sulfate proteoglycan 4 (CSPG4), EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin 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 alpha (IL-22Rα), kinase insert domain receptor (kdr), kappa light chain, leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, preferentially expressed antigen in melanoma (PRAME), survivin, tumor-associated glycoprotein 72 (TAG7 2), 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, biantigen, cancer-testis antigen, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), natural killer group 2 member D (NKG2D) ligand, cancer / testis antigen 1B (CTAG;also known as NY-ESO-1 and LAGE-2), melan A (MART-1), glycoprotein 100 (gp100), glypican 3 (GPC3), G protein-coupled receptor 5D (GPRC5D), fetal tumor antigen, TAG72, tyrosinase-related protein 1 (TRP1; also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2; also known as dopachrome tautomerase, dopachrome delta-isomerase, or DCT), vascular endothelial growth factor receptor 2 (VEGF-R2), carcinoembryonic antigen (CEA), selected from among estrogen receptor, progesterone receptor, prostate specific antigen, ephrin B2, CD123, CD133, c-Met, O-acetylated GD2 (OGD2), CE7 epitope of L1-CAM, Wilms' tumor 1 (WT-1), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD138, pathogen specific 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;

[0023] The antigen targeted by the receptor, in some embodiments, comprises an antigen associated with a B cell malignancy, such as any of a number of known B cell markers. In some embodiments, the antigen is or comprises CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igκ, Igλ, CD79a, CD79b, or CD30.

[0024] In some embodiments, the antigen is or comprises a pathogen-specific or pathogen-expressed antigen. In some embodiments, the antigen is a viral antigen (e.g., a viral antigen from HIV, HCV, HBV, etc.), a bacterial antigen, and / or a parasitic antigen.

[0025] In some embodiments, the recombinant receptor is or contains a functional non-TCR antigen receptor or TCR or an antigen-binding fragment thereof. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR). In some cases, the chimeric antigen receptor contains an extracellular domain that contains an antigen-binding domain. In some cases, the antigen-binding domain is or contains an antibody, or an antibody fragment thereof, optionally a single-chain fragment. In some embodiments, the fragment contains an antibody variable region joined by a flexible linker. In some aspects, the fragment contains an scFv.

[0026] In some embodiments, the chimeric antigen receptor further contains a spacer and / or hinge region. In some embodiments, the chimeric antigen receptor contains an intracellular signaling region. In some cases, the intracellular signaling region contains an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or contains a primary signaling domain, a signaling domain capable of inducing a primary activation signal in T cells, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain is or contains the intracellular signaling domain of a CD3 chain, optionally a CD3-zeta (CD3ζ) chain, or a signaling portion thereof.

[0027] In some embodiments, the CAR comprises an antigen-specific scFv, a transmembrane domain, optionally a cytoplasmic signaling domain derived from a costimulatory molecule that is or comprises 4-1BB, and optionally a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule that is or comprises a CD3 zeta signaling domain, and optionally further comprises a spacer between the transmembrane domain and the scFv; the CAR comprises, in order, an antigen-specific scFv, a transmembrane domain, optionally a cytoplasmic signaling domain derived from a costimulatory molecule that is or comprises a 4-1BB signaling domain, and optionally a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule that is a CD3 zeta signaling domain; or the CAR comprises, in order, an antigen-specific scFv, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule that may be a 4-1BB signaling domain, and optionally a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule that is or comprises a CD3 zeta signaling domain.

[0028] In some embodiments, the chimeric antigen receptor further comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling region. In some embodiments, the intracellular signaling region further comprises a costimulatory signaling region. In some aspects, the costimulatory signaling region comprises the intracellular signaling domain of a T cell costimulatory molecule, or a signaling portion thereof. In some examples, the costimulatory signaling region comprises the intracellular signaling domain of CD28, 4-1BB or ICOS, or a signaling portion thereof. In some embodiments, the costimulatory signaling region is between the transmembrane domain and the intracellular signaling region.

[0029] In some of any such embodiments, the subject has a disease or condition, and optionally the recombinant receptor specifically recognizes or specifically binds to an antigen associated with the disease or condition or an antigen expressed or present on cells of the disease or condition.

[0030] In some embodiments, the method produces an output composition in which the ratio of recombinant receptor-expressing CD4+ T cells to recombinant receptor-expressing CD8+ T cells, optionally the ratio of viable cells thereof, varies from the average of the ratios in the plurality of T cell compositions produced by the method by no more than 20%, or no more than 10%, or no more than 5%, and / or varies from such average by no more than one standard deviation. In some embodiments, the method produces an output composition in which the ratio of recombinant receptor-expressing CD4+ T cells to recombinant receptor-expressing CD8+ T cells, optionally the ratio of viable cells thereof, is between 0.5:1 and 2:1, or between 0.8:1 and 1.6:1, or between 1:1 and 1.5:1, or about 0.5:1 and 2:1, or about 0.8:1 and 1.6:1, or about 1:1 and 1.5:1, inclusive. In some instances, the ratio of recombinant receptor-expressing CD4+ T cells to recombinant receptor-expressing CD8+ T cells, optionally viable cells thereof, in the output composition is or is about 1.2:1, 1.1:1, 1:1, 0.9:1 or 0.8:1. In some cases, the ratio of recombinant receptor-expressing CD4+ T cells to recombinant receptor-expressing CD8+ T cells, optionally viable cells thereof, in the output composition is 1:1 or is about 1:1.

[0031] In some embodiments, viable cells comprise cells that are apoptotic marker negative (-), optionally where the apoptotic marker is annexin V or active caspase-3.

[0032] In some of any such embodiments, the method is performed in vitro or ex vivo.

[0033] Provided herein is an output composition produced by any of the methods described herein.Also provided is a pharmaceutical composition containing the output composition described.In some embodiments, the pharmaceutical composition further contains a pharmaceutical carrier.

[0034] Provided herein are methods of treatment comprising administering to a mammalian subject an output composition produced by any of the described methods or any of the described pharmaceutical compositions. In some embodiments, the cells are derived from the subject to which the cells are administered.

[0035] In certain embodiments of the provided methods, the naive-like CD4+ cells and / or the naive-like CD8+ cells are surface positive for CD45RA and CCR7. In some embodiments of the provided methods, the naive-like CD4+ cells and / or the naive-like CD8+ cells are surface positive for CD27 and CCR7. In certain embodiments of the provided methods, the naive-like CD4+ cells and the naive-like CD8+ cells are surface positive for CD45RA, CD27 and CCR7, and surface negative for CD45RO.

[0036] In certain embodiments of the provided methods, the naive-like CD4+ cells and / or naive-like CD8+ cells are surface positive for CCR7 and surface negative for CD62L. In some embodiments of the provided methods, the input composition comprises a ratio of 1.1:1 or about 1.1:1 naive-like CD4+ cells to naive-like CD8+ cells that are surface positive for CD45RA and CCR7. In certain embodiments of the provided methods, the input composition comprises a ratio of 1.69:1 or about 1.69:1 naive-like CD4+ cells to naive-like CD8+ cells that are surface positive for CD45RA and CD27. In some embodiments, the input cell composition comprises a ratio of 1.69:1 or about 1.69:1 naive-like CD4+ cells to naive-like CD8+ cells that are surface positive for CD27 and CCR7.

[0037] In certain embodiments, provided herein are methods for generating a cell composition, the methods comprising the steps of: combining a first cell composition comprising CCR7+CD45RA+CD4+ T cells with a second cell composition comprising CCR7+CD45RA+CD8+ T cells to produce an input cell composition having a ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells that is between 0.8:1 and 2.2:1 or about 0.8:1 and 2.2:1, inclusive. In some embodiments of the provided methods, 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 the biological sample obtained from the subject.

[0038] In certain embodiments of the provided methods, the method comprises determining the number, number per volume, number per weight and / or percentage of CCR7+CD45RA+CD4+ T cells in the first cell composition and / or the number, number per volume, number per weight and / or percentage of CCR7+CD45RA+CD8+ T cells in the second composition prior to combining. In certain embodiments of the provided methods, the method comprises determining the number, number per volume, number per weight and / or percentage of CCR7+CD45RA+CD4+ T cells in a biological sample from the subject prior to combining. In some embodiments of the provided methods, 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 a biological sample from the subject.

[0039] In certain aspects, provided herein is a method for generating a cell composition 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 a biological sample obtained from a subject or one or more samples derived therefrom; and producing an input composition comprising CD4+ T cells and CD8+ T cells, wherein the ratio of CCR7+CD45RA+CD4+ T cells to naive-like CD8+ T cells is between or about 2.2:1 and 0.8:1, inclusive, wherein said ratio in the input composition is adjusted or altered compared to the ratio of CCR7+CD45RA+CD4+ T cells to naive-like CD8+ T cells in the biological sample from the subject.

[0040] Certain embodiments of the provided methods further include contacting the input composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the input composition.

[0041] Provided herein is a method of generating a cell composition, comprising the steps of: contacting an input composition comprising CCR7+CD45RA+CD4+T cells and CCR7+CD45RA+CD8+T cells from a biological sample from a subject, wherein the input composition has a ratio of CCR7+CD45RA+CD4+T cells to CCR7+CD45RA+CD8+T cells present in the input composition of between or about 0.8:1 and 2.2:1, inclusive, with an agent comprising a nucleic acid molecule encoding a recombinant receptor, under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the composition. In some embodiments, the provided method further comprises stimulating the cells before, during and / or after said contacting step, wherein stimulating comprises incubating the cells in the presence of one or more stimuli, wherein stimulating results in activation and / or proliferation of the cells.

[0042] In certain aspects, provided herein is a method of generating a cell composition comprising: combining a first cell composition comprising CCR7+CD45RA+CD4+ T cells with a second cell composition comprising CCR7+CD45RA+CD8+ T cells to produce an input cell composition having a ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells that is between 0.8:1 and 2.2:1, or about 0.8:1 and 2.2:1, inclusive; contacting the input composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor, under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the composition; and stimulating the cells before, during and / or after said contacting, wherein stimulating comprises incubating the cells in the presence of one or more stimuli, wherein stimulating results in activation and / or proliferation of the cells.

[0043] In certain embodiments of the provided methods, the number, volume, weight and / or percentage of CCR7+CD45RA+CD4+T cells and / or the number, volume, weight and / or percentage of CCR7+CD45RA+CD8+T cells are determined by flow cytometry. In some embodiments of the provided methods, the ratio of CCR7+CD45RA+CD4+T cells to CCR7+CD45RA+CD8+T cells is adjusted compared to the ratio of CCR7+CD45RA+CD4+T cells to CCR7+CD45RA+CD8+T cells in a biological sample from the subject. In certain embodiments of the provided methods, the input composition comprises a ratio of CCR7+CD45RA+CD4+ cells to CCR7+CD45RA+CD8+ cells of, or about, 0.8:1 to 2.0:1, 0.8:1 to 1.6:1, 0.8:1 to 1.4:1, 0.8:1 to 1.2:1, or 1.0:1 to 1.2:1, inclusive. In certain embodiments of the provided methods, the input composition comprises a ratio of CCR7+CD45RA+CD4+ cells to CCR7+CD45RA+CD8+ cells of, or about, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, or 1.0:1.

[0044] In some embodiments of the provided methods, the input composition comprises a ratio of CCR7+CD45RA+CD4+ cells to CCR7+CD45RA+CD8+ cells of 1.1:1 or about 1.1:1. In certain embodiments, provided herein are methods for generating a cell composition, the methods comprising: combining a first cell composition comprising CD27+CCR7+CD4+T cells with a second cell composition comprising CD27+CCR7+CD8+T cells to produce an input cell composition having a ratio of CD27+CCR7+CD4+T cells to CD27+CCR7+CD8+T cells that is between 1.2:1 and 2.4:1 or about 1.2:1 and 2.4:1, inclusive.

[0045] In certain embodiments of the provided methods, 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 the biological sample obtained from the subject.In some embodiments of the provided methods, the method comprises determining the number, volume, weight and / or percentage of CD27+CCR7+CD4+ T cells in the first cell composition and / or the number, volume, weight and / or percentage of CD27+CCR7+CD8+ T cells in the second composition before combining.In certain embodiments of the provided methods, the method comprises determining the number, volume, weight and / or percentage of CD27+CCR7+CD4+ T cells in a biological sample from a subject before combining.

[0046] In certain embodiments of the provided methods, 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 the subject. In certain embodiments, provided herein is a method for generating a cell composition 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 a biological sample obtained from a subject or one or more samples derived therefrom; and producing an input composition comprising CD4+T cells and CD8+T cells, wherein the ratio of CD27+CCR7+CD4+T cells to naive-like CD8+T cells is between or about 2.2:1 and 0.8:1, inclusive, wherein the ratio in the input composition is adjusted or altered compared to the ratio of CD27+CCR7+CD4+T cells to naive-like CD8+T cells in the biological sample from the subject.

[0047] Some embodiments of the methods provided further include contacting the input composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the input composition.

[0048] In certain embodiments, provided herein is a method of generating a cell composition, comprising contacting an input composition comprising CD27+CCR7+CD4+T cells and CD27+CCR7+CD8+T cells from a biological sample from a subject with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions for introducing 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 about 0.8:1 and 2.2:1, inclusive. Certain embodiments of the provided methods further comprise stimulating the cells before, during and / or after said contacting step, where the stimulating comprises incubating the cells in the presence of one or more stimuli, where the stimulating results in activation and / or proliferation of the cells.

[0049] In some embodiments, provided herein are methods of 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 produce an input cell composition having a ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells that is between or about 0.8:1 and 2.2:1, inclusive; contacting the input composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the composition; and stimulating the cells before, during and / or after said contacting, wherein the stimulating comprises incubating the cells in the presence of one or more stimuli, wherein the stimulating results in activation and / or proliferation of the cells.

[0050] In certain embodiments of the provided methods, the number, volume, weight and / or percentage of CD27+CCR7+CD4+T cells and / or the number, volume, weight and / or percentage of CD27+CCR7+CD8+T cells are determined by flow cytometry.In certain embodiments of the provided methods, the ratio of CD27+CCR7+CD4+T cells to CD27+CCR7+CD8+T cells is adjusted compared to the ratio of CD27+CCR7+CD4+T cells to CD27+CCR7+CD8+T cells in a biological sample from a subject.

[0051] In some embodiments of the provided methods, the input composition comprises a ratio of CD27+CCR7+CD4+ cells to CD27+CCR7+CD8+ cells of between 0.8:1 and 2.0:1, between 0.8:1 and 1.6:1, between 0.8:1 and 1.4:1, between 0.8:1 and 1.2:1, or between 1.0:1 and 1.2:1, inclusive, or between about 0.8:1 and 2.0:1, between about 0.8:1 and 1.6:1, between about 0.8:1 and 1.4:1, between about 0.8:1 and 1.2:1, or between about 1.0:1 and 1.2:1, inclusive. In certain embodiments of the provided methods, the input composition comprises a ratio of CD27+CCR7+CD4+ cells to CD27+CCR7+CD8+ cells of or about 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, or 1.0:1. In certain embodiments of the provided methods, the input composition comprises a ratio of CD27+CCR7+CD4+ cells to CD27+CCR7+CD8+ cells of or about 1.1:1. In some embodiments, the input cell composition comprises a ratio of CD27+CCR7+CD4+ cells to CD27+CCR7+CD8+ cells of or about 1.69:1.

[0052] In some embodiments, provided herein are methods for generating a cell composition comprising combining a first cell composition comprising CD62L-CCR7+CD4+ T cells with a second cell composition comprising CD62L-CCR7+CD8+ T cells to produce an input cell composition having a ratio of CD62L-CCR7+CD4+ T cells to CD62L-CCR7+CD8+ T cells that is between 0.5:1 and 2:1, or about 0.5:1 and 2:1, inclusive.

[0053] In certain aspects, provided herein is a method for generating a cell composition 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 a subject or one or more samples derived therefrom; and producing an input composition comprising CD4+T cells and CD8+T cells, wherein the ratio of CD62L-CCR7+CD4+T cells to naive-like CD8+T cells is between 0.5:1 and 2:1 or about 0.5:1 and 2:1, inclusive, wherein said ratio in the input composition is adjusted or altered compared to the ratio of CD62L-CCR7+CD4+T cells to naive-like CD8+T cells in the biological sample from the subject.

[0054] Certain embodiments of the provided methods further include contacting the input composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the input composition. In some embodiments, provided herein are methods of generating a cell composition comprising: combining a first cell composition comprising CD62L-CCR7+CD4+ T cells with a second cell composition comprising CD62L-CCR7+CD8+ T cells to produce an input cell composition having a ratio of CD62L-CCR7+CD4+ T cells to CD62L-CCR7+CD8+ T cells that is between 0.5:1 and 2:1, or about 0.5:1 and 2:1, inclusive; contacting the input composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor, under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the composition; and stimulating the cells before, during and / or after said contacting, wherein the stimulating comprises incubating the cells in the presence of one or more stimuli, wherein the stimulating results in activation and / or proliferation of the cells.

[0055] In certain embodiments of the provided methods, the number, volume, weight and / or percentage of CD62L-CCR7+CD4+T cells and / or the number, volume, weight and / or percentage of CD62L-CCR7+CD8+T cells are determined by flow cytometry. In certain embodiments of the provided methods, the ratio of CD62L-CCR7+CD4+T cells to CD62L-CCR7+CD8+T cells is adjusted compared to the ratio of CD62L-CCR7+CD4+T cells to CD62L-CCR7+CD8+T cells in a biological sample from the subject. In some embodiments of the provided methods, the input composition comprises a ratio of CD62L-CCR7+CD4+ cells to CD62L-CCR7+CD8+ cells of at or about 0.5:1 to 1.5:1, 1:1 to 2:1, 0.75:1 to 1.5:1, or 0.8:1 to 1.2:1, inclusive. In certain embodiments of the provided methods, the input composition comprises a ratio of CD62L-CCR7+CD4+ cells to CD62L-CCR7+CD8+ cells of, or about, 1.2:1, 1.1:1, 1.0:1, 0.9:1, or 0.8:1.

[0056] In certain embodiments of the provided methods, the biological sample is or is obtained from a blood, plasma or serum sample.In some embodiments of the provided methods, the biological sample is or includes a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product or a leukapheresis product.In certain embodiments of the provided methods, the biological sample is or is obtained from an apheresis sample or a leukapheresis sample. [Brief description of the drawings]

[0057] [Figure 1]FIG. 1A depicts a plot of a bivariate fit analysis of the ratio of viable CD4+ cells to viable CD8+ cells (viable CD4+ / CD8+ ratio) in apheresis samples compared to the ratio of CAR+CD4+ T cells to CAR+CD8+ T cells (CAR+CD4+ / CD8+ ratio) in the T cell composition after T cell activation, transduction and expansion with a chimeric antigen receptor (CAR) construct. The curves represent the boundaries of the bivariate normal ellipse at p=0.990. Data points represent the average ratio of four samples from each subject, including healthy subjects (circles) and subjects with myeloma (plus signs). FIG. 1B depicts a plot of a bivariate fit analysis of the CD45RA+ / CCR7+CD4 / CD8 ratio in the 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 a chimeric antigen receptor (CAR) construct, and expansion. The curves represent the boundaries of the bivariate normal ellipse at p=0.990. Data points represent the average ratios of four samples from each subject, including healthy subjects (circles) and subjects with myeloma (plus signs). [Figure 2A]2A-2C represent plots of bivariate fit analysis of the ratio of cells of various phenotypes in the starting mixture of selected CD4 and CD8 cells compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+T cell composition. FIG. 2A represents plots of bivariate fit analysis of the ratio of CD45RA+ / CCR7+ / CD4+ cells 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. FIG. 2B represents plots of 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 depicts a plot of a bivariate fit analysis of the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / 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. The curves represent the boundaries of the bivariate normal ellipse at p=0.950. Data points represent the average ratios of multiple compositions from each subject, including healthy donors (circles) and patients with multiple myeloma (plus signs). [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 3A]3A-3C represent plots of bivariate fit analysis of the ratio of cells of various phenotypes in the starting mixture of selected CD4 and CD8 cells from seven donors with multiple myeloma compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+T cell composition generated. FIG. 3A represents plots of bivariate fit analysis of the ratio of CD27+ / CCR7+ / CD4+ cells 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. FIG. 3B represents plots of bivariate fit analysis of the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / 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 3C depicts a plot of 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. The curves represent the boundaries of the bivariate normal ellipse at p=0.950. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] Detailed Description Provided herein are methods for preparing cell compositions, e.g., input cell compositions, for use in genetically engineering cells to express recombinant receptors. In some embodiments, the methods include one or more steps to produce an input cell composition containing a predetermined, controlled, or desired ratio of naive-like CD4+ T cells to naive-like CD8+ T cells. In some cases, the provided input composition can be produced by mixing or combining a cell composition containing CD4+ T cells, where the number or percentage of naive-like CD4+ T cells is known or determined, with a cell composition containing CD8+ T cells, where the number or percentage of naive-like CD8+ T cells is known or determined, so that a selected or desired ratio is achieved. Also provided are methods for stimulating, expanding, and / or inducing the proliferation of cells in the input composition. The provided methods can also include methods related to genetic engineering of cells, e.g., transduction methods, e.g., methods for introducing recombinant receptors, such as chimeric antigen receptors, into such cells for use in connection with adoptive cell therapy.

[0059] In one embodiment, processing of the generated input composition includes incubating the cells under stimulatory conditions, for example, in some aspects, to activate the cells to manipulate or transduce or expand the cells. In some embodiments, the method includes a step for manipulating multiple cell types, for example CD4+ cells and CD8+ cells, for example, isolated and present in the input composition. In some aspects, the manipulating step is carried out to introduce an engineered antigen receptor, such as a TCR, for example a high affinity TCR, or a functional non-TCR antigen receptor, for example a chimeric antigen receptor (CAR), into the cells. In some aspects, the method includes further processing, for example further incubation, for example at or about 37°C±2°C, and / or formulation of the cells and compositions containing them. In some embodiments, the processing produces a resultant output composition containing engineered cells, for example engineered CD4+ cells and CD8+ cells, for example cells in which engineered CD4+ cells 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 the present methods to a patient, such as in connection with adoptive cell therapy.

[0060] In some aspects, it is advantageous to have an output composition containing engineered cells, e.g., CAR+ T cells, in which the engineered CD4+ and CD8+ cells are present in a desired ratio or within a certain degree of tolerance of the desired ratio. For example, multiple different cell populations or cell types, e.g., isolated CD4 + and CD8 +Engineering the T cell populations and cells enriched for T cell subpopulations can improve efficacy or reduce or avoid unwanted effects. In some cases, such ratios include output ratios that are considered appropriate or optimal for administration to a patient, for example, in the context of a therapeutic application, for example, in the context of adoptive cell therapy. In some embodiments, the desired ratio of CD4+ T cells to CD8+ T cells in an output composition for administration to a subject, such as in the context of adoptive cell therapy, is 2:1 to 0.5:1 or about 2:1 to 0.5:1, for example 1:1 or about 1:1. In some aspects, a composition containing isolated CD8+ and CD4+ T cells, for example a composition containing a desired ratio of such cells, increases the ability of the cells ultimately administered to a subject to persist, expand, become activated, and / or engraft in vivo or when administered to a subject. In some aspects, this improves or increases one or more effector functions or activation phenotypes. For example, such benefits can in some aspects be achieved by administering CD4+ and CD8+ populations, rather than just CD8+ populations.

[0061] In some embodiments, the method has one or more advantages, such as reduced cost, time and / or resources, compared to other preparation, isolation, incubation and manipulation methods. Such advantages may include that multiple cell populations that are present at or near a desired ratio can be isolated, processed, e.g., incubated, and / or manipulated with improved efficiency and / or reduced complexity, time, cost and / or resource usage compared to other methods.

[0062] In some embodiments, the provided method is based on the finding that in certain cell production processes for engineering cells, the input ratio of CD4+ cells to CD8+ cells, or the ratio of their viable cells, may not correlate with the output ratio of engineered CD4+ cells to CD8+ cells, or the ratio of their viable cells.As shown herein, it is found that the desired ratio of engineered CD4+ T cells to CD8+ T cells (e.g., CAR+ CD4+ T cells to CAR+ CD8+ T cells, or the ratio of their viable cells) in the output composition correlates or relates to the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells, or the ratio of their viable cells, present in the input composition before the cells are subjected to a production process involving, for example, one or more steps of stimulating, activating, expanding, expanding and / or transducing cells to produce the output composition.A typical example of such naive-like cells is the ratio of CD4+ cells to CD8+ cells that are CD45RA+ and CCR7+, CD62L- / CCR7+ or CD27+ / CCR7+. In some aspects, such a correlation between the ratio of naive-like CD4 / CD8 cells in the input composition and the ratio of engineered CD4+ cells to CD8+ cells (e.g., CAR+CD4+T cells to CAR+CD8+T cells, or viable cells thereof) in the output composition is observed to exist despite variability in the process used to generate or produce the output composition. The source of this variability may include a number of different factors, such as specific steps or conditions of the process that may result in variability between cell compositions, or may result from differences in the individuals or samples from which the engineered cells are isolated, selected, derived, or obtained.

[0063] In certain embodiments, the methods provided are based on the finding that a desired ratio of engineered CD4+ T cells to CD8+ T cells (e.g., CAR+ CD4+ T cells to CAR+ CD8+ T cells, or viable cells thereof) in an output composition correlates or relates to the ratio of CD45RA+ / CCR7+ CD4+ T cells to CD45RA+ / CCR7+ CD8+ T cells, or viable cells thereof, present in an input composition prior to subjecting the cells to a production process involving, for example, one or more steps of stimulating, activating, expanding, expanding, and / or transducing the cells to produce the output composition. In certain aspects, such correlation between the ratio of CD45RA+ / CCR7+ CD4+ cells to CD45RA+ / CCR7+ / CD8+ cells in the input composition and the ratio of engineered CD4+ cells to CD8+ cells (e.g., CAR+ CD4+ T cells to CAR+ CD8+ T cells, or viable cells thereof) in the output composition is observed to exist despite variations in the donors and / or processes used to generate or produce the output composition.

[0064] In certain embodiments, the methods provided are based on the finding that a desired ratio of engineered CD4+ T cells to CD8+ T cells (e.g., CAR+ CD4+ T cells to CAR+ CD8+ T cells, or viable cells thereof) in the output composition correlates or relates to the ratio of CD62L- / CCR7+ CD4+ T cells to CD62L- / CCR7+ CD8+ T cells, or viable cells thereof, present in the input composition before the cells are subjected to a production process involving, for example, one or more steps of stimulating, activating, expanding, expanding and / or transducing the cells to produce the output composition. In certain aspects, such correlation between the ratio of CD62L- / CCR7+ CD4+ cells to CD62L- / CCR7+ / CD8+ cells in the input composition and the ratio of engineered CD4+ cells to CD8+ cells (e.g., CAR+ CD4+ T cells to CAR+ CD8+ T cells, or viable cells thereof) in the output composition is observed to exist despite variations in the donors and / or processes used to generate or produce the output composition.

[0065] In some embodiments, the methods provided are based on the finding that a desired ratio of engineered CD4+ T cells to CD8+ T cells (e.g., CAR+ CD4+ T cells to CAR+ CD8+ T cells, or viable cells thereof) in the output composition correlates or relates to the ratio of CD27+ / CCR7+ CD4+ T cells to CD27+ / CCR7+ CD8+ T cells, or viable cells thereof, present in the input composition before the cells are subjected to a production process involving, for example, one or more steps of stimulating, activating, expanding, expanding, and / or transducing the cells to produce the output composition. In certain aspects, such correlation between the ratio of CD27+ / CCR7+ CD4+ cells to CD27+ / CCR7+ / CD8+ cells in the input composition and the ratio of engineered CD4+ cells to CD8+ cells (e.g., CAR+ CD4+ T cells to CAR+ CD8+ T cells, or viable cells thereof) in the output composition is observed to exist despite variations in the donors and / or processes used to generate or produce the output composition.

[0066] In some aspects, the provided methods ensure that the output composition of genetically engineered cells, e.g., CAR+ T cells, generated by the cell production process achieves a relatively consistent and / or controlled desired ratio of engineered CD4+ T cells to CD8+ T cells, or viable cell ratios thereof, that exhibit low variance or variance in such ratios that is below an acceptable threshold variance among compositions produced by the process, including those derived from samples from many different subjects, e.g., subjects with different characteristics, e.g., age, number and / or type of prior treatments, and indications and their subtypes or severity or grades. In some aspects, such processes produce ratios of such engineered CD4+ to CD8+ (e.g., of CAR+CD4+ T cells to CAR+CD8+ T cells, or viable cells thereof) that vary by no more than 20%, or no more than 10%, or no more than 5% from the average of said ratios in a plurality of T cell compositions produced by the process, and / or that vary by no more than one standard deviation from such average, or that vary by no more than 20%, or no more than 10%, or no more than 5% between such different samples and patients, and among a plurality of T cell compositions produced by the process.

[0067] In some embodiments, the use of processes that result in greater consistency between cell production processes and between resulting output compositions, e.g., with respect to engineered CD4 T cell to CD8 T cell ratios, can be advantageous in ensuring consistency in subject dosing, which in some aspects can optimize the efficacy, titer, and / or safety of the administered composition between treated subjects. + T cell populations and CD8 +Engineering cells that are enriched in T cell population with desired output ratio can improve efficacy or reduce or avoid unwanted effects.In some aspects, this isolation or enrichment increases the ability of the cells that are ultimately administered to the subject to persist, grow, become activated, and / or engraft in vivo or when administered to the subject.In some aspects, this improves or increases one or more effector functions or activation phenotypes.Such results can be achieved even when there is donor variability between the starting cell samples for cell engineering.

[0068] In some embodiments, the methods provided herein allow for the production of therapeutic cell compositions of engineered cells with a desired output ratio in the output composition, without the need to process and / or administer CD4+ T cells and CD8+ T cells separately. Thus, the methods provided provide a simpler and / or more controlled way to prepare compositions with engineered CD4+ T cell populations and CD8+ T cell populations at or near a desired output ratio. In certain aspects, after producing an input composition containing a desired ratio of naive-like CD4 and CD8 cells as described, the methods provided can be used in cell production processes where CD4+ T cells and CD8+ T cells are processed together in a single stream process, e.g., activated, stimulated, expanded and / or transduced. Thus, in some aspects, the methods allow for the introduction of engineered antigen receptors for use in adoptive cell therapy, where cell populations are combined and isolated, incubated and / or manipulated, with increased efficiency and / or reduced complexity, time, cost and / or resource use compared to methods where the populations are isolated, incubated and / or manipulated separately.

[0069] Also provided are cells and compositions prepared by the methods, including pharmaceutical compositions and formulations, as well as kits, systems and devices for carrying out the methods. Methods of using the cells and compositions prepared by the methods, including therapeutic methods such as methods of adoptive cell therapy, and pharmaceutical compositions for administration to a subject are also provided.

[0070] All publications, including patent documents, scientific papers, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. In the event that the definitions set forth herein conflict or are otherwise inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein shall take precedence over the definitions incorporated herein by reference.

[0071] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0072] I. Methods for generating engineered cells in which the ratio of CD4+ to CD8+ T cells or specific subtypes thereof is controlled Provided herein is a method for generating a cell composition, e.g., an input cell composition, for use in genetically engineering cells to express a recombinant receptor. In some embodiments, the input cell composition contains CD4+ T cells and CD8+ T cells. In certain embodiments, the input cell composition contains one or more subtypes or populations of CD4+ T cells and / or CD8+ T cells. In certain embodiments, the one or more subtypes or populations are naive cells and / or naive-like cells. In particular embodiments, 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 particular embodiments, the input cell composition contains and / or has a fixed, preferred, target, predetermined, and / or controlled ratio of naive-like CD4+ T cells to naive-like CD8+ T cells.

[0073] In certain embodiments, the method includes one or more steps of mixing or combining cells or compositions of cells to generate or produce an input cell composition. In certain embodiments, the cells or cell compositions are selected and / or isolated from a sample. In some embodiments, the cells of the input cell composition are selected and / or isolated from a sample. In some embodiments, the CD4+ T cells and / or the composition of CD4+ T cells are selected or isolated from a sample. In some embodiments, the sample is a biological sample, such as a blood sample, an apheresis sample and / or a leukapheresis sample. In certain embodiments, the sample is a sample from a subject, for example, a human subject. In particular embodiments, the composition of CD4+ T cells and the composition of CD8+ T cells are isolated and / or selected from the same sample. In certain embodiments, the composition of CD4+ T cells and the composition of CD8+ T cells are isolated and / or selected from a sample taken or obtained from the same subject.

[0074] In some embodiments, the generation and production of the input cell composition includes one or more steps of evaluating, characterizing and / or identifying the cells. In certain embodiments, the cells are evaluated, characterized and / or identified in a composition of CD4+ T cells. In some embodiments, the cells are evaluated, characterized and / or identified in a composition of CD8+ T cells. In some embodiments, the cells are evaluated for cells that are indicative of a naive-like state in T cells and / or positive for a marker associated with a naive-like state in T cells. In certain embodiments, the cells are evaluated for cells that are indicative of a non-naive-like state in T cells and / or negative for a marker associated with a non-naive-like state in T cells. In certain embodiments, the cells from the CD4+ T cell composition and the CD8+ T cell composition, such as cells from a cell composition isolated from a biological sample from a subject, are evaluated, characterized and / or identified to determine the amount, level, proportion and / or percentage of 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. In certain embodiments, cells from a CD4+ T cell composition and a CD8+ T cell composition, such as cells from a cell composition isolated from a biological sample from a subject, are evaluated, characterized and / or identified to determine the amount, level, proportion and / or percentage of cells that are naive-like cells.

[0075] In some embodiments, the methods include 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 predetermined ratio of naive-like CD4+ T cells to naive-like CD8+ T cells. In some embodiments, the generation or production of the input cell composition comprises one or more steps of: (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, proportion and / or percentage of cells positive for one or more markers associated with a naive-like state and / or cells negative for one or more markers associated with a non-naive-like state in the composition of CD4+ T cells and / or CD8+ T cells; and / or (iii) mixing or combining cells of the composition of CD4+ T cells with cells of the composition of CD8+ T cells at a predetermined, fixed and / or preferred ratio of naive-like CD4+ T cells to naive-like CD8+ T cells. In certain embodiments, the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells in the input cell composition is different from the ratio present in the sample.

[0076] In certain embodiments, the content, make-up and / or constitution of the input cell composition correlates with, controls, corresponds to and / or is associated with the content, structure and / or constitution of the output cell composition. In certain embodiments, the amount, proportion, percentage, number, number per volume, number per weight and / or ratio of naive and / or naive-like cells, e.g., naive-like T cells, in the input cell composition correlates with, controls, corresponds to and / or is associated with the content, structure and / or constitution of the output cell composition. In certain embodiments, the amount, proportion, number, number per volume, number per weight and / or ratio of naive-like CD4+ T cells and / or naive-like CD8+ T cells in the input cell composition correlates with, controls, corresponds to and / or is associated with the content, structure and / or constitution of the output cell composition.

[0077] In some embodiments, the method includes one or more steps of genetically engineering the cells of the input composition. In some embodiments, the genetic engineering includes one or more steps of incubating the cells of the input cell composition under conditions that activate and / or stimulate the cells, delivering genes, e.g., recombinant and / or heterologous genes, to the cells, expanding the cells by incubating the cells under activating or stimulating conditions, harvesting the cells, and / or storing the cells by freezing, e.g., by cryopreservation. In some embodiments, the one or more genetic engineering steps produce an output cell composition containing the engineered cells. In certain embodiments, the engineered cells of the output composition have a fixed, predetermined, and / or target ratio of CD4+ cells to CD8+ cells.

[0078] In some embodiments, the methods provided herein include one or more steps to generate engineered cells having a predetermined ratio of CD4+ T cells to CD8+ T cells, such as cells expressing a recombinant receptor. In certain embodiments, the methods provided herein include one or more steps to genetically engineer cells from a starting cell composition and / or an input cell composition to generate a resultant cell composition and / or an output cell composition having a predetermined, genetically engineered ratio of CD4+ T cells to CD8+ T cells. In certain embodiments, the genetically engineering step is or includes transfection or transduction of cells from an input cell composition to introduce an agent containing a nucleic acid into the cells of the input cell composition. In some embodiments, the nucleic acid encodes a recombinant receptor, such as a chimeric antigen receptor (CAR). In some embodiments, the process of generating the output cell composition includes one or more steps of activating or stimulating cells of the input cell composition, genetically engineering, transducing or transfecting cells from the input cell composition, and / or expanding transfected cells, thereby resulting in an output cell composition having a predetermined ratio of genetically engineered CD4+ T cells to CD8+ T cells, for example a 1:1 ratio.

[0079] A. Cells and Preparation of Input Cell Composition In some embodiments, the method provided herein includes one or more steps to prepare cells for genetic manipulation. In certain embodiments, the one or more steps include isolating cells from a biological sample to prepare a composition of cells to be genetically manipulated, such as an input cell composition. In some embodiments, the preparation of the input cell composition includes one or more steps of isolating two or more cell types and / or compositions of specific cell types or cell subtypes, and mixing or combining the cell types and / or compositions of specific cell types into a single input cell composition. In certain embodiments, the cell composition of a specific cell type is evaluated to determine the presence, amount and / or ratio of additional subtypes in the composition. In certain embodiments, the cell composition of a specific cell type is mixed or combined to achieve an input cell composition with a fixed or determined ratio of cell types or cell subtypes. In some embodiments, the cell type and / or cell subtype correlates with, controls, corresponds to and / or is associated with the content, structure and / or organization of the output cell composition.

[0080] In certain embodiments, the preparation of the input cell composition includes one or more steps of isolating a composition of CD4+ T cells or a composition comprising CD4+ T cells, isolating a composition of CD8+ T cells or a composition comprising CD8+ T cells, and mixing or combining a CD4+ T cell composition and a CD8+ T cell composition of a particular cell type into a single input cell composition. In certain 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+ T cell and / or CD8+ T cell subtypes of the cell composition. In certain embodiments, the CD4+ T cell and / or CD8+ T cell subtypes are or include naive and / or naive-like CD4+ T cells and / or CD8+ T cells. In some embodiments, the proportion or amount of naive-like cells in the input cell composition correlates with, controls, corresponds to and / or is associated with the content, structure and / or composition of the output cell composition.

[0081] In some embodiments, the cells isolated from the sample, such as CD4+ T cells 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 or lymphoid organs, and / or are cells of the immune system, such as cells of innate or adaptive immunity. In some embodiments, the cells are lymphocytes. In certain embodiments, the lymphocytes are T lymphocytes or T cells. In some embodiments, the cells include CD4+ T cells and CD8+ T cells.

[0082] In certain embodiments, the composition of T cells, e.g., CD4+ T cell composition and / or CD8+ T cell composition, contains subtypes of cells further classified by function, activation state, maturity, differentiation potential, expansion potential, marker or cytokine secretion profile, and / or differentiation degree. For example, in some embodiments, the cells are or include naive and / or naive-like CD4+ T cells and / or CD8+ T cells. With respect to the subject to be treated, the cells can be allogeneic and / or autologous. Among the methods are off-the-shelf methods. In some embodiments, the method includes the steps of isolating cells from a subject, preparing, treating, culturing and / or manipulating the cells, and reintroducing the cells into the same subject before or after cryopreservation.

[0083] In some embodiments, the preparation of engineered cells comprises one or more culture steps and / or preparation steps.The cells used in the input cell composition, such as the composition of cells that undergo genetic engineering for purposes such as expressing recombinant receptors such as CAR, can contain cells isolated from a sample such as a biological sample, such as cells obtained from a subject or derived from a subject.In some embodiments, the subject from which the cells are isolated is the subject that has a disease or condition or requires cell therapy or is administered cell therapy.In some embodiments, the subject is a human that requires a particular therapeutic intervention, such as adoptive cell therapy, for which the cells are isolated, processed and / or engineered.

[0084] In some aspects, the sample from which the cells are derived or isolated, for example, the biological sample, is blood or blood-derived sample, or is derived from apheresis product or leukapheresis product.Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), white blood cells, bone marrow, thymus, and / or cells derived therefrom.In some embodiments, the cells are derived from, isolated from, and / or selected from a sample or biological sample that may include a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product.Samples include samples from autologous and allogeneic sources in the context of cell therapy, for example adoptive cell therapy.

[0085] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. In some embodiments, the cells are obtained from xenogeneic sources, e.g., mouse, rat, non-human primate, and pig.

[0086] In some embodiments, the isolation of cells or cell compositions, such as T lymphocytes or CD4+T cell compositions and / or CD8+T cell compositions, comprises one or more preparation steps and / or cell separation steps that are not based on affinity.In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, to enrich desired components, to lyse or remove cells that are sensitive to a particular reagent.In some examples, cells are separated based on one or more characteristics, such as density, adhesive properties, size, sensitivity and / or resistance to a particular component.

[0087] In some instances, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. The sample, in some aspects, contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects, contains cells other than red blood cells and platelets.

[0088] In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and to place the cells in a buffer or medium appropriate for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution does not contain calcium and / or magnesium, and / or many or all divalent cations. In some aspects, the washing step is accomplished in a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are washed after washing, e.g., with Ca ++ / Mg ++ The cells are resuspended in a variety of biocompatible buffers, such as PBS free of erythrocytes. In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0089] In some embodiments, the methods involve density-based cell separation methods, such as preparation of white blood cells from peripheral blood by lysing red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0090] In some embodiments, the isolation method comprises the separation of different cell types based on the expression or presence in cells of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method for such marker-based separation may be used. In some embodiments, the separation is affinity or immunoaffinity-based separation. For example, the isolation in some aspects comprises the separation of cells and cell populations based on the expression or expression level in cells of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such marker, and then generally followed by a washing step and separation of the cells that are bound to the antibody or binding partner from the cells that are not bound to the antibody or binding partner.

[0091] Such a separation step can be based on positive selection, where the cells that bind to the reagent are retained for further use, and / or negative selection, where the cells that do not bind to the antibody or binding partner are retained. In some instances, both fractions are retained for further use. In some aspects, negative selection can be particularly useful when antibodies that specifically identify cell types in a heterogeneous population are not available, so that separation is best performed based on markers expressed by cells other than the desired population.

[0092] Separation does not necessarily result in the enrichment or removal of 100% of a particular cell population or cells expressing a particular marker.For example, positive selection or enrichment of a particular type of cell, such as cells expressing a marker, refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker.Similarly, negative selection, removal, or depletion of a particular type of cell, such as cells expressing a marker, refers to decreasing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0093] In some cases, multiple separation steps are carried out, in which the positively or negatively selected fraction from one step is subjected to another separation step, such as subsequent positive or negative selection.In some cases, a single separation step can simultaneously deplete the cells that express multiple markers, for example, by incubating cells with multiple antibodies or binding partners, each specific to the marker that is targeted for negative selection.Similarly, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding partners that are expressed on various cell types.

[0094] For example, in some aspects, specific subpopulations or subtypes of T cells, such as CD4+ T cells and / or CD8+ T cells, are isolated by positive or negative selection techniques. In certain embodiments, CD4+ T cells and / or cell compositions of CD4+ T cells or cell compositions comprising CD4+ T cells are isolated by positive or negative selection techniques. In particular embodiments, CD8+ T cells and / or cell compositions of CD8+ T cells or cell compositions comprising CD8+ T cells are isolated by positive or negative selection techniques. In particular embodiments, CD4+ T cell compositions and / or CD8+ T cell compositions are isolated by positive or negative selection techniques.

[0095] In some embodiments, isolation is performed by enrichment of a particular cell population by positive selection, or depletion of a particular cell population by negative selection. In some embodiments, positive or negative selection is performed by isolating cells from a marker expressed or expressed on the cells that is positively or negatively selected, respectively. + or marker+), or expressed at relatively high levels (marker 高 ), is accomplished by incubating with one or more antibodies or other binding agents that specifically bind to one or more surface markers.

[0096] In some embodiments, T cells are separated from the PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes, e.g., CD14. + or CD8 + The selection process is + Helper T cells and CD8 + It is used to separate cytotoxic T cells. Such CD4 + Composition and CD8 + The compositions can contain cells that can be further classified or sorted into subpopulations based on positive or negative expression of markers and / or relative expression levels of markers. Such subtypes can include naive, naive-like and / or non-naive subtypes or subpopulations.

[0097] In certain embodiments, CD4+ T cells, e.g., CD4+ T helper cells, are classified into naive and / or naive-like cells and non-naive and / or non-naive-like cells by identifying cell populations with cell surface antigens. In particular embodiments, CD8+ T cells, e.g., CD8+ T helper cells, are classified into naive and / or naive-like cells and non-naive and / or non-naive-like cells by identifying cell populations with cell surface antigens.

[0098] In certain embodiments, T lymphocytes are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques. In particular embodiments, CD4+ T cells and / or compositions of CD4+ T cells are separated or isolated using immunomagnetic separation techniques. In some embodiments, CD8+ T cells and / or compositions of CD8+ T cells are separated or isolated using immunomagnetic separation techniques. Separation and isolation using immunomagnetic (or affinity magnetic) separation techniques is reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p17-25, edited by SA Brooks and U. Schumacher (c) Humana Press Inc., Totowa, NJ.

[0099] In some aspects, a sample or composition of cells to be separated is incubated with a small magnetizable or magnetically responsive material, such as a magnetically responsive particle or microparticle, such as a paramagnetic bead (such as, for example, Dynalbeads or MACS beads). The magnetically responsive material, such as a particle, is generally attached, directly or indirectly, to a binding partner, such as an antibody, that specifically binds to a molecule, such as a surface marker, present on the cell, cells, or population of cells that is desired to be separated, e.g., negatively or positively selected.

[0100] In some embodiments, magnetic particles or beads comprise magnetically responsive materials bound to specific binding members, such as antibodies or other binding partners.There are many known magnetically responsive materials that are used in magnetic separation methods.Suitable magnetic particles include those described in U.S. Patent No. 4,452,773 to Molday and European Patent Specification EP 452342 B, which are incorporated herein by reference.Colloidal-sized particles, such as those described in U.S. Patent No. 4,795,698 to Owen and U.S. Patent No. 5,200,084 to Liberti et al., are other examples.

[0101] Incubation is generally carried out under conditions whereby the antibody or binding partner attached to the magnetic particle or bead, or a molecule such as a secondary antibody or other reagent that specifically binds to such an antibody or binding partner, specifically binds to a cell surface molecule if present on cells in the sample.

[0102] In some aspects, the sample is placed in a magnetic field, and the cells with magnetically responsive or magnetizable particles attached are attracted to the magnet and separated from the unlabeled cells. For positive selection, the cells that are attracted to the magnet are retained; for negative selection, the cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subjected to further separation steps.

[0103] In certain embodiments, magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin.In certain embodiments, magnetic particles are attached to cells via coating with a primary antibody specific for one or more markers.In certain embodiments, cells, rather than beads, are labeled with a primary antibody or binding partner, and then magnetic particles coated with a cell type-specific secondary antibody or other binding partner (e.g., streptavidin) are added.In certain embodiments, streptavidin-coated magnetic particles are used in combination with biotinylated primary or secondary antibodies.

[0104] In some embodiments, the magnetically responsive particles remain attached to the cells that are then incubated, cultured, and / or manipulated; in some aspects, the particles remain attached to the cells for administration to patients. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known, including, for example, the use of competing unlabeled antibodies and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0105] In some embodiments, affinity-based selection is via magnetic activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). The magnetic activated cell sorting (MACS) system allows for high purity selection of cells to which magnetized particles are attached. In certain embodiments, MACS operates in a mode in which non-target and target species are eluted sequentially after application of an external magnetic field. That is, cells attached to magnetized particles are held in place while non-attached species are eluted. Then, after this first elution step is completed, species that were captured in the magnetic field and prevented from eluting are liberated in some manner so that they can be eluted and collected. In certain embodiments, non-target cells are labeled and depleted from a heterogeneous population of cells.

[0106] In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the method of the present invention.In some aspects, the system is used to perform each of these steps in a closed or sterile environment, for example, to minimize error, user handling, and / or contamination.In one example, the system is the system described in International Patent Application Publication No. WO2009 / 072003, or US 20110003380 A1.

[0107] In some embodiments, the system or device performs one or more, e.g., all, of the isolation, processing, manipulation, and formulation steps in an integrated or self-contained system and / or in an automated or programmable manner. In some aspects, the system or device includes a computer and / or a computer program connected to the system or device, which allows a user to program, control, evaluate the outcome of, and / or adjust various aspects of the processing, isolation, manipulation, and formulation steps.

[0108] In some aspects, the separation and / or other steps are performed using, for example, the CliniMACS system (Miltenyi Biotec) for automated separation of cells 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 pinch valves. The integrated computer in some aspects controls all components of the instrument and commands the system to perform repetitive procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selected column. The peristaltic pump controls the flow rate of the entire tubing set and, together with the pinch valves, ensures a controlled flow of buffer through the system and continuous suspension of the cells.

[0109] The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labeling of cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tubing set, which is then connected to a bag containing buffer and a cell collection bag. The tubing set is made of pre-assembled sterile tubing including a pre-column and a separation column, and is for single use only. After the start of the separation program, the system automatically applies the cell sample onto the separation column. The labeled cells are retained in the column while the unlabeled cells are removed by a series of washing steps. In some embodiments, the cell population for use in the methods described herein is unlabeled and is not retained in the column. In some embodiments, the cell population for use in the methods described herein is labeled and is retained in the column. In some embodiments, the cell population for use in the methods described herein is eluted from the column after removal of the magnetic field and collected in a cell collection bag.

[0110] In certain embodiments, the separation and / or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system in some aspects includes a cell processing unit that allows for automatic washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an on-board camera and image recognition software that determines optimal cell fractionation endpoints by identifying macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into red blood cells, white blood cells, and plasma layers. The CliniMACS Prodigy system can also include an integrated cell culture chamber that accomplishes cell culture protocols such as, for example, cell differentiation and expansion, antigen loading, and long-term cell culture. An input port can allow for aseptic removal and replenishment of culture medium, and cells can be monitored using an integrated microscope. See, e.g., 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.

[0111] In some embodiments, the cell populations described herein are collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluid stream. In some embodiments, the cell populations described herein are collected and enriched (or depleted) via preparative-scale (FACS) sorting. In certain embodiments, the cell populations described herein are collected and enriched (or depleted) by using a microelectromechanical system (MEMS) chip combined with a FACS-based detection system (see, for example, WO 2010 / 033140, Cho et al. (2010) Lab Chip 10:1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376). In either case, cells can be labeled with multiple markers, which allows for the isolation of clearly defined T cell subsets with high purity.

[0112] In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate separation for positive and / or negative selection.For example, separation may be based on binding to fluorescently labeled antibody.In some examples, separation of cells based on binding of antibody or other binding partner specific to one or more cell surface markers is performed in fluid flow, for example by fluorescence activated cell sorting (FACS), including preparative scale (FACS), and / or by microelectromechanical system (MEMS) chips, for example, combined with flow cytometry detection system.Such methods allow positive and negative selection based on multiple markers at the same time.

[0113] Input cell composition In certain embodiments, the present invention provides a method for producing and / or preparing an input cell composition.In certain embodiments, the input cell composition is a composition of cells for use in genetic engineering, such as cells that will be genetically engineered with a nucleic acid encoding a recombinant protein, such as a recombinant receptor, such as CAR, and / or cells that will undergo a process to produce a genetically engineered cell that expresses a recombinant protein, such as a recombinant receptor, such as CAR.In certain embodiments, the cells of the input composition will be treated with, contacted with, and / or incubated with a nucleic acid encoding a recombinant receptor.In certain embodiments, the input cell composition contains CD4+ T cells and CD8+ T cells.In certain embodiments, the input cell composition contains CD4+ T cells and CD8+ T cells, including naive and / or naive-like T cells, in a specific desired ratio, fixed ratio, and / or controlled ratio of CD4+ T cells to CD8+ T cells.

[0114] In some embodiments, a desired, fixed and / or controlled ratio of CD4+ T cells to CD8+ T cells that are naive and / or naive-like T cells is a ratio or number of cells exhibited by the two types of cells or isolated cell populations contained in an input cell composition designed to result in an output cell composition at or within a desired, predetermined and / or controlled ratio of engineered CD4+ T cells to CD8+ T cells upon completion of incubation and / or manipulation or other processing steps and / or upon thawing and / or immediately prior to administration to a subject.

[0115] In certain embodiments, the input cell composition contains a ratio of CD4+ naive-like T cells to CD8+ naive-like T cells, such as a predetermined ratio, a controlled ratio, and / or a fixed ratio. In particular embodiments, the ratio of CD4+ naive-like T cells to CD8+ naive-like T cells is 10:1 to 0.05:1, 8:1 to 0.1:1, 5:1 to 0.2:1, 2.5:1 to 0.25:1, 2.2:1 to 0.8:1, 2:1 to 0.5:1, or 1.5:1 to 1:1, inclusive. In particular embodiments, the ratio of CD4+ naive-like T cells to CD8+ naive-like T cells is 2:1 to 0.8:1, 1.6:1 to 0.8:1, 1.4:1 to 0.8:1, 1.2:1 to 0.8:1, or 1.2:1 to 0.8:1, inclusive. In some embodiments, the ratio is between 2.2:1 and 0.8:1, inclusive. In certain embodiments, the ratio of CD4+ naive-like T cells to CD8+ naive-like T cells is 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, or is about 2.2:1, about 2.1:1, about 2.0:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1.1:1, about 1.0:1, about 0.9:1, or about 0.8:1. In certain embodiments, the ratio is 1.1:1 or about 1.1:1.

[0116] In certain embodiments, the input cell composition has an amount of total cells or total viable cells of 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8, 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×10 8 , 4.5×10 8 , 5×10 8 , 5×10 8 Or 1×10 9 1 x 10 pieces or approx. 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×10 8 , about 5×10 8 Or about 1 x 10 9cells. In certain embodiments, the input cell composition has an amount of cells expressing CD4 or CD8 that is 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 , 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×10 8 , 4.5×10 8 , 5×10 8 , 5×10 8 or 1×10 9 cells or about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×108 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×10 8 , about 5×10 8 Or about 1 x 10 9 In some embodiments, the input cell composition comprises naive-like CD4+ T cells and naive-like CD8+ T cells in an amount of 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 , 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×10 8 , 4.5×10 8 , 5×10 8 , 5×10 8 Or 1×10 9 1 x 10 pieces or approx. 6 , about 5×106 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×10 8 , about 5×10 8 Or about 1 x 10 9 There are 10 pieces.

[0117] In certain embodiments, the input cell composition comprises 1×10 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 or 1×10 8 ~3×10 8 In certain embodiments, the input cell composition has an amount of cells expressing CD4 or CD8 of 1×10 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×108 ~3×10 8 1 x 10 pieces or approx. 6 ~1×10 10 , about 1×10 7 ~1×10 9 , about 5×10 7 ~5×10 8 Or about 1 x 10 8 ~3×10 8 In some embodiments, the input cell composition comprises naive-like CD4+ T cells and naive-like CD8+ T cells in an amount of 1×10 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 1 x 10 pieces or approx. 6 ~1×10 10 , about 1×10 7 ~1×10 9 , about 5×10 7 ~5×10 8 Or about 1 x 10 8 ~3×10 8 There are 10.

[0118] 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% naive-like cells. In certain embodiments, the input cell composition contains or comprises 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 90% or less, or 85% or less naive-like cells.

[0119] In certain embodiments, the methods provided herein include one or more steps of producing, generating, and / or creating an input cell composition. In certain embodiments, the steps of producing, generating, and / or creating an input cell composition include one or more steps of mixing or combining cells of a composition of CD4+ T cells with cells of a composition of CD8+ T cells.

[0120] In some embodiments, the cells of the input composition, such as CD4+ T cells CD8+ T cells, are isolated and / or selected from a sample, such as a biological sample. In certain embodiments, the source of the cells of the input composition is a composition of cells, such as a composition of CD4+ T cells and a composition of CD8+ T cells, isolated and / or selected from a sample, such as a composition of CD4+ T cells and a composition of CD8+ T cells, which are isolated and / or selected, such as a composition of CD4+ T cells and a composition of CD8+ T cells, which are isolated and / or selected, such as a composition of CD4+ T cells and a composition of CD8+ T cells, which are isolated and / or selected, such as a composition of CD8 ... from the same sample. In certain embodiments, the composition of CD4+ T cells and the composition of CD8+ T cells are isolated and / or selected from a sample taken or obtained from the same subject.

[0121] In certain embodiments, a composition of CD4+ T cells contains or comprises 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% CD4+ T cells. In some embodiments, a composition of CD4+ T cells contains or comprises up to 100%, up to 99%, up to 98%, up to 97%, up to 96%, up to 95%, up to 90%, or up to 85% CD4+ T cells.

[0122] In certain embodiments, a composition of CD8+ T cells contains or comprises 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% CD8+ T cells. In particular embodiments, a composition of CD8+ T cells contains or comprises up to 100%, up to 99%, up to 98%, up to 97%, up to 96%, up to 95%, up to 90%, or up to 85% CD8+ T cells.

[0123] In certain embodiments, the process of producing, generating and / or making an input cell composition comprises one or more steps of measuring, determining and / or quantifying the amount, proportion, number, number per volume, number per weight and / or percentage of viable CD4+T cells and / or viable CD8+T cells present in the composition of CD4+T cells and / or the composition of CD8+T cells, e.g., before combining or mixing the cells of the cell composition. In certain embodiments, the process of producing, generating and / or making an input cell composition comprises one or more steps of measuring, determining and / or quantifying the amount, proportion, number, number per volume, number per weight and / or percentage of naive-like CD4+T cells and / or naive-like CD8+T cells present in the composition of CD4+T cells and / or the composition of CD8+T cells. In some embodiments, the naive-like CD4+T cells and / or naive-like CD8+T cells are viable naive-like cells.

[0124] In certain embodiments, the process of producing, generating and / or making an input cell composition comprises one or more steps of measuring, determining and / or quantifying the amount, proportion, number, number per volume, number per weight and / or percentage of viable CD4+T cells and / or viable CD8+T cells present in a sample, e.g., a biological sample. In certain embodiments, the process of producing, generating and / or making an input cell composition comprises one or more steps of measuring, determining and / or quantifying the amount, proportion, number, number per volume, number per weight and / or percentage of naive-like CD4+T cells and / or naive-like CD8+T cells present in a sample. In some embodiments, the naive-like CD4+T cells and / or naive-like CD8+T cells are viable naive-like cells.

[0125] In some embodiments, the cells of the input composition are isolated and / or selected from a sample, e.g., a biological sample. In certain embodiments, the proportion of naive-like cells, e.g., the proportion of naive-like CD4+ T cells and CD8+ T cells, in the sample is known or has been determined, measured or assessed. In some embodiments, cells from the sample are isolated and / or selected to directly produce a cell composition, e.g., an input cell composition, with a predetermined, fixed or controlled ratio of naive-like CD4+ T cells to naive-like CD8+ T cells. In certain embodiments, cells are isolated and / or selected by immunoaffinity bead selection. In some embodiments, cells are isolated and / or selected by affinity column. In certain embodiments, cells from the sample are isolated or selected according to any of the methods described in WO 2015 / 164675 to produce a cell composition with a predetermined, controlled and / or fixed ratio of naive-like CD4+ cells to naive-like CD8+ cells.

[0126] In certain embodiments, the input cell composition contains cells isolated and / or selected directly from a sample by a first and second isolation or selection. In certain embodiments, the input composition is produced by performing a first and second selection to isolate a sufficient amount, number or concentration of CD4+ T cells and CD8+ T cells to result in a predetermined, fixed and / or controlled ratio of naive-like CD4+ T cells to naive-like CD8+ T cells.

[0127] In some embodiments, cells from a sample are directly isolated, selected and / or enriched to produce an input cell composition enriched for CD4+ and CD8+ cells. In some embodiments, the amount, number, percentage, number by volume and / or number by weight of naive-like CD4+ and naive-like CD8+ cells in a sample are measured, assessed and / or determined, and the CD4+ and CD8+ cells are isolated, selected and / or enriched in sufficient amounts to achieve an input cell composition with a predetermined, fixed or controlled ratio of naive-like CD4+ T cells to naive-like CD8+ T cells. In some embodiments, the cells directly isolated, selected and / or enriched from a sample are the input cell composition and are used in subsequent processing steps, such as those involving incubation, stimulation, activation, manipulation and / or formulation of the enriched cells.

[0128] In some embodiments, the cells isolated, selected and / or enriched from the sample, e.g., the input cell composition, contain a ratio of CD4+ cells to CD8+ cells that is a predetermined, fixed or controlled ratio of naive-like CD4+ cells to naive-like CD8+ cells. In embodiments of the methods provided herein, the first and / or second selection of the sample or selection of a subpopulation thereof can be performed to result in an input cell composition with a desired ratio of naive-like CD4+ T cells to naive-like CD8+ cells.

[0129] In some embodiments, the ratio of CD4+T cells to CD8+T cells in a sample, e.g., a biological sample, is determined before performing the first and / or second selection from the sample. In certain embodiments, the ratio of naive-like CD4+T cells to naive-like CD8+T cells in a sample is determined before performing the first and / or second selection. Based on the ratio of CD4+T cells to CD8+T cells and / or the ratio of naive-like CD4+T cells to CD8+T cells in a sample, which may vary between samples, a specific selection mode can be tailored to the sample, e.g., by sizing a chromatography column or selecting the amount or concentration of immunoaffinity reagents, to achieve a desired, fixed or controlled ratio. The relative level or frequency of various cell populations in a subject can be determined based on the evaluation of the surface expression of one or more markers present on such populations or subpopulations. Several well-known methods can be used to evaluate the expression level of surface markers or surface proteins, such as affinity-based methods, e.g., immunoaffinity-based methods, e.g., in the context of cell surface proteins, detection by flow cytometry.

[0130] Depending on the situation, the appropriate ratio of naive-like CD4+ T cells and CD8+ T cells may vary, for example, depending on the specific disease, condition or pretreatment of the subject from which the cells are derived, and / or the specific antigen specificity of the cells, the relative representation between the specific types of cells (e.g., CD4+ cells) of various subpopulations, such as effector vs. memory vs. naive cells, and / or one or more conditions under which the cells are incubated, such as medium, stimulants, incubation time, buffer, oxygen content, carbon dioxide content, antigens, cytokines, antibodies and other components. Thus, a cell type that is typically or generally known to grow or expand faster than other cell types may not always have such properties in every situation. Thus, in some aspects, the ratio of naive-like CD4+ T cells and naive-like CD8+ T cells is determined based on the known ability of the cell type in normal or typical situations, combined with evaluation of the phenotype or condition of the cell or of the subject from which the cell is derived, and / or experimental evidence.

[0131] In some embodiments, the separation and / or isolation steps are carried out using immunomagnetic beads. In some embodiments, a cell sample containing CD4+ cells and CD8+ cells is contacted with magnetic beads containing a first immunoaffinity reagent that binds to CD4 or CD8 and a second immunoaffinity reagent that binds to the other of CD4 or CD8. The separation and / or isolation steps can be performed simultaneously or sequentially.

[0132] In some embodiments, the first and / or second immunoaffinity reagents are present in the incubation composition at a sub-optimal yield concentration, in which case the enriched composition contains, e.g., 70% but not all of the total CD4+ cells in the incubation composition and / or, e.g., 70% but not all of the CD8+ cells in the incubation composition, thereby producing a composition enriched in CD4+ T cells and CD8+ T cells.

[0133] In some embodiments, a concentration of an affinity reagent that is less than the optimal yield concentration is a concentration that is less than that used or required to optimize or maximize the yield of bound cells in a given selection or enrichment that involves incubating cells with the reagent and recovering or separating cells that are bound to the reagent (where "yield" refers, for example, to the number of cells so recovered or selected compared to the total number of cells in the incubation that the reagent targets, or the total number of cells in the incubation for which the reagent exhibits specificity, or the total number of cells in the incubation that have a marker for which the reagent exhibits specificity and can bind). A concentration that is less than the optimal yield concentration is generally a concentration or amount of reagent in such a process or step that, when the cells that are bound to the reagent are recovered, the yield of bound cells, e.g., CD4+ T cells and / or CD8+ T cells, is less than 100%, e.g., 70% or less. In some embodiments, a yield of 50% or less, 45% or less, 40% or less, 30% or less, or 25% or less, or about 50% or less, about 45% or less, about 40% or less, about 30% or less, or about 25% or less is achieved by a concentration of affinity reagent below the optimal concentration. The concentration can be expressed in terms of the number or mass of particles or surfaces per cell and / or the number of mass or molecules of reagent (e.g., antibody, such as antibody fragment) per cell. In certain embodiments, the concentration below the optimal yield concentration is sufficient to elicit or achieve a fixed, controlled, and / or predetermined ratio of naive-like CD4+ T cells to naive-like CD8+ T cells.

[0134] In some embodiments, for example when working at a concentration less than the optimal yield concentration for each or one or more of two or more selection reagents with affinity for CD4+T cells and / or CD8+T cells, one or more of such reagents are used at a higher concentration than one or more of the other such reagents to bias the ratio of the cell type recognized by that reagent compared to the cell type recognized by the other reagent. For example, a reagent that specifically binds to a marker for which it is desired to bias the ratio can be included at a concentration (e.g., agent or mass per cell) that is increased by 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more compared to the other, depending on how much it is desired to increase the ratio.

[0135] In some embodiments, the amount of immunoaffinity reagent is proportional to the approximate yield of enriched cells when working in a suboptimal range and / or with enough cells to saturate the reagent. In certain embodiments, the appropriate amount or concentration of immunoaffinity reagent, depending on the desired ratio of the resulting composition containing enriched or selected CD4+ T cells and CD8+ T cells, can be determined by routine methods.

[0136] In some embodiments, the separation and / or isolation steps are performed using magnetic beads to which the immunoaffinity reagents are reversibly bound, for example via peptide ligand interactions with streptavidin muteins as described in WO 2015 / 164675. Exemplary of such magnetic beads are Streptamers®. In some embodiments, the separation and / or isolation steps are performed using magnetic beads such as those commercially available from Miltenyi Biotec.

[0137] In some embodiments, the first selection or enrichment of CD4+ and CD8+ cells from the sample is performed using an immunoaffinity-based reagent comprising at least a first and a second affinity chromatography matrix, respectively, on which antibodies are immobilized. In some embodiments, one or both of the first and / or second selections can use multiple affinity chromatography matrices and / or antibodies, in which case the multiple matrices and / or antibodies used in the same selection, i.e., the first or second selection, are connected in series. In some embodiments, the one or more affinity chromatography matrices used in the first and / or second selections have a concentration of at least about 50×10 6 cells / mL, 100×10 6 cells / mL, 200×10 6 cells / mL or 400 x 10 6 Cells / mL can be adsorbed or selected or concentrated. In some embodiments, the adsorption capacity can be adjusted based on the diameter and / or length of the column. In some embodiments, the culture-initiating ratio of the selected or concentrated composition is achieved by choosing a sufficient amount of matrix to achieve the culture-initiating ratio, and / or in a sufficient relative amount to achieve the culture-initiating ratio, based on, for example, the adsorption capacity of one or more columns for selecting cells.

[0138] In an exemplary embodiment, the CD4+ T cells and CD8+ T cells have equal or similar proportions of naive-like cells, and the adsorptive capacity of the matrix or matrices is the same between the first and second selections, e.g., 1×10 for both. 8 cells / mL or approximately 1 x 10 8cells / mL, where the enrichment or selection of cells in the first and second selection results in a composition containing CD4+ cells to CD8+ cells with a naive-like CD4+ cell to CD8+ T cell ratio of 1:1 or about 1:1. In certain embodiments, the appropriate volume, diameter or number of affinity matrix chromatography columns for the first and / or second selection can be routinely selected or determined depending on the proportion of naive-like cells and the desired ratio of the input cell composition to be generated.

[0139] In some embodiments, the adsorption capacity of one or more column matrices is adjusted to take into account the difference in frequency of naive-like cells, such as naive-like CD4+ or CD8+ cells, compared with the frequency of the cells of the respective CD4+ or CD8+ parent population in the starting sample from the subject.The relative level or frequency of various cell populations in the subject can be determined based on the evaluation of the surface expression of one or more markers present on such populations or subpopulations.A number of well-known methods can be used to evaluate the expression level of surface markers or surface proteins, such as affinity-based methods, such as immunoaffinity-based methods, such as flow cytometry detection in the context of cell surface proteins.

[0140] In some embodiments, naive-like cells, such as naive-like CD4+T cells and / or CD8+T cells, are evaluated, measured and / or detected in a cell composition, such as a CD4+T cell composition and / or a CD8+T cell composition, or a sample, such as a biological sample. In some embodiments, a naive-like T cell is a T cell that is positive for the expression of one or more markers that indicate that the cell is naive and / or a naive-like cell. In certain embodiments, a naive-like T cell is a cell that is positive for the expression of a marker associated with a naive or naive-like state in a T cell. In certain embodiments, a naive-like T cell is a T cell that is negative for the expression of one or more markers that indicate that the cell is not naive and / or a naive-like cell. In certain embodiments, a naive-like T cell is a cell that is negative for the expression of a marker associated with a non-naive or non-naive-like state in a T cell. In certain embodiments, non-naive or non-naive-like states in T cells include, but are not limited to, effector T (TEFF) cells, memory T cells, central memory T cells (TCM), effector memory T (TEM) cells, and combinations thereof.

[0141] In some embodiments, the naive-like T cell is positive for the expression of at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten markers that indicate that the cell is naive and / or a naive-like cell and / or that are associated with a naive or naive-like state in T cells. In some embodiments, the markers are expressed on the cell surface. In certain embodiments, the naive-like T cell is negative for the expression of at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten markers that indicate that the cell is non-naive and / or a non-naive-like cell and / or that are associated with a non-naive or naive-like state in T cells.

[0142] Markers that indicate that T cells are naive and / or naive-like T cells and / or are associated with naive or naive-like state in T cells include, but are not limited to, CD27, CD28, CD45RA, CD62L and / or CCR7. In some embodiments, naive-like T cells, such as naive-like CD4+T cells and / or CD8+T cells, are positive for the expression of CD27, CD28, CD45RA and / or CCR7. In certain embodiments, naive-like T cells are positive for the surface expression of one or more of CD27, CD28, CD45RA and / or CCR7. In some embodiments, naive-like T cells, such as naive-like CD4+T cells and / or CD8+T cells, are negative for the expression of CD62L.

[0143] Markers that indicate that a cell is non-naive and / or a non-naive-like T cell and / or are associated with a non-naive or non-naive-like state in T cells include, but are not limited to, CD25, CD45RO, CD56, KLRG1 and / or CD95. In some embodiments, naive-like T cells, such as naive-like CD4+ T cells and / or CD8+ T cells, are negative for the expression of CD25, CD45RO, CD56 and / or KLRG1. In certain embodiments, naive-like T cells, such as naive-like CD4+ T cells and / or CD8+ T cells, have low expression of markers associated with non-naive or non-naive-like cells. In certain embodiments, naive-like T cells have low expression of CD95. In certain embodiments, naive-like T cells are negative for the surface expression of one or more of CD25, CD45RO, CD56 and / or KLRG1.

[0144] In some embodiments, low expression of a marker associated with non-naive or non-naive-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% lower than expression of the marker in a cell that is a non-naive-like cell and / or in a cell that is indicative of the cell being a non-naive and / or non-naive-like T cell and / or is positive for one or more markers associated with a non-naive or non-naive-like state in T cells. In certain embodiments, low expression of a marker associated with non-naive or non-naive-like cells is indicative of effector T (T EFF ) cells, memory T cells, central memory T cells (T CM ) and / or Effector Memory T(T EM ) is or contains 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% lower than expression of that marker in the cell.

[0145] In some embodiments, the markers that indicate that the cell is non-naive and / or non-naive-like T cell and / or that are associated with the non-naive or non-naive-like state in T cells include one or more cytokines.For example, in certain embodiments, non-naive or non-naive-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 certain embodiments, the one or more cytokines are expressed intracellularly by non-naive-like T cells, for example, during or after treatment with an agent that prevents, inhibits or reduces secretion.

[0146] In certain embodiments, naive-like T cells are positive for the expression, e.g., surface expression, of CD45RA and CCR7. In certain embodiments, naive-like CD4+ T cells are positive for the expression, e.g., surface expression, of CD45RA and CCR7. In some embodiments, naive-like CD8+ T cells are positive for the expression, e.g., surface expression, of CD45RA and CCR7. In certain embodiments, naive-like 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 certain embodiments, naive-like 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 embodiments, naive-like 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.

[0147] In certain embodiments, the CD4+T cells and / or CD8+T cells are viable cells. In certain embodiments, the CD4+T cells and / or CD8+T cells are viable naive-like cells. Viable cells are positive for the expression of markers that indicate that the cells undergo normal functional cellular processes and / or are not undergoing or are not in the process of undergoing necrosis or programmed cell death. In some embodiments, viability can be assessed by the redox potential of cells, 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 signs of cell death in assays.

[0148] In certain embodiments, cell viability is assessed by assays that 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, viable cells are negative for the expression of one or more apoptotic markers, such as annexin V or active caspase 3. In some embodiments, viable cells are negative for the expression or activation of one or more apoptotic markers, such as, but are not limited to, caspases, such as caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, and caspase 10, Bcl-2 family members, such as Bax, Bad, and Bid, annexin V, and / or TUNEL staining.

[0149] In some embodiments, expression is or includes the amount, level, concentration, and / or presence of a marker. In particular embodiments, the marker is a polypeptide. In some embodiments, the marker is an mRNA. In some embodiments, expression is or includes the amount, level, concentration, and / or presence of a polypeptide, e.g., a marker polypeptide. In certain embodiments, expression is or includes the amount, level, concentration, and / or presence of a polynucleotide encoding a marker, e.g., an mRNA or a cDNA derived from that mRNA. In certain embodiments, expression is or includes the amount, level, concentration, and / or presence of a marker on the cell surface or exposed on the cell surface or in the cell membrane. In certain embodiments, expression is or includes the amount, level, concentration, and / or presence of a marker on the cell surface or exposed on the cell surface or in the cell membrane. In particular embodiments, expression is or includes intracellular expression, e.g., the amount, level, concentration, and / or presence of a marker within a cell, e.g., in the cytosol, nucleus, endoplasmic reticulum, and / or Golgi apparatus.

[0150] In some embodiments, the markers are measured, evaluated and / or quantified by performing an in vitro assay. In some examples, the in vitro assay is an immunoassay, an aptamer-based assay, a histological or cytological assay, or an mRNA expression level assay. In some cases, the in vitro assay used can be an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry assay, surface plasmon resonance (SPR), chemiluminescence assay, lateral flow immunoassay, inhibition assay or avidity assay. In some embodiments, the expression of the markers is measured, evaluated and / or quantified by RNA-seq. In certain embodiments, the expression of the markers is measured, evaluated and / or quantified by immunostaining techniques. In certain embodiments, the expression of the markers is measured, evaluated and / or quantified by flow cytometry analysis. In some embodiments, the expression of the markers is measured, evaluated and / or quantified by intracellular cytokine staining.

[0151] In some embodiments, markers are measured, assessed and / or quantified in cells of the CD4+ T cell composition. In certain 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-like CD4+ T cells. In certain embodiments, 10%-50%, 20%-60%, 25%-75%, 30%-80%, 40%-90%, 50%-100%, 30%-50%, 40%-60%, 50%-70%, 60%-80%, 70%-90%, 80%-100%, 5%-25%, 25%-50%, 50%-75%, or 75%-99% of the CD4+ T cells are naive-like CD4+ T cells. In certain embodiments, the naive-like CD4+ T cells are viable naive-like CD4+ T cells.

[0152] In certain embodiments, markers are measured, assessed and / or quantified in cells of the CD8+ T cell composition. In certain 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-like CD8+ T cells. In certain embodiments, 10%-50%, 20%-60%, 25%-75%, 30%-80%, 40%-90%, 50%-100%, 30%-50%, 40%-60%, 50%-70%, 60%-80%, 70%-90%, 80%-100%, 5%-25%, 25%-50%, 50%-75%, or 75%-99% of the CD8+ T cells are naive-like CD4+ T cells. In certain embodiments, the naive-like CD8+ T cells are viable naive-like CD8+ T cells.

[0153] In some embodiments, cells from the composition of CD4+ T cells are mixed or combined with cells from the composition of CD8+ T cells in an amount and / or ratio sufficient to produce an input cell composition having a ratio of CD4+ naive-like T cells to CD8+ naive-like T cells of between 10:1 and 0.05:1, 8:1 and 0.1:1, 5:1 and 0.2:1, 2.5:1 and 0.25:1, 2.2:1 and 0.8:1, 2:1 and 0.5:1, or 1.5:1 and 1:1, inclusive. In some embodiments, the cells are mixed in an amount and / or ratio sufficient for a ratio of CD4+ naive-like T cells to CD8+ naive-like T cells of between 2.2:1 and 0.8:1, inclusive. In certain embodiments, the cells are mixed or combined to provide a ratio of CD4+ naive-like T cells to CD8+ naive-like T cells of 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 or about 2.2:1, about 2.1:1, about 2.0:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1.1:1, about 1.0:1, about 0.9:1 or about 0.8:1. In certain embodiments, the cells are mixed or combined to provide a ratio of 1.1:1 or about 1.1:1.

[0154] In some embodiments, to produce an input cell composition with a predetermined ratio of CD4+ naive-like T cells to CD8+ naive-like T cells, 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8, 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×10 8 , 4.5×10 8 , 5×10 8 , 5×10 8 Or 1×10 9 Pieces or about 5 x 10 5 , about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×10 8 , about 5×10 8 Or about 1 x 10 9The amount of total CD4+ T cells or total viable CD4+ T cells is 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 , 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×10 8 , 4.5×10 8 , 5×10 8 , 5.5×10 8 Or 1×10 9 Pieces or about 5 x 10 5 , about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×108 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×10 8 , about 5.5×10 8 Or about 1 x 10 9 In certain embodiments, 1×10 to 1×10 total CD8+ T cells or total viable CD8+ T cells are mixed or combined with 1×10 total CD8+ T cells to produce an input cell composition with a predetermined ratio of CD4+ naive-like T cells to CD8+ naive-like T cells. 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 1 × 10 total CD4+ T cells or total viable CD4+ T cells 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 pcs or approx. 1×10 6 ~1×10 10 , about 1×10 7 ~1×10 9 , about 5×10 7 ~5×10 8 Or about 1 x 10 8 ~3×10 8 The amount of total CD8+ T cells or total viable CD8+ T cells is mixed or combined with the amount of total CD8+ T cells.

[0155] In some embodiments, to produce an input cell composition with a predetermined ratio of CD4+ naive-like T cells to CD8+ naive-like T cells, 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 , 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×10 8 , 4.5×10 8 , 5×10 8 , 5×10 8 Or 1×10 9 Pieces or about 5 x 10 5 , about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8, about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×10 8 , about 5×10 8 Or about 1 x 10 9 5 × 10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 , 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×10 8 , 4.5×10 8 , 5×10 8 , 5.5×10 8 Or 1×10 9Pieces or about 5 x 10 5 , about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×10 8 , about 5.5×10 8 Or about 1 x 10 9 In certain embodiments, to produce an input cell composition with a predetermined ratio of CD4+ naive-like T cells to CD8+ naive-like T cells, 5×10 5 ~1×10 10 , 1×10 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 5 × 10 naive-like CD4 T cells 5 ~1×10 10 , 1×106 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 Pieces or about 5 x 10 5 ~1×10 10 , about 1×10 6 ~1×10 10 , about 1×10 7 ~1×10 9 , about 5×10 7 ~5×10 8 Or about 1 x 10 8 ~3×10 8 The cells are mixed or combined with an amount of naive-like CD8+ T cells.

[0156] In certain embodiments, the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells of the input cell composition is adjusted, modified and / or altered as compared to the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells of a sample, e.g., a biological sample. In certain embodiments, the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells is adjusted, modified and / or altered as compared to the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells of a sample, e.g., a biological sample. In certain embodiments, the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells is adjusted, modified and / or altered as compared to the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells of a biological sample, e.g., ... %, about 90%, about 95%, about 100%, about 1 fold, about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 20 fold, about 50 fold, about 100 fold or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold. In certain embodiments, the sample is the sample from which the cells of the input cell composition were derived and from which they were isolated, selected and / or obtained.

[0157] In certain embodiments, the input cell composition contains a ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells, e.g., a predetermined ratio, a controlled ratio, and / or a fixed ratio. In particular embodiments, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells is 10:1 to 0.05:1, 8:1 to 0.1:1, 5:1 to 0.2:1, 2.5:1 to 0.25:1, 2.2:1 to 0.8:1, 2:1 to 0.5:1, or 1.5:1 to 1:1, inclusive. 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, 1.6:1 and 0.8:1, 1.4:1 and 0.8:1, 1.2:1 and 0.8:1, or 1.2:1 and 0.8:1, inclusive. In some embodiments, the ratio is between 2.2:1 and 0.8:1, inclusive. In certain embodiments, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells is 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, or is about 2.2:1, about 2.1:1, about 2.0:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1.1:1, about 1.0:1, about 0.9:1 or about 0.8:1. In certain embodiments, the ratio is 1.1:1 or about 1.1:1.

[0158] In certain embodiments, the input cell composition has an amount of total cells or total viable cells of 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×108 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 , 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×10 8 , 4.5×10 8 , 5×10 8 , 5×10 8 Or 1×10 9 pcs or approx. 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×108 , about 5×10 8 Or about 1 x 10 9 In certain embodiments, the input cell composition has an amount of cells expressing CD4 or CD8 of 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 , 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×10 8 , 4.5×10 8 , 5×10 8 , 5×10 8 Or 1×10 9 Pieces or about 5 x 10 5 , about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×108 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×10 8 , about 5×10 8 Or about 1 x 10 9 In some embodiments, the input cell composition has an amount of CD45RA+ / CCR7+ / CD4+ T cells and CD45RA+ / CCR7+ / CD8+ T cells of 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 , 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×108 , 4.5×10 8 , 5×10 8 , 5×10 8 Or 1×10 9 Pieces or about 5 x 10 5 , about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×10 8 , about 5×10 8 Or about 1 x 10 9 There are 10.

[0159] In certain embodiments, the input cell composition comprises 1×10 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 or 1×10 8 ~3×10 8In certain embodiments, the input cell composition has an amount of cells expressing CD4 or CD8 of 5×10 5 ~1×10 10 , 1×10 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 Pieces or about 5 x 10 5 ~1×10 10 , about 1×10 6 ~1×10 10 , about 1×10 7 ~1×10 9 , about 5×10 7 ~5×10 8 Or about 1 x 10 8 ~3×10 8 In some embodiments, the input cell composition has an amount of CD45RA+ / CCR7+ / CD4+ T cells and CD45RA+ / CCR7+ / CD8+ T cells of 5×10 5 ~1×10 10 , 1×10 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 Pieces or about 5 x 10 5 ~1×10 10 , about 1×10 6 ~1×10 10 , about 1×10 7 ~1×10 9 , about 5×10 7 ~5×10 8 Or about 1 x 10 8 ~3×10 8 There are 10.

[0160] 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% CD45RA+ / CCR7+ cells. In certain embodiments, the input cell composition contains or comprises 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 90% or less, or 85% or less CD45RA+ / CCR7+ cells.

[0161] In some embodiments, to produce an input cell composition with a predetermined ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells, 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 , 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×108 , 4.5×10 8 , 5×10 8 , 5×10 8 Or 1×10 9 Pieces or about 5 x 10 5 , about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×10 8 , about 5×10 8 Or about 1 x 10 9 The amount of total CD4+ T cells or total viable CD4+ T cells is 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×108 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 , 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×10 8 , 4.5×10 8 , 5×10 8 , 5.5×10 8 Or 1×10 9 Pieces or about 5 x 10 5 , about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×108 , about 5×10 8 , about 5.5×10 8 Or about 1 x 10 9 In certain embodiments, 5×10 to 5×10 total CD8+ T cells or total viable CD8+ T cells are mixed or combined with an input cell composition having a predetermined ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells. 5 ~1×10 10 , 1×10 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 5 × 10 total CD4+ T cells or total viable CD4+ T cells 5 ~1×10 10 , 1×10 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 Pieces or about 5 x 10 5 ~1×10 10 , about 1×10 6 ~1×10 10 , about 1×10 7 ~1×10 9 , about 5×10 7 ~5×10 8 Or about 1 x 10 8 ~3×10 8 The amount of total CD8+ T cells or total viable CD8+ T cells is mixed or combined with the amount of total CD8+ T cells.

[0162] In certain embodiments, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells of the input cell composition has been adjusted, modified and / or altered as compared to the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells of the sample, e.g., biological sample. In certain embodiments, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells is determined from a biological sample by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1x, 1.5x, 2x, 2.5x, 3x, 4x, 5x, 10x, 20x, 50x, 100x or about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60% , about 70%, about 80%, about 90%, about 95%, about 100%, about 1 fold, about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 20 fold, about 50 fold, about 100 fold or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold. In certain embodiments, the sample is the sample from which the cells of the input cell composition were derived and from which they were isolated, selected and / or obtained.

[0163] In certain embodiments, the input cell composition contains a ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells, e.g., a predetermined ratio, a controlled ratio, and / or a fixed ratio. In particular embodiments, the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells is 10:1 to 0.05:1, 8:1 to 0.1:1, 5:1 to 0.2:1, 2.5:1 to 0.25:1, 2.2:1 to 0.8:1, 2:1 to 0.5:1, or 2:1 to 1:1, inclusive. 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, inclusive. In some embodiments, the ratio is between 1.8:1 and 1.6:1, inclusive. In certain 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 certain embodiments, the ratio is or is about 1.69:1.

[0164] In certain embodiments, the input cell composition has an amount of total cells or total viable cells of 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8, 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×10 8 , 4.5×10 8 , 5×10 8 , 5×10 8 or 1×10 9 , about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×10 8 , about 5×10 8 or about 1×10 9cells. In certain embodiments, the input cell composition has an amount of cells expressing CD4 or CD8 that is 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 , 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×10 8 , 4.5×10 8 , 5×10 8 , 5×10 8 or 1×10 9 cells or about 5×10 5 , about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8, about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×10 8 , about 5×10 8 Or about 1 x 10 9 In some embodiments, the input cell composition has an amount of CD27+ / CCR7+ / CD4+ T cells and CD27+ / CCR7+ / CD8+ T cells of 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 , 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×10 8 , 4.5×10 8 , 5×10 8 , 5×108 Or 1×10 9 Pieces or about 5 x 10 5 , about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×10 8 , about 5×10 8 Or about 1 x 10 9 There are 10.

[0165] In certain embodiments, the input cell composition comprises 1×10 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 or 1×10 8 ~3×10 8 In certain embodiments, the input cell composition has an amount of cells expressing CD4 or CD8 of 5×10 5 ~1×10 10, 1×10 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 Pieces or about 5 x 10 5 ~1×10 10 , about 1×10 6 ~1×10 10 , about 1×10 7 ~1×10 9 , about 5×10 7 ~5×10 8 Or about 1 x 10 8 ~3×10 8 In some embodiments, the input cell composition has an amount of CD27+ / CCR7+ / CD4+ T cells and CD27+ / CCR7+ / CD8+ T cells of 5×10 5 ~1×10 10 , 1×10 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 Pieces or about 5 x 10 5 ~1×10 10 , about 1×10 6 ~1×10 10 , about 1×10 7 ~1×10 9 , about 5×10 7 ~5×10 8 Or about 1 x 10 8 ~3×10 8 There are 10 pieces.

[0166] 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% CD27+ / CCR7+ cells. In certain embodiments, the input cell composition contains or comprises 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 90% or less, or 85% or less CD27+ / CCR7+ cells.

[0167] In some embodiments, to produce an input cell composition with a predetermined ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells, 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 , 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×108 , 4.5×10 8 , 5×10 8 , 5×10 8 Or 1×10 9 Pieces or about 5 x 10 5 , about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×10 8 , about 5×10 8 Or about 1 x 10 9 The amount of total CD4+ T cells or total viable CD4+ T cells is 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×108 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 , 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×10 8 , 4.5×10 8 , 5×10 8 , 5.5×10 8 Or 1×10 9 Pieces or about 5 x 10 5 , about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×108 , about 5×10 8 , about 5.5×10 8 Or about 1 x 10 9 In certain embodiments, 5×10 to 5×10 total CD8+ T cells or total viable CD8+ T cells are mixed or combined with 5×10 total CD8+ T cells to produce an input cell composition with a predetermined ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells. 5 ~1×10 10 , 1×10 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 5 × 10 total CD4+ T cells or total viable CD4+ T cells 5 ~1×10 10 , 1×10 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 Pieces or about 5 x 10 5 ~1×10 10 , about 1×10 6 ~1×10 10 , about 1×10 7 ~1×10 9 , about 5×10 7 ~5×10 8 Or about 1 x 10 8 ~3×10 8 The amount of total CD8+ T cells or total viable CD8+ T cells is mixed or combined with the amount of total CD8+ T cells.

[0168] In certain embodiments, the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells of the input cell composition has been adjusted, modified and / or altered as compared to the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells of the sample, e.g., biological sample. In certain embodiments, the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells is determined from a biological sample by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold or about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about Adjusted, modified or altered by 70%, about 80%, about 90%, about 95%, about 100%, about 1 fold, about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 20 fold, about 50 fold, about 100 fold or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold. In certain embodiments, the sample is the sample from which the cells of the input cell composition were derived and from which they were isolated, selected and / or obtained.

[0169] In certain embodiments, the input cell composition contains a ratio of certain CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells, such as a predetermined ratio, a controlled ratio, and / or a fixed ratio. In certain embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells is from 10:1 to 0.05:1, from 8:1 to 0.1:1, from 5:1 to 0.2:1, from 2.5:1 to 0.25:1, from 2.2:1 to 0.8:1, from 2:1 to 0.5:1 or from 1.5:1 to 1:1, including the values at both ends. In certain embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells is from 2:1 to 0.8:1, from 1.6:1 to 0.8:1, from 1.4:1 to 0.8:1, from 1.2:1 to 0.8:1 or from 1.2:1 to 0.8:1, including the values at both ends. In some embodiments, the ratio is from 2.2:1 to 0.8:1, including the values at both ends. In certain embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells is 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, or about 2.2:1, about 2.1:1, about 2.0:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1.1:1, about 1.0:1, about 0.9:1 or about 0.8:1. In certain embodiments, the ratio is 1.1:1, or about 1.1:1.

[0170] In certain embodiments, the input cell composition is 1×10 6 ~1×10 10 、1×10 7 ~1×10 9 、5×10 7 ~5×10 8 or 1×10 8 ~3×10 8 total cells or total viable cells. In certain embodiments, the input cell composition has an amount of cells expressing CD4 or CD8 of 1×10 6 ~1×1010 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 1 x 10 pieces or approx. 6 ~1×10 10 , about 1×10 7 ~1×10 9 , about 5×10 7 ~5×10 8 Or about 1 x 10 8 ~3×10 8 In some embodiments, the input cell composition has an amount of CD62L- / CCR7+ / CD4+ T cells and CD62L- / CCR7+ / CD8+ T cells of 1×10 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 1 x 10 pieces or approx. 6 ~1×10 10 , about 1×10 7 ~1×10 9 , about 5×10 7 ~5×10 8 Or about 1 x 10 8 ~3×10 8 There are 10.

[0171] 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% CD62L- / CCR7+ cells. In certain embodiments, the input cell composition contains or comprises 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 90% or less, or 85% or less CD62L- / CCR7+ cells.

[0172] In some embodiments, to produce an input cell composition with a predetermined ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells, 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 , 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×10 8 , 4.5×108 , 5×10 8 , 5×10 8 Or 1×10 9 1 x 10 pieces or approx. 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×10 8 , about 5×10 8 Or about 1 x 10 9 Total CD4+ T cells or total viable CD4+ T cells in an amount of 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1.0×10 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×108 , 2.0×10 8 , 2.1×10 8 , 2.2×10 8 , 2.3×10 8 , 2.4×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3.0×10 8 , 3.5×10 8 , 4.0×10 8 , 4.5×10 8 , 5×10 8 , 5.5×10 8 Or 1×10 9 pcs or approx. 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4.0×10 8 , about 4.5×10 8 , about 5×10 8 , about 5.5×10 8 Or about 1 x 10 9In certain embodiments, 1×10 to 1×10 total CD8+ T cells or total viable CD8+ T cells are mixed or combined with 1×10 total CD8+ T cells to produce an input cell composition with a predetermined ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells. 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 1 × 10 total CD4+ T cells or total viable CD4+ T cells 6 ~1×10 10 , 1×10 7 ~1×10 9 , 5×10 7 ~5×10 8 Or 1×10 8 ~3×10 8 pcs or approx. 1×10 6 ~1×10 10 , about 1×10 7 ~1×10 9 , about 5×10 7 ~5×10 8 Or about 1 x 10 8 ~3×10 8 The amount of total CD8+ T cells or total viable CD8+ T cells is mixed or combined with the amount of total CD8+ T cells.

[0173] In certain embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells of the input cell composition has been adjusted, modified and / or altered as compared to the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells of the sample, e.g., biological sample. In certain embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells is determined from a biological sample by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold or about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, About 70%, about 80%, about 90%, about 95%, about 100%, about 1 fold, about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 20 fold, about 50 fold, about 100 fold or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold. In certain embodiments, the sample is the sample from which the cells of the input cell composition were derived and from which they were isolated, selected and / or obtained.

[0174] In some embodiments, the step of producing, generating and / or creating the input cell composition comprises one or more steps of mixing or combining cells of the CD4+ T cell composition with cells of the 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 that is between 2.2:1 and 0.8:1. In certain embodiments, prior to the mixing or combining, the number, number per volume, number 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, assessed and / or quantified. In some embodiments, the amount, level, number, number per volume, number per weight and / or percentage of CD45RA+ / CCR7+ cells are measured, assessed and / or quantified by detecting CD45RA+;CCR7+ T cells. In certain embodiments, the input cell composition has a ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells of 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 1.1:1 or about 1.1:1.

[0175] In some embodiments, the step of producing, generating and / or creating the input cell composition comprises one or more steps of mixing or combining cells of the CD4+ T cell composition with cells of the 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 that is between 2.4:1 and 1:1. In certain embodiments, prior to mixing or combining, the number, number per volume, number 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, assessed and / or quantified. In some embodiments, the amount, level, number, number per volume, number per weight and / or percentage of CD27+ / CCR7+ cells are measured, assessed and / or quantified by detecting CD45RA+;CCR7+ T cells. In certain embodiments, the input cell composition has a ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells of between 2.4:1 and 1:1. In some embodiments, the input cell composition has a ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells of at or about 1.69:1.

[0176] In some embodiments, the step of producing, generating and / or creating the input cell composition comprises one or more steps of mixing or combining cells of the CD4+ T cell composition with cells of the 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 that is between 2.2:1 and 0.8:1. In certain embodiments, prior to the mixing or combining, the number, number per volume, number 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, assessed and / or quantified. In some embodiments, the amount, level, number, number per volume, number per weight and / or percentage of CD62L- / CCR7+ cells are measured, assessed and / or quantified by detecting CD62L- / CCR7+ T cells. In certain embodiments, the input cell composition has a ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells of between 2.2:1 and 0.8:1. In some embodiments, the input cell composition has a ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells of 1.1:1 or about 1.1:1.

[0177] In some embodiments, the step of producing, generating and / or creating the input cell composition comprises one or more steps of mixing or combining cells of the CD4+ T cell composition with cells of the CD8+ T cell composition to produce an input cell composition having a ratio of naive-like CD4+ T cells to naive-like CD8+ T cells that is between 2.2:1 and 0.8:1. In certain embodiments, prior to the mixing or combining, the number, number per volume, number per weight, and / or amount, level or percentage of naive-like cells in the CD4+ T cell composition and the CD8+ T cell composition are measured, assessed and / or quantified. In some embodiments, the amount, level, number, number per volume, number per weight and / or percentage of naive-like cells are measured, assessed and / or quantified by detecting CD45RA+;CCR7+ T cells. In certain embodiments, the input cell composition has a ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells of 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 1.1:1 or about 1.1:1.

[0178] B. Cell Activation or Stimulation In some embodiments, the cells are incubated and / or cultured before and / or in association with and / or after the process for genetic engineering. In certain embodiments, the cells are incubated and / or cultured before, during, or immediately after one or more steps associated with a genetic engineering protocol or process. The incubation step can include culturing, cultivating, stimulating, activating, and / or expanding. The incubation and / or manipulation can be performed in a culture vessel, such as a unit, chamber, well, column, tube, tube set, valve, vial, culture dish, bag, or other vessel for culturing or cultivating cells. In some embodiments, the composition or cells are incubated under stimulatory conditions or in the presence of a stimulant. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in a population, and / or to mimic antigen exposure, and / or to prime cells for genetic engineering, such as for the introduction of recombinant antigen receptors.

[0179] In some embodiments, the method provided herein comprises one or more steps of incubating cells under stimulating or activating conditions.In some embodiments, cells are incubated for 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 or about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 12 hours, about 16 hours, about 18 hours, about 24 hours, about 36 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days or more than 7 days. In some embodiments, the cells are incubated for 5-90 minutes, 1-4 hours, 2-8 hours, 6-24 hours, 12-48 hours, 16-32 hours, 18-30 hours, 1-4 days, or 2-7 days. In some embodiments, the cells are incubated for 2-15 days, 2-12 days, 2-10 days, 2-8 days, 2-6 days, 2-4 days, 4-12 days, 4-10 days, 4-8 days, 4-6 days, 6-12 days, 6-10 days, 6-8 days, 8-12 days, 8-10 days, or 10-12 days. In some embodiments, the cells are incubated for at least 2 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.

[0180] In some embodiments, the cells are genetically engineered by a method or process that includes one or more steps for gene transfer. In some embodiments, gene transfer is accomplished by first stimulating the cells, for example by incubating the cells under stimulatory conditions, for example by incubating the cells with a stimulus that induces a response such as proliferation, survival and / or activation, and then performing gene transfer. In some embodiments, the cells are incubated under stimulatory conditions prior to gene transfer to activate the cells. In some embodiments, gene transfer is or includes transduction of activated cells. In certain embodiments, the stimulatory agent or stimulatory conditions induce and / or can induce primary signal, signal transduction, stimulation, activation and / or expansion of cells.

[0181] In some embodiments, cells, such as the cells of the input cell composition, are incubated under stimulating or activating conditions before gene transfer.In some embodiments, cells are incubated under stimulating or activating conditions for 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 or about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 12 hours, about 16 hours, about 18 hours, about 24 hours, about 36 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days or more than 7 days. In some embodiments, the cells of the input cell composition are incubated under stimulatory or activating conditions for 5-90 minutes, 1-4 hours, 2-8 hours, 6-24 hours, 12-48 hours, 16-32 hours, 18-30 hours, 1-4 days, or 2-7 days prior to gene transfer. In certain embodiments, the cells of the input cell composition are incubated under stimulatory or activating conditions for 2-15 days, 2-12 days, 2-12 days, 2-8 days, 2-6 days, 2-4 days, 4-12 days, 4-10 days, 4-8 days, 4-6 days, 6-12 days, 6-10 days, 6-8 days, 8-12 days, 8-10 days, or 10-12 days. In some embodiments, the cells are incubated for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 days.

[0182] In some embodiments, cells are incubated under stimulating or activating conditions during part or all of gene transfer.For example, in certain embodiments, cells are incubated under stimulating conditions during part or all of the transduction or transfection of cells that is carried out to introduce heterologous nucleic acid.In certain embodiments, cells are incubated under stimulating or activating conditions in the presence of viral vectors, such as gamma retroviral vectors or lentiviral vectors.

[0183] In certain embodiments, the cells are incubated under stimulatory or activating conditions after gene transfer, e.g., immediately after gene transfer. In some embodiments, the cells are incubated under stimulatory or activating conditions after gene transfer, e.g., to expand the cells to sufficient numbers for clinical application. In some embodiments, the cells are incubated under stimulating or activating conditions after gene transfer for 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 or for about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 12 hours, about 16 hours, about 18 hours, about 24 hours, about 36 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days or more than about 7 days. In some embodiments, the cell composition is incubated under stimulating or activating conditions after gene transfer for 5-90 minutes, 1-4 hours, 2-8 hours, 6-24 hours, 12-48 hours, 16-32 hours, 18-30 hours, 1-4 days, or 2-7 days before gene transfer. In some embodiments, the cells of the composition are incubated under stimulating or activating conditions after gene transfer for 2-15 days, 2-12 days, 2-12 days, 2-8 days, 2-6 days, 2-4 days, 4-12 days, 4-10 days, 4-8 days, 4-6 days, 6-12 days, 6-10 days, 6-8 days, 8-12 days, 8-10 days, or 10-12 days. In some embodiments, the cells are incubated for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 days. In some embodiments, the cells are expanded for a sufficient time to generate cell numbers suitable for clinical application.

[0184] The conditions can include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions and / or stimuli such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells.

[0185] In some embodiments, the stimulatory condition or agent induces and / or is capable of inducing a primary signal, signaling, stimulation, activation and / or expansion of a cell.

[0186] In some embodiments, the stimulatory conditions or stimuli include one or more agents, e.g., ligands, that can activate the intracellular signaling domain of the TCR complex. In some aspects, the agents activate or initiate the TCR / CD3 intracellular signaling cascade in T cells. Such agents can include antibodies, such as antibodies specific for the TCR, e.g., anti-CD3. In some embodiments, the stimulatory conditions include one or more agents, e.g., ligands, e.g., anti-CD28, that can stimulate a costimulatory receptor. In some embodiments, such agents and / or ligands may be bound to a solid support, such as beads, and / or one or more cytokines. Optionally, the expansion method may further include adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulatory agents include IL-2, IL-15, and / or IL-7. In certain embodiments, the agents are antibodies that specifically bind to the recombinant receptor. In certain embodiments, the recombinant receptor is an antigen-binding receptor, e.g., a CAR, and the agent is an anti-idiotypic antibody that specifically binds to the antigen-binding receptor.

[0187] In some embodiments, the agent and / or ligand is attached to magnetic beads, such as paramagnetic beads (e.g., Dynalbeads or MACS beads). In certain embodiments, the beads can be removed from the cells by placing the cells in a magnetic field to remove and / or separate the beads from the cells, e.g., debeading the cells. In certain embodiments, the beads are removed immediately after incubation to activate the cells. In some embodiments, the beads are removed prior to gene delivery, such as by transduction or transfection. In certain embodiments, the beads are removed during or immediately after gene delivery. In particular embodiments, the beads are removed prior to or during incubation to expand the cells. In some embodiments, the beads are removed prior to harvesting and / or cryoprotecting the cells.

[0188] In some aspects, the incubation is carried out according to techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother., 35(9):651-660, Terakura et al. (2012) Blood. 1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0189] 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 the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded) and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells can include gamma-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000-3600 rads to block cell division. In some aspects, the feeder cells are added to the culture medium prior to the addition of the population of T cells.

[0190] In some embodiments, the stimulatory conditions include a temperature suitable for the proliferation of human T lymphocytes, such as at least about 25°C, generally at least about 30°C, and generally at or about 37°C. Optionally, the incubation may further include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. The LCL can be irradiated with gamma radiation in the range of about 6000-10,000 rads. The LCL feeder cells in some aspects are provided in any suitable amount, such as at a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.

[0191] In embodiments, antigen-specific T cells, such as antigen-specific CD4+ T cells and / or CD8+ T cells, are obtained by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, antigen-specific T cell lines or antigen-specific T cell clones can be generated against a cytomegalovirus antigen by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen.

[0192] C. Genetic Engineering In some embodiments, the provided method involves the step of generating or producing a composition of engineered cells, such as a cell composition that contains cells expressing recombinant antigen receptors.Various methods are well known for introducing genetically engineered components, such as recombinant receptors, such as CAR or TCR, and can be used with the provided method and composition.Exemplary methods include the method for transferring the nucleic acid encoding the receptor, such as viral transduction, such as retroviral or lentiviral transduction, transposon and electroporation.

[0193] In certain embodiments, the cells of the input cell composition are contacted with one or more agents containing a nucleic acid, e.g., a nucleic acid encoding a recombinant receptor such as a CAR, and / or the agents are introduced into the cells of the input cell composition. In some embodiments, the agent is a vector, e.g., a viral vector, a plasmid, or a transposon. In some embodiments, the cells are CD4+ T cells. In certain embodiments, the cells are CD8+ T cells. In particular embodiments, the step of introducing a nucleic acid into the cells and / or contacting the cells with a nucleic acid results in an engineered cell with a predetermined, desired, or 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 the cell or sample obtained from the cell, e.g., a nucleic acid obtained from another organism or cell that is not normally found in the cell being engineered and / or the organism from which such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, and is not found in nature, e.g., a nucleic acid that includes a chimeric combination of nucleic acids encoding various domains from multiple different cell types.

[0194] In some embodiments, the preparation method includes a step for freezing, e.g., cryopreserving, the cells before, e.g., immediately before, between, or after, e.g., immediately after, any of the steps or stages for genetic engineering and / or the steps or stages for producing an output cell composition containing genetically engineered cells. In certain embodiments, the cells are cryofreezed before, during, or after the steps of isolating or selecting the cells, mixing or combining the cells into an input cell composition, incubation, activation, gene transfer, transduction, transfection, expansion, 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 step or step during the process of genetically engineering the cells for later or subsequent analysis, e.g., for analysis after the composition of engineered cells is administered to a subject.

[0195] In some embodiments, the freezing and subsequent thawing process removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, for example after a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters may be used in some aspects. One example involves using PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing medium. This is then diluted 1:1 with medium so that the final concentrations of DMSO and HSA are 10% and 4%, respectively. The cells are then typically frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0196] 1. Vectors and methods for genetic manipulation In some embodiments, the recombinant nucleic acid is transferred into the cell using a recombinant infectious viral particle, such as a vector derived from Simian Virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, the recombinant nucleic acid is transferred into the T cell using a recombinant lentiviral or retroviral vector, such as a gamma-retroviral vector (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi:10.1038 / gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10):1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2,e93; Park et al., Trends Biotechnol. 2011 November 29(11):550-557). In some embodiments, the virus is an adeno-associated virus (AAV).

[0197] In some embodiments, retroviral vectors, such as those derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), spleen focus forming virus (SFFV), have long terminal repeats (LTRs). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell source. Retroviruses are typically amphotropic, meaning that they can infect 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. Several examples of retroviral systems have been described (e.g., U.S. Pat. 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).

[0198] Methods of lentiviral transduction are known. Exemplary methods are described, for example, in Wang et al. (2012) J. Immunother. 35(9): 689-701, Cooper et al. (2003) Blood. 101: 1637-1644, Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114 and Cavalieri et al. (2003) Blood. 102(2): 497-505.

[0199] In some embodiments, the viral vector particle contains a genome derived from a retroviral genome-based vector, such as a lentivirus genome or a gamma retrovirus-based vector.In some aspects of the provided viral vector, the heterologous nucleic acid encoding a recombinant receptor, such as an antigen receptor, such as CAR, is contained and / or located between the 5'LTR sequence and the 3'LTR sequence of the vector genome.

[0200] In some embodiments, the viral vector genome is a lentiviral genome, such as HIV-1 genome or SIV genome. For example, lentiviral vectors have been produced by multiple attenuation of virulence genes, for example, deletion of genes env, vif, vpu and nef, which can make 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 constructed to carry the essential sequences for the integration, selection and transfer of foreign nucleic acid into host cells. Known lentiviruses are readily available from depositories or collections such as the American Type Culture Collection ("ATCC", 10801 University Boulevard, Manassas, VA 20110-2209), or can be isolated from known sources using publicly available techniques.

[0201] 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 infectious anemia virus (E1AV). For example, lentiviral vectors have been generated by multiple attenuation of HIV virulence genes, such as deletion of genes env, vif, vpr, vpu and nef to make the vector 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 viral-based and are constructed to carry the necessary sequences for integration of foreign nucleic acid, selection and transfer of the nucleic acid into a host cell. Known lentiviruses are readily available from depositories or collections such as the American Type Culture Collection ("ATCC", 10801 University Boulevard, Manassas, VA 20110-2209) or can be isolated from known sources using publicly available techniques.

[0202] In some embodiments, the viral genome vector can contain the sequence of 5'LTR and 3'LTR of retrovirus such as lentivirus.In some aspects, the viral genome construct can contain the sequence from 5'LTR and 3'LTR of lentivirus, and in particular, can contain the R sequence and U5 sequence from 5'LTR of lentivirus and the inactivated or self-inactivated 3'LTR of lentivirus.The LTR sequence can be the LTR sequence from any species of lentivirus.For example, they can be the LTR sequence from HIV, SIV, FIV or BIV.Typically, the LTR sequence is the HIV LTR sequence.

[0203] In some embodiments, the nucleic acid of a viral vector, such as an HIV viral vector, lacks additional transcription units. The vector genome can contain an inactivated or self-inactivating 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 produce the viral vector RNA can be used to generate a self-inactivating (SIN) vector. This deletion can then be transferred to the 5'LTR of the proviral DNA during reverse transcription. Self-inactivating vectors generally have a deletion of enhancer and promoter sequences from the 3' long terminal repeat (LTR), which are copied to the 5'LTR upon vector integration. In some embodiments, sufficient sequences can be removed, including removal of the TATA box, to abolish the transcriptional activity of the LTR. This can prevent the production of full-length vector RNA in transduced cells. In some aspects, the U3 element of the 3'LTR contains deletion of its enhancer sequence, TATA box, Sp1 and NF-kappa B site. As a result of the self-inactivating 3'LTR, the provirus generated after entry and reverse transcription contains an inactivated 5'LTR. This can improve safety by reducing the risk of mobilization of the vector genome and the effect of LTR on nearby cellular promoters. The self-inactivating 3'LTR can be constructed by any method known in the art. In some embodiments, it does not affect the titer of the vector or the properties of the vector in vitro or in vivo.

[0204] Optionally, the U3 sequence from lentivirus 5'LTR can be replaced with a promoter sequence in the virus construct, for example, a heterologous promoter sequence. This can increase the titer of the virus that is recovered from packaging cell line. Enhancer sequences can also be included. Any enhancer / promoter combination that increases the expression of viral RNA genome in packaging cell line can be used. In one example, the CMV enhancer / promoter sequence is used (US Pat. No. 5,385,839 and US Pat. No. 5,168,062).

[0205] In certain embodiments, the risk of insertional mutagenesis can be minimized by constructing a retroviral vector genome, such as a lentiviral vector genome, to be integration-deficient. Various approaches can be implemented to produce a non-integrating vector genome. In some embodiments, the integrase enzyme component of the pol gene can be engineered with a mutation such that it encodes a protein with an inactive integrase. In some embodiments, the vector genome itself can be modified to prevent integration, for example, by mutating or deleting one or both attachment sites, or by deleting or modifying the 3'LTR-proximal polypurine tract (PPT) to make it non-functional. In some embodiments, non-genetic approaches can be utilized, including pharmacological agents that inhibit one or more functions of integrase. These approaches are not mutually exclusive; that is, two or more of them can be used simultaneously. For example, both the integrase and attachment site can be non-functional, the integrase and PPT sites can be non-functional, the attachment site and PPT site can be non-functional, or all of them can 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).

[0206] In some embodiments, the methods provided involve transducing cells by contacting, e.g., incubating, a cell composition comprising a plurality of cells with a viral particle. In some embodiments, the cells to be transfected or transduced are or include primary cells obtained from a subject, e.g., cells enriched and / or selected from a subject.

[0207] In some embodiments, the concentration of transduced cells in the composition is 1.0×10 5 cells / mL~1.0×10 8 cells / mL or approximately 1.0 x 10 5 cells / mL~1.0×10 8 cells / mL, e.g., at least 1.0 x 10 5 cells / mL, 5×10 5 cells / mL, 1×10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL or 1 x 10 8 cells / mL, or at least about 1.0 x 10 5 cells / mL, approximately 5×10 5 cells / mL, approximately 1×10 6 cells / mL, approximately 5×10 6 cells / mL, approximately 1×10 7 cells / mL, approximately 5×10 7 cells / mL or approximately 1 x 10 8 cells / mL, or approximately 1.0 x 10 5 cells / mL, approximately 5×10 5 cells / mL, approximately 1×10 6 cells / mL, approximately 5×10 6 cells / mL, approximately 1×10 7 cells / mL, approximately 5×10 7 cells / mL or approximately 1 x 10 8 In cells / mL.

[0208] In some embodiments, the viral particles are provided at a certain ratio of copies of viral vector particles or their infectious units (IU) per total number of cells to be transduced (IU / cell). For example, in some embodiments, the viral particles are present during the contacting step at an amount of viral vector particles of 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50 or 60 IU or about 0.5, about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 30, about 40, about 50 or about 60 IU per cell or at least about 0.5, about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 30, about 40, about 50 or about 60 IU per cell.

[0209] In some embodiments, the titer of viral vector particles is 1×10 6 IU / mL ~ 1×10 8 IU / mL or approximately 1 × 10 6 IU / mL ~ 1×10 8 IU / mL, e.g. 5×10 6 IU / mL ~ 5 × 10 7 IU / mL or approximately 5 × 10 6 IU / mL ~ 5 × 10 7 IU / mL, e.g., at least 6 × 10 6 IU / mL, 7×10 6 IU / mL, 8×10 6 IU / mL, 9×10 6 IU / mL, 1×10 7 IU / mL, 2×10 7 IU / mL, 3×10 7 IU / mL, 4×10 7 IU / mL or 5 × 10 7 IU / mL.

[0210] In some embodiments, transduction is achieved at a multiplicity of infection (MOI) of less than 100, such as typically less than 60, less than 50, less than 40, less than 30, less than 20, less than 10, less than 5 or less.

[0211] In some embodiments, the method involves contacting or incubating the cells with the viral particles. In some embodiments, the contacting is for a period of 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 at least about 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, or more.

[0212] In some embodiments, the contacting step can be accomplished by centrifugation, e.g., spinoculation (e.g., centrifugal inoculation). In some embodiments, the composition containing the cells, viral particles, and reagents can be spun, generally with relatively little force or at a low speed, e.g., at a speed lower than that used to pellet the cells, e.g., at or about 600 rpm to 1700 rpm (e.g., 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm, or about 600 rpm, about 1000 rpm, or about 1500 rpm, or about 1700 rpm, or at least 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm). In some embodiments, the rotation is between 100g and 3200g or about 100g and 3200g (e.g., 100g, 200g, 300g, 400g, 500g, 1000g, 1500g, 2000g, 2500g, 3000g, or 3200g or about 100g, about 200g, about 300g, about 400g, about 500g, about 1000g, about 1500g, about 2000g, about The rotation is performed with a force such as a relative centrifugal force of 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. The term "relative centrifugal force" or RCF is generally understood to be the inertial force imparted to an object or material (e.g., a cell, sample or pellet and / or a point in a chamber or other container being rotated) at a particular point in space relative to the axis of rotation, relative to the gravity of the earth. Its value can be determined using well known formulas, taking into account gravity, the speed of rotation and the radius of rotation (the distance from the axis of rotation to the object, material or particle whose RCF is being measured).

[0213] In certain embodiments, the input cells are treated with, incubated with, or contacted with particles that contain binding molecules that bind to or recognize the recombinant receptor encoded by the viral DNA.

[0214] In some embodiments, the recombinant nucleic acid is transferred into the T cell by electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3):e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16):1431-1437). In some embodiments, the recombinant nucleic acid is transferred into the T cell by transposition (see, e.g., 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 genetic material into and expressing it in immune cells include calcium phosphate transfection (e.g., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), protoplast fusion, cationic liposome-mediated transfection, tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346:776-777 (1990)), and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)).

[0215] Other approaches and vectors for the transfer of nucleic acids encoding recombinant products are described, for example, in International Patent Application Publication No. WO2014055668 and U.S. Patent No. 7,446,190.

[0216] In some embodiments, recombinant nucleic acid is transferred into T cells by transposon. Examples of transposons suitable for use with mammalian cells, such as 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, this system includes a transposon engineered to contain foreign DNA (also referred to herein as cargo DNA), such as a gene encoding a recombinant receptor, flanked by inverted / direct repeat (IR / DR) sequences that are recognized by the associated transposase. In some embodiments, a non-viral plasmid encodes the transposase under the control of a promoter. In some embodiments, transfection of the plasmid into a host cell results in the transient expression of the transposase at a level sufficient to integrate the transposon into genomic DNA.

[0217] Sleeping Beauty (SB) is a synthetic member of the Tc / 1-mariner superfamily of transposons reconstructed from a dormant element harbored in the salmonid fish genome. SB transposon-based transfection is a two-component system consisting of a transposase and a transposon containing inverted repeat / direct repeat (IR / DR) sequences that result in precise integration into TA dinucleotides. The transposon is designed with an expression cassette of interest flanked by the IR / DR. SB transposase binds to a specific binding site located on the IR of the Sleeping beauty transposon. SB transposase mediates the integration of the transposon, a mobile element (SB) that encodes a cargo sequence flanked on both sides by inverted terminal repeats that harbor binding sites for catalytic enzymes. Appropriate expression occurs when SB inserts the gene sequence into the TA target dinucleotide in the vertebrate chromosome by a cut-and-paste mechanism. This system has been used to engineer various vertebrate cell types, including primary human peripheral blood leukocytes. In some embodiments, cells are contacted with, incubated with, and / or treated with SB transposon, which comprises a cargo gene flanked by SB IR sequences, such as a gene encoding a recombinant receptor or CAR. In certain embodiments, cells to be transfected are contacted with, incubated with, and / or treated with a plasmid comprising a SB transposon, which comprises a cargo gene flanked by SB IR sequences, such as a gene encoding a CAR. In certain embodiments, the plasmid further comprises a gene encoding SB transposase that is not flanked by SB IR sequences.

[0218] PiggyBac (PB) is another transposon system that can be used to integrate cargo DNA into host, e.g., human, genomic DNA. PB transposase recognizes PB transposon-specific inverted terminal repeats (ITRs) located at both ends of the transposon, and efficiently moves its contents from its original location and efficiently integrates them into the TTAA chromosomal site. The PB transposon system allows the gene of interest between the two ITRs in the PB vector to be mobilized into the target genome. The PB system has been used to engineer various vertebrate cell types, including primary human cells. In some embodiments, the cells to be transfected are contacted with, incubated with, and / or treated with a PB transposon that contains a cargo gene, e.g., a gene encoding a CAR, flanked by PB IR sequences. In certain embodiments, cells to be transfected are contacted, incubated with, and / or treated with a plasmid containing a PB transposon that contains a cargo gene, such as a gene encoding a CAR, flanked by PB IR sequences. In certain embodiments, the plasmid further contains a gene encoding an SB transposase that is not flanked by PB IR sequences.

[0219] In some embodiments, transposon-mediated transduction is performed using a plasmid containing a transposase gene and a plasmid containing a transposon containing a cargo DNA sequence flanked by inverted / direct repeat (IR / DR) sequences recognized by the transposase. In certain embodiments, the cargo DNA sequence encodes a heterologous protein, such as a recombinant T cell receptor or CAR. In some embodiments, the plasmid contains a transposase and a transposon. In some embodiments, the transposase is under the control of a ubiquitous promoter or any promoter suitable for driving the expression of the transposase in target cells. Ubiquitous 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 for the selection of cells in which the cargo DNA is stably integrated into genomic DNA. Suitable selection cassettes include, but are not limited to, selection cassettes encoding a kanamycin resistance gene, a spectinomycin resistance gene, a streptomycin resistance gene, an ampicillin resistance gene, a carbenicillin resistance gene, a hygromycin resistance gene, a bleomycin resistance gene, an erythromycin resistance gene, and a polymyxin B resistance gene.

[0220] In some embodiments, a plasmid containing components for transposon-mediated transduction, such as SB transposase and SB transposon, is introduced into the target cell. Any convenient protocol can be used, which may result in in vitro or in vivo introduction of the components of the system into the target cell, depending on the location of the target cell. For example, if the target cell is an isolated cell, the system can be directly introduced into the cell, such as by using standard transformation techniques, under cell culture conditions that allow the survival of the target cell. Such techniques include, but are not limited to, viral infection, transformation, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, viral vector delivery, and the like. The choice of method generally depends on the type of cell to be transformed and the context in which the transformation is performed (i.e., in vitro, ex vivo, or in vivo). A review of these methods can be found in Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley&Sons, 1995.

[0221] In some embodiments, the SB transposon and the SB transposase source are introduced into a target cell of a multicellular organism, for example, a mammal or a human, under conditions sufficient for the excision of the nucleic acid flanked by the inverted repeat sequences from the vector carrying the transposon, and the subsequent integration of the excised nucleic acid into the genome of the target cell. Some embodiments further comprise a step of ensuring that the required transposase activity is present in the target cell together with the introduced transposon. Depending on the structure of the transposon vector itself, i.e., whether the vector contains a region that codes for a product with transposase activity, the method may further comprise a step of introducing a second vector that codes for the required transposase activity into the target cell.

[0222] In some embodiments, the amount of vector nucleic acid containing transposon and the amount of vector nucleic acid encoding transposase that are introduced into cells are sufficient to cause the desired excision and insertion of transposon nucleic acid into the genome of target cells.Thus, the amount of vector nucleic acid introduced should cause a sufficient amount of transposase activity and a sufficient copy number of the nucleic acid that is desired to be inserted into target cells.The amount of vector nucleic acid introduced into target cells will vary depending on the efficiency of the specific introduction protocol used, for example the specific ex vivo administration protocol used.

[0223] Once the vector DNA is combined with the required transposase and enters the target cell, the nucleic acid region of the vector flanked by the inverted repeats, i.e., the vector nucleic acid located between the inverted repeats recognized by the Sleeping Beauty transposase, is excised from the vector by the provided transposase and inserted into the genome of the target cell. Thus, the introduction of the vector DNA into the target cell is followed by transposase-mediated excision and insertion of the exogenous nucleic acid carried by the vector into the genome of the target cell. In certain embodiments, the vector is integrated into the genome of 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 cells transfected with the SB transposon 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 continues to exist in the target cell genome, not transiently, and is transmitted to the progeny of the target cell as part of the chromosomal genetic material.

[0224] In certain embodiments, transposons are used to integrate nucleic acids, i.e., polynucleotides, of various sizes into a target cell genome. In some embodiments, the size of the DNA inserted into a target cell genome using the method ranges from about 0.1 kb to about 200 kb, about 0.5 kb to about 100 kb, about 1.0 kb to about 8.0 kb, about 1.0 to about 200 kb, about 1.0 to about 10 kb, about 10 kb to about 50 kb, about 50 kb to about 100 kb, or about 100 kb to about 200 kb. In some embodiments, the size of the DNA inserted into a target cell genome using the method ranges from about 1.0 kb to about 8.0 kb. In some embodiments, the size of the DNA inserted into a target cell genome using the method ranges from about 1.0 to about 200 kb. In certain embodiments, the size of the DNA inserted into a target cell genome using the method ranges from about 1.0 kb to about 8.0 kb.

[0225] In some embodiments, cells, such as T cells, can be transfected with a T cell receptor (TCR) or a chimeric antigen receptor (CAR) either during or after expansion. This transfection to introduce the gene of the desired receptor can be carried out, for example, with any suitable retroviral vector. The genetically modified cell population can then be released from the first stimulation (e.g., anti-CD3 / anti-CD28 stimulation) and then stimulated with a second type of stimulation, for example, via the newly introduced receptor. This second type of stimulation can include antigen stimulation in the form of a peptide / MHC molecule, a cognate (bridged) ligand of the genetically introduced receptor (e.g., the natural ligand of the CAR), or any ligand (such as an antibody) that directly binds to the framework of the new receptor (e.g., by recognizing the constant region in the receptor). See, e.g., 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).

[0226] In some cases, vectors can be used that do not require activating cells, such as T cells. In some such cases, cells can be selected and / or transduced prior to activation. Thus, cells can be manipulated prior to culturing the cells, or after culturing the cells, and in some cases, simultaneously with at least a portion of the culturing.

[0227] Among the additional nucleic acids, e.g., genes for transfer, are those to improve the efficacy of therapy, e.g., by enhancing the survival and / or function of the transferred cells, those to provide genetic markers for selecting and / or evaluating cells, e.g., for assessing survival or localization in vivo, and those to improve safety, e.g., by rendering cells susceptible to negative selection in vivo, as described in Lupton SD et al., Mol. and Cell Biol., 11:6 (1991) and Riddell et al., Human Gene Therapy 3:319-338 (1992). See also PCT / US91 / 08442 and PCT / US94 / 05601 publications by Lupton et al., which describe the use of bifunctional selectable fusion genes derived by fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., U.S. Patent No. 6,040,177 to Riddell et al., columns 14-17.

[0228] 2. Recombinant Receptors In some embodiments, the cells used in the provided methods or the cells administered in the context of the provided methods contain or are engineered to contain engineered receptors, such as engineered antigen receptors, such as chimeric antigen receptors (CARs) or T cell receptors (TCRs). Also provided are populations of such cells, compositions containing such cells and / or enriched in such cells, such as compositions enriched or selected for certain types of cells, such as T cells or CD8+ or CD4+ T cells. Compositions include pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are methods for administering cells and compositions to subjects, such as patients, according to the provided methods and / or the provided articles of manufacture or compositions.

[0229] In some embodiments, the cells contain one or more nucleic acids introduced by genetic engineering, thereby expressing recombinant or genetically engineered products of such nucleic acids. In some embodiments, gene transfer is achieved by first stimulating the cells, such as by combining with a stimulus that induces a response, such as proliferation, survival and / or activation, as measured by expression of a cytokine or activation marker, and then transducing and expanding the activated cells to sufficient numbers for clinical use.

[0230] The cells generally 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). Receptors also include other chimeric receptors.

[0231] a. Chimeric antigen receptor (CAR) In some embodiments of the provided methods and uses, the chimeric receptor, e.g., chimeric antigen receptor, contains one or more domains that combine a ligand binding domain (e.g., an antibody or antibody fragment) that confers specificity 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 that provides a primary activation signal, e.g., a T cell activation domain. In some embodiments, the intracellular signaling domain contains, or additionally contains, a costimulatory signaling domain to enhance effector function. In some embodiments, the chimeric receptor, when engineered into immune cells, can regulate the activity of T cells and, in some cases, regulate the differentiation or homeostasis of T cells, thereby resulting in engineered cells with improved in vivo longevity, survival and / or persistence, e.g., for use in gene cell therapy.

[0232] In some embodiments, engineered cells, e.g., T cells, are provided that express a CAR with specificity for a particular antigen (or marker or ligand), e.g., an antigen expressed on the surface of a particular cell type. In some embodiments, the antigen is a polypeptide. In some embodiments, the antigen is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on disease or condition cells, e.g., tumor or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or expressed on engineered cells.

[0233] In certain embodiments, the recombinant receptor, e.g., the chimeric receptor, contains an intracellular signaling region, which includes a cytoplasmic signaling domain (also interchangeably referred to as an intracellular signaling domain), such as a cytoplasmic (intracellular) region that can induce a primary activation signal in a T cell, e.g., a cytoplasmic signaling domain of a T cell receptor (TCR) component (e.g., the cytoplasmic signaling domain of the ζ chain of the CD3 zeta (CD3ζ) chain, or a functional variant or signaling portion thereof), and / or includes an immunoreceptor tyrosine-based activation motif (ITAM).

[0234] In some embodiments, the chimeric receptor further comprises an extracellular ligand-binding domain that specifically binds to the ligand (e.g., antigen) antigen. In some embodiments, the chimeric receptor is a CAR that comprises an extracellular antigen recognition domain that specifically binds to the antigen. In some embodiments, the ligand, such as an antigen, is a protein that is expressed on the surface of a cell. In some embodiments, the CAR is a TCR-like CAR, and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which is recognized on the cell surface in association with a major histocompatibility complex (MHC) molecule, like a TCR.

[0235] Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells are described in, for example, International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. 2002131960, 2013287748, 20130149337, U.S. Patent Nos. 6,451, 6,515, 6,525, 6,535, 6,545, 6,555, 6,565, 6,575, 6,585, 6,590, 6,665, 6,770, 6,785, 6,790, 6,810, 6,825, 6,910, 6,925, 6,930, 6,945, 6,950, 6,965, 6,970, 6,985, 6,990, 7,102, 7,110, 7,125, 7,130, 7,145, 7,150, 7,160, 7,170, 7,185, 7,190, 7,192, 7,174, 7,196, 7,185, 7,197, 7,198, 7,199, 8,103, 8,104, 8,105, 8,106, 8 Nos. 1,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP 2537416, and / or those described in Sadelain et al., Cancer Discov. 2013 April;3(4):388-398; Davila et al. (2013) PLoS ONE 8(4):e61338; Turtle et al., Curr.Opin.Immunol., 2012 October;24(5):633-39; Wu et al., Cancer, 2012 March 18(2):160-75. In some aspects, the antigen receptor includes CARs described in U.S. Patent No. 7,446,190 and those described in International Patent Application Publication No. WO / 2014055668 A1.Examples of CARs include CARs disclosed in any of the above publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, U.S. Pat. No. 7,446,190, U.S. Pat. No. 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, U.S. Patent No. 7,446,190, and U.S. Patent No. 8,389,282.

[0236] In some embodiments, CARs are constructed with specificity for a particular antigen (or marker or ligand) intended to induce an attenuated response, such as an antigen expressed in a particular cell type targeted by adoptive therapy, e.g., a cancer marker, and / or an antigen expressed on a normal or non-diseased cell type. Thus, a CAR typically comprises, in its extracellular portion, one or more antigen-binding molecules, such as one or more antigen-binding fragments, domains, or portions, or one or more antibody variable domains, and / or antibody molecules. In some embodiments, a CAR comprises a variable heavy chain (VH) of a monoclonal antibody (mAb), H ) and variable light chain (V L ) and / or antigen-binding portions of an antibody molecule, such as single chain antibody fragments (scFv) derived from

[0237] In some embodiments, the antibody or its antigen-binding portion is expressed on cells as part of a recombinant receptor, such as an antigen receptor. Among antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). In general, CARs containing antibodies or antigen-binding fragments that exhibit TCR-like specificity for peptide-MHC complexes can also be called TCR-like CARs. In some embodiments, the extracellular antigen-binding domain specific for the MHC-peptide complex of the TCR-like CAR is linked to one or more intracellular signaling components, in some aspects via a linker and / or a transmembrane domain. In some embodiments, such molecules can typically mimic or approximate the signal through natural antigen receptors, such as TCRs, and optionally the signal through such receptors in combination with costimulatory receptors.

[0238] In some embodiments, recombinant receptors, such as chimeric receptors (e.g., CARs), comprise a ligand-binding domain that binds, e.g., specifically binds, to an antigen (or ligand). Some of the antigens targeted by chimeric receptors are expressed in association with the disease, condition, or cell type that is targeted via adoptive cell therapy. Some of the diseases and conditions include proliferative, neoplastic, and malignant diseases and disorders, including blood cancers, immune system cancers, for example, B, T, and myeloid leukemias, lymphomas, and cancers and tumors, including lymphomas, leukemias, and / or myelomas, such as multiple myeloma.

[0239] In some embodiments, the antigen (or ligand) is a polypeptide. In some embodiments, the antigen is a carbohydrate or other molecule. In some embodiments, the antigen (or ligand) is selectively expressed or overexpressed on disease or condition cells, e.g., tumor or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or expressed on engineered cells.

[0240] In some embodiments, the CAR contains an antibody or antigen-binding fragment (e.g., scFv) that specifically recognizes an antigen expressed on the surface of a cell, such as an intact antigen.

[0241] In some embodiments, the antigen (or ligand) is a tumor antigen or a cancer marker. In some embodiments, the antigen (or ligand) is selected from the group consisting of 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, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, chondroitin sulfate proteoglycan 4 (CSPG4), EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin 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 alpha (IL-22Rα), kinase insert domain receptor (kdr), kappa light chain, leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, preferentially expressed antigen in melanoma (PRAME), survivin, tumor-associated glycoprotein 72 (TAG72), 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 antigen, cancer-testis antigen, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), natural killer group 2 member D (NKG2D) ligand, cancer / testis antigen 1B (CTAG; also known as NY-ESO-1 and LAGE-2), melan A (MART-1), glycoprotein 100 (gp100), glypican 3 (GPC3), G protein-coupled receptor 5D (GPRC5D), tumor embryonic antigen, TAG72, tyrosinase-related protein 1 (TRP1; also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2; dopachrome The antigen targeted by the receptor may be or include an antigen associated with a B-cell malignancy, such as any of a number of known B-cell markers, including any of a number of known B-cell markers. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igκ, Igλ, CD79a, CD79b, or CD30.

[0242] In some embodiments, the antigen is a pathogen-specific antigen. In some embodiments, the antigen is a viral antigen (e.g., a viral antigen from HIV, HCV, HBV, etc.), a bacterial antigen, and / or a parasitic antigen.

[0243] 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 for CD19.In some embodiments, the antibody or antibody fragment that binds to CD19 is a mouse-derived antibody, such as FMC63 and SJ25C1.In some embodiments, the antibody or antibody fragment is a human antibody, such as described in US Patent Publication No. 2016 / 0152723.

[0244] In some embodiments, the scFv is derived from FMC63. FMC63 generally refers to a murine monoclonal IgG1 antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, NR, et al. (1987). Leucocyte typing III. 302). The FMC63 antibody comprises CDRH1 and H2 as shown in SEQ ID NO:38, 39, and CDRH3 as shown in SEQ ID NO:40 or 54, respectively, 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 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO:41. H ), and a light chain variable region (V LIn 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 variable heavy chain region of FMC63 depicted in SEQ ID NO:41, and the variable light chain region of FMC63 depicted in SEQ ID NO:42. In some embodiments, the variable heavy chain and the variable light chain are connected by a linker. In some embodiments, the linker is depicted in SEQ ID NO:56. In some embodiments, the scFv comprises, in order, V H , linker, and V L In some embodiments, the scFv comprises, in order, V L , linker, and V H In some embodiments, the svFc is encoded by a sequence of nucleotides set forth in SEQ ID NO:57, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:57. In some embodiments, the scFv comprises a sequence of amino acids set forth in SEQ ID NO:43, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:43.

[0245] In some embodiments, the scFv is derived from SJ25C1. SJ25C1 is a murine monoclonal IgG1 antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, NR, et al. (1987). Leucocyte typing III. 302). The SJ25C1 antibody comprises CDRH1, H2, and H3 sequences as set forth in SEQ ID NOs:47-49, respectively, and CDRL1, L2, and L3 sequences as set forth in SEQ ID NOs:44-46, respectively. The SJ25C1 antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO:50. H ), and a light chain variable region (V 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 variable heavy chain region of SJ25C1 depicted in SEQ ID NO:50, and the variable light chain region of SJ25C1 depicted in SEQ ID NO:51. In some embodiments, the variable heavy chain and the variable light chain are connected by a linker. In some embodiments, the linker is depicted in SEQ ID NO:52. In some embodiments, the scFv comprises, in order, V H , linker, and V L In some embodiments, the scFv comprises, in order, V L , linker, and V H In some embodiments, the scFv comprises a sequence of amino acids set forth in SEQ ID NO:53, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:53.

[0246] In some embodiments, the antigen or antigen-binding domain is BCMA. In some embodiments, the scFv contains a VH and a VL derived from an antibody or antibody fragment specific for BCMA. In some embodiments, the antibody or antibody fragment that binds to BCMA is or contains a VH and a VL derived from an antibody or antibody fragment shown in International Patent Application Publication Nos. WO 2016 / 090327 and WO 2016 / 090320.

[0247] 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 for GPRC5D.In some embodiments, the antibody or antibody fragment that binds to GPRC5D is or contains VH and VL derived from the antibody or antibody fragment shown in International Patent Application Publication Nos. WO 2016 / 090329 and WO 2016 / 090312.

[0248] In some embodiments, the CAR contains a TCR-like antibody, for example, an antibody or antigen-binding fragment (e.g., scFv) that specifically recognizes an intracellular antigen, such as a tumor-associated antigen that is presented as an MHC-peptide complex on the cell surface. In some embodiments, the antibody or antigen-binding portion thereof that recognizes the MHC-peptide complex can be expressed on a cell as part of a recombinant receptor, for example, an antigen receptor. Among the antigen receptors are functional non-TCR antigen receptors, for example, chimeric antigen receptors (CARs). In general, a CAR that contains an antibody or antigen-binding fragment that shows TCR-like specificity for peptide-MHC complexes can also be called a TCR-like CAR.

[0249] References to the "major histocompatibility complex" (MHC) generally refer to proteins, generally glycoproteins, that contain polymorphic peptide-binding sites or grooves that can form complexes with peptide antigens of polypeptides that include peptide antigens processed by cellular machinery. In some cases, MHC molecules can be displayed or expressed on the cell surface, including as complexes with peptides, i.e., MHC-peptide complexes, for presentation of antigens in a conformation recognizable by antigen receptors on T cells, such as TCRs or TCR-like antibodies. Generally, MHC class I molecules are heterodimers having a transmembrane α chain with, in some cases, three α domains, and non-covalently associated β2-microglobulin. Generally, MHC class II molecules are composed of two transmembrane glycoproteins, α and β, both of which typically span the membrane. MHC molecules can include one or more antigen-binding sites for binding peptides, and the active portion of the MHC that contains the sequences necessary for recognition by appropriate antigen receptors. In some embodiments, MHC class I molecules deliver peptides of cytosolic origin to the cell surface, where the MHC-peptide complex is generally recognized by CD8 + T cells, although in some cases by T cells such as CD4+ T cells. In some embodiments, MHC class II molecules deliver peptides of endocytic origin to the cell surface, where they are typically recognized by CD4 + T cells. Generally, MHC molecules are encoded by a cluster of linked loci collectively referred to as H-2 in mice and human leukocyte antigen (HLA) in humans. Thus, typically, the human MHC can also be referred to as the human leukocyte antigen (HLA).

[0250] The term "MHC-peptide complex" or "peptide-MHC complex" or variations thereof refer to a complex or association of a peptide antigen with an MHC molecule, for example, generally by non-covalent interaction of the peptide in the binding groove or cleft of the MHC molecule. In some embodiments, the MHC-peptide complex is present or displayed on the surface of a cell. In some embodiments, the MHC-peptide complex can be specifically recognized by an antigen receptor, such as a TCR, a TCR-like CAR, or an antigen-binding portion thereof.

[0251] In some embodiments, a peptide, such as a peptide antigen or epitope of a polypeptide, can associate with an MHC molecule, for example for recognition by an antigen receptor. Generally, a peptide is derived from or based on a fragment of a longer biological molecule, such as a polypeptide or protein. In some embodiments, a peptide is typically about 8 to about 24 amino acids in length. In some embodiments, a peptide has a length of 9 to 22 amino acids or about 9 to 22 amino acids for recognition in an MHC class II complex. In some embodiments, a peptide has a length of 8 to 13 amino acids or about 8 to 13 amino acids for recognition in an MHC class I complex. In some embodiments, upon recognition of a peptide in association with an MHC molecule, such as an MHC-peptide complex, an antigen receptor, such as a TCR or a TCR-like CAR, generates or triggers an activation signal to a T cell, which induces a T cell response, such as T cell proliferation, cytokine production, a cytotoxic T cell response, or other response.

[0252] In some embodiments, the TCR-like antibodies or antigen-binding portions are known or can be made by known methods (see, e.g., U.S. Patent Application Publication Nos. 2002 / 0150914; 2003 / 0223994; 2004 / 0191260; 2006 / 0034850; 2007 / 00992530; 20090226474; 20090304679; and International PCT Publication No. WO 03 / 068201).

[0253] In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to an MHC-peptide complex can be produced by immunizing a host with an effective amount of an immunogen that contains a specific MHC-peptide complex. In some cases, the peptide of the MHC-peptide complex is an epitope of an antigen that can bind to MHC, such as a tumor antigen, such as a universal tumor antigen, a myeloma antigen, or other antigens described below. In some embodiments, an effective amount of the immunogen is then administered to the host to elicit an immune response, where the immunogen retains its three-dimensional form for a period of time sufficient to elicit an immune response to the three-dimensional presentation of the peptide in the binding groove of the MHC molecule. The serum collected from the host is then assayed to determine whether the desired antibody that recognizes the three-dimensional presentation of the peptide in the binding groove of the MHC molecule has been generated. In some embodiments, the generated antibody can be evaluated to confirm that the antibody can distinguish the MHC-peptide complex from the MHC molecule alone, the peptide of interest alone, and the complex with an unrelated peptide to MHC. The desired antibody can then be isolated.

[0254] In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to MHC-peptide complexes can be generated by using antibody library display methods, such as phage antibody libraries. In some embodiments, for example, a phage display library of mutant Fab, scFv, or other antibody types can be generated, in which members of the library are mutated at one or more residues in one or more CDRs. See, for example, US Patent Application Publication Nos. 20020150914, 2014 / 0294841; and Cohen CJ. et al. (2003) J Mol. Recogn. 16:324-332.

[0255] The term "antibody" is used herein in the broadest sense and includes intact antibodies, as well as fragment antigen-binding (Fab) fragments, F(ab') 2fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chains (V H ) regions, single chain antibody fragments including single chain variable fragments (scFv), and single domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0256] In some embodiments, the antigen-binding proteins, antibodies, and antigen-binding fragments thereof specifically recognize the antigen of the full-length antibody. In some embodiments, the heavy and light chains of the antibody can be full-length or can be antigen-binding portions (Fab, F(ab')2, Fv, or single-chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly, for example, IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g., human IgG1). In another embodiment, the antibody light chain constant region is selected from, for example, kappa or lambda, particularly kappa.

[0257] Among the antibodies provided are antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab') 2; diabody; single-chain antibody; variable heavy chain (V H ) region, scFv and single-domain V H Single-chain antibody molecules such as single antibodies; and multispecific antibodies formed from antibody fragments, including but not limited to those. In certain embodiments, the antibody is a single-chain antibody fragment comprising a variable heavy chain region and / or a variable light chain region, such as an scFv.

[0258] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ), respectively) generally have similar structures, and each domain comprises four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single V H or V L domain may be sufficient to confer antigen-binding specificity. Furthermore, an antibody that binds a particular antigen can be isolated using the V H or V L domain from an antibody that binds the antigen, and a library of complementary V L or V H domains can be screened. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0259] Single domain antibody is an antibody fragment that comprises all or a part of the heavy chain variable domain of an antibody, or all or a part of the light chain variable domain.In certain embodiments, single domain antibody is a human single domain antibody.In some embodiments, CAR comprises an antibody heavy chain domain that specifically binds to an antigen, such as a cancer marker or a cell surface antigen of a targeted cell or disease, such as a tumor cell or cancer cell, such as any of the target antigens described herein or known in the art.

[0260] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, e.g., a fragment that contains a non-naturally occurring configuration, such as having two or more antibody regions or chains linked by synthetic linkers, e.g., peptide linkers, and / or a configuration that cannot be generated by enzymatic digestion of naturally occurring intact antibodies. In some embodiments, the antibody fragment is an scFv.

[0261] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody may optionally include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody typically refers to a variant of a non-human antibody that has undergone humanization to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0262] Thus, in some embodiments, the chimeric antigen receptor comprising the TCR-like CAR comprises an extracellular portion containing an antibody or an antibody fragment. In some embodiments, the antibody or fragment comprises an scFv. In some aspects, the chimeric antigen receptor comprises an extracellular portion containing an antibody or a fragment, and an intracellular signaling region. In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain capable of inducing a primary activation signal in T cells, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM).

[0263] In some embodiments, the recombinant receptor, e.g., CAR, e.g., an antibody portion thereof, further comprises a spacer, which comprises a hinge region, e.g., an IgG4 hinge region, and / or a C H 1 / C LThe recombinant receptor may be or may comprise at least a portion of an immunoglobulin constant region or a variant or modified version thereof, such as an Fc region. In some embodiments, the recombinant receptor further comprises a spacer and / or a hinge region. In some embodiments, the constant region or portion is that of a human IgG, such as IgG4 or IgG1. In some aspects, the portion of the constant region serves as a spacer region between the antigen recognition component, e.g., scFv, and the transmembrane domain. The spacer can be of a length that provides increased cellular responsiveness after antigen binding compared to the absence of the spacer. In some examples, the spacer is 12 amino acids or about 12 amino acids in length, or is 12 amino acids or less in length. Exemplary spacers include those having at least about 10-229 amino acids, about 10-200 amino acids, about 10-175 amino acids, about 10-150 amino acids, about 10-125 amino acids, about 10-100 amino acids, about 10-75 amino acids, about 10-50 amino acids, about 10-40 amino acids, about 10-30 amino acids, about 10-20 amino acids, or about 10-15 amino acids, and including any integer between either of the endpoints of the recited ranges. In some embodiments, the spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include an IgG4 hinge alone, an IgG4 hinge linked to a CH2 and CH3 domain, or an IgG4 hinge linked to a CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153 or International Patent Application Publication No. WO2014031687. In some embodiments, the spacer has a sequence as set forth in SEQ ID NO:1 and is encoded by a sequence as set forth in SEQ ID NO:2. In some embodiments, the spacer has a sequence as set forth in SEQ ID NO:3. In some embodiments, the spacer has a sequence as set forth in SEQ ID NO:4.

[0264] In some embodiments, the constant region or portion is of IgD. In some embodiments, the spacer has a sequence set forth in SEQ ID NO:5. In some embodiments, the spacer has an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs:1, 3, 4, and 5. In some embodiments, the spacer has a sequence set forth in SEQ ID NOs:23-31. In some embodiments, the spacer has an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs:23-31.

[0265] In some embodiments, the antigen receptor comprises an intracellular domain directly or indirectly linked to the extracellular domain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an ITAM. For example, in some aspects, the antigen recognition domain (e.g., the extracellular domain) is linked to one or more intracellular signaling components, such as a signaling component that mimics activation through an antigen receptor complex, such as a TCR complex in the case of a CAR, and / or a signal through another cell surface receptor. In some embodiments, the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g., an scFv) and the intracellular signaling domain. Thus, in some embodiments, the antigen binding component (e.g., an antibody) is linked to one or more transmembrane regions and an intracellular signaling region. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the receptor, such as a CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domain to transmembrane domains of the same or different surface membrane proteins and minimize interactions with other members of the receptor complex.

[0266] The transmembrane domain in some embodiments is derived from either natural or synthetic sources. When the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., at least including) 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, CD154. Alternatively, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises primarily hydrophobic residues such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain. In some embodiments, the linkage is by a linker, spacer, and / or transmembrane domain. In some aspects, the transmembrane domain comprises the transmembrane portion of CD28.

[0267] In some embodiments, the extracellular domain and the transmembrane domain can be directly or indirectly linked. In some embodiments, the extracellular domain and the transmembrane domain are linked by a spacer, such as any of those described herein. In some embodiments, the receptor contains the extracellular portion of the molecule from which the transmembrane domain is derived, such as the extracellular portion of CD28.

[0268] Some intracellular signaling domains mimic or approximate signals through natural antigen receptors, signals through such receptors in combination with costimulatory receptors, and / or signals through costimulatory receptors alone. In some embodiments, a short oligopeptide or polypeptide linker, e.g., a linker 2-10 amino acids in length, such as one containing glycine and serine, e.g., a glycine-serine doublet, is present to form a link between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0269] Receptors, e.g., CARs, generally comprise at least one or more intracellular signaling components. In some embodiments, receptors comprise intracellular components of the TCR complex, such as the TCR CD3 chain, e.g., the CD3ζ chain, which mediates T cell activation and cytotoxicity. Thus, in some aspects, ROR1-binding antibodies are linked to one or more cell signaling modules. In some embodiments, the cell signaling modules comprise a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, receptors, e.g., CARs, further comprise a portion of one or more additional molecules, such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, CARs comprise a chimeric molecule between CD3 zeta (CD3ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

[0270] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of immune cells, such as T cells engineered to express the CAR. For example, in some situations, the CAR induces the function of T cells, such as cytolytic activity or T helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling region of an antigen receptor component or a costimulatory molecule is used instead of an intact immunostimulatory chain, for example, when it transmits an effector function signal. In some embodiments, the one or more intracellular signaling domains include the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects also include that of a co-receptor that acts in concert with such receptor in a natural context to initiate signal transduction after antigen receptor binding, and / or any derivative or variant of such molecule, and / or any synthetic sequence with the same functional capability.

[0271] In relation to natural TCR, full activation generally requires not only signal transduction through TCR, but also costimulatory signal.Thus, in some embodiments, CAR also includes components for generating secondary or costimulatory signals to promote full activation.In other embodiments, CAR does not include components for generating costimulatory signals.In some aspects, additional CAR is expressed in the same cell to provide components for generating secondary or costimulatory signals.

[0272] T cell activation, in some aspects, is described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some aspects, the CAR contains one or both of such signaling components.

[0273] In some aspects, CAR comprises a primary cytoplasmic signaling sequence that controls the primary activation of TCR complex.The primary cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif known as immune receptor tyrosine-based activation motif or ITAM.Examples of ITAM-containing primary cytoplasmic signaling sequences include those derived from TCR or CD3zeta, FcRgamma or FcRbeta.In some embodiments, the cytoplasmic signaling molecule in CAR contains a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3zeta.

[0274] In some embodiments, CAR comprises the signaling region and / or transmembrane portion of costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10 and ICOS. In some aspects, the same CAR comprises both signaling region and costimulatory component. In some embodiments, chimeric antigen receptor comprises intracellular domain or its functional variant derived from T cell costimulatory molecule, for example, between transmembrane domain and intracellular signaling domain. In some aspects, T cell costimulatory molecule is CD28 or 41BB.

[0275] In some embodiments, the signaling region and / or activation domain are included in one CAR, while the costimulatory component is provided by another CAR that recognizes another antigen. In some embodiments, the CAR includes an activating or stimulating CAR and a costimulatory CAR, both expressed on the same cell (see WO2014 / 055668). In some aspects, the cell includes one or more stimulating or activating CARs and / or costimulatory CARs. In some embodiments, the cell further includes an inhibitory CAR (iCAR, see Fedorov et al., Sci.Transl.Medicine, 5(215) (December, 2013)), e.g., a CAR that recognizes an antigen other than an antigen associated with and / or specific to a disease or condition, such that the activation signal delivered via the disease-targeting CAR is reduced or inhibited by the inhibitory CAR binding to its ligand, e.g., reducing off-target effects.

[0276] In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane domain and signaling domain linked to a CD3 (e.g., CD3ζ) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) costimulatory domain linked to a CD3ζ intracellular domain.

[0277] In some embodiments, the CAR includes one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., a primary activation domain, in the cytoplasmic portion. Exemplary CARs include the intracellular components CD3zeta, CD28, and 4-1BB.

[0278] In some embodiments, the antigen receptor further comprises a marker, and / or the cells expressing CAR or other antigen receptor further comprise a surrogate marker, such as a cell surface marker that can be used to confirm the transduction or manipulation of the cells to express the receptor. In some aspects, the marker comprises all or a part (e.g., truncated) of CD34, NGFR, or epidermal growth factor receptor, such as a truncated form of such a cell surface receptor (e.g., tEGFR). Exemplary surrogate markers can include truncated cell surface polypeptides, such as truncated forms that are non-functional and do not or cannot transmit a signal, or do not or cannot transmit a signal normally transmitted by the full-length cell surface polypeptide, and / or do not or cannot internalize. Exemplary truncated cell surface polypeptides include truncated growth factor receptors or other receptors, such as truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (tEGFR, an exemplary tEGFR sequence is shown in SEQ ID NO:7 or 16), or prostate specific membrane antigen (PSMA), or modified forms thereof. tEGFR can contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibodies or binding molecules, which can be used to identify or select cells engineered with the tEGFR construct and the encoded exogenous protein, and / or to eliminate or isolate cells expressing the encoded exogenous protein. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April;34(4):430-434. In some aspects, the marker, e.g., surrogate marker, comprises all or a portion of CD34 (e.g., a truncated form), all or a portion of NGFR (e.g., a truncated form), CD19 or a truncated CD19, e.g., a truncated non-human CD19, or all or a portion of epidermal growth factor receptor (e.g., a truncated form) (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 super-fold 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, such as species variants, monomeric variants, and codon-optimized and / or high-sensitivity variants of fluorescent proteins. In some embodiments, the marker is or comprises an enzyme, such as luciferase, the lacZ gene from Escherichia coli (E. coli), alkaline phosphatase, secreted placental alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT). Exemplary luminescent reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or variants thereof.

[0279] In some embodiments, the marker is a selection marker. In some embodiments, the selection marker is or comprises a polypeptide that confers resistance to an exogenous 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 blasticidin resistance gene, a neomycin resistance gene, a geneticin resistance gene, or a zeocin resistance gene, or modified versions thereof.

[0280] In some embodiments, the nucleic acid encoding the marker is operably linked to a linker sequence, e.g., a cleavable linker sequence, e.g., a polynucleotide encoding T2A. For example, the marker, and optionally the linker sequence, can be any of those disclosed in PCT Publication No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR), which may be linked to a linker sequence, such as a T2A cleavable linker sequence. Exemplary polypeptides of truncated EGFR (e.g., tEGFR) include the amino acid sequence set forth in SEQ ID NO:7 or 16, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:7 or 16.

[0281] In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a linker sequence, e.g., a cleavable linker sequence, e.g., an MMP-cleavable linker sequence. For example, the marker, and optionally the linker sequence, can be any of those disclosed in Patent Application Publication No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR), optionally linked to a linker sequence, such as a T2A-cleavable linker sequence.

[0282] In some embodiments, the marker is a molecule that is not naturally found on or on the surface of T cells, e.g., a cell surface protein, or a portion thereof. In some embodiments, the molecule is a non-self molecule, e.g., a non-self protein, i.e., one that is not recognized as "self" by the immune system of the host into which the cells are to be adoptively transferred.

[0283] In some embodiments, the marker does not perform a therapeutic function and / or has no effect other than being used, for example, to select cells in which the manipulation has been successful, as a marker for genetic manipulation. In another embodiment, the marker is a therapeutic molecule or a molecule that otherwise exerts some desirable effect, such as a ligand for cells encountered in vivo, for example, a costimulatory molecule or an immune checkpoint molecule that enhances and / or attenuates the response of cells when engrafted and encountered with the ligand.

[0284] In some cases, the CARs are referred to as first-generation, second-generation, and / or third-generation CARs. In some aspects, a first-generation CAR provides only a CD3-chain-induced signal upon antigen binding; in some aspects, a second-generation CAR provides such a signal and a costimulatory signal, for example, one that includes an intracellular signaling domain derived from a costimulatory receptor such as CD28 or CD137; in some aspects, a third-generation CAR in some aspects includes multiple costimulatory domains of different costimulatory receptors.

[0285] In some embodiments, the chimeric antigen receptor includes an extracellular portion that contains an antibody or fragment described herein. In some aspects, the chimeric antigen receptor includes an extracellular portion that contains an antibody or fragment described herein, and an intracellular signaling domain. In some embodiments, the antibody or fragment is a scFv or single-domain V H contains an antibody, and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain includes the 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.

[0286] In some aspects, the transmembrane domain contains the transmembrane portion of CD28. The extracellular domain and the transmembrane domain can be directly or indirectly linked. In some embodiments, the extracellular domain and the transmembrane domain are linked by a spacer, such as any described herein. In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule, for example, between the transmembrane domain and the intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

[0287] In some embodiments, the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that contains a signaling portion of CD28 or a functional variant thereof and a signaling portion of CD3ζ or a functional variant thereof. In some embodiments, the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that contains 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 comprises a portion of an Ig molecule, such as a human Ig molecule, e.g., a spacer that contains an Ig hinge, e.g., an IgG4 hinge, e.g., a spacer that is just a hinge.

[0288] In some embodiments, the transmembrane domain of a receptor, e.g., a CAR, is the transmembrane domain of human CD28 or a variant thereof, e.g., the 27 amino acid transmembrane domain of human CD28 (Accession No. P10747.1), or is a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8; in some embodiments, the transmembrane domain-containing portion of a recombinant receptor is the transmembrane domain of human CD28 or a variant thereof, e.g., the 27 amino acid transmembrane domain of human CD28 (Accession No. P10747.1), or is a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8; The amino acid sequence set forth in SEQ ID NO:9, or an amino acid sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0289] In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

[0290] In some embodiments, the intracellular signaling region comprises the intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as the 41 amino acid domain thereof and / or such a domain having an LL to GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular signaling domain can comprise an amino acid sequence as set forth in SEQ ID NO:10 or 11, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:10 or 11. In some embodiments, the intracellular region comprises the intracellular costimulatory signaling domain of 4-1BB or a functional variant or portion thereof, e.g., the 42 amino acid cytoplasmic domain of human 4-1BB (Accession No. Q07011.1), or a functional variant or portion thereof, e.g., the amino acid sequence set forth in SEQ ID NO:12, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:12.

[0291] In some embodiments, the intracellular signaling region comprises a human CD3 chain, optionally a CD3 stimulatory signaling domain or a functional variant thereof, such as the 112 AA cytoplasmic domain of human CD3 isoform 3 (Accession No. P20963.2), or a CD3 signaling domain as described in U.S. Patent No. 7,446,190 or U.S. Patent No. 8,911,993. In some embodiments, the intracellular signaling region comprises an amino acid sequence as set forth in SEQ ID NO:13, 14, or 15, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:13, 14, or 15.

[0292] In some aspects, the spacer contains only the hinge region of an IgG, e.g., an IgG4 or IgG1 hinge only, e.g., the hinge only spacer shown in SEQ ID NO:1. H 2 and / or C H In some embodiments, the spacer is an Ig hinge, such as an IgG4 hinge, linked to the C3 domain. H 2 and C H In some embodiments, the spacer is an Ig hinge, such as an IgG4 hinge, linked to the C3 domain. H An Ig hinge linked to only 3 domains, such as an IgG4 hinge. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker, such as a known flexible linker.

[0293] For example, in some embodiments, the CAR comprises an antibody, e.g., an antibody fragment, including an scFv, a spacer, e.g., a portion of an immunoglobulin molecule, e.g., a spacer containing a hinge region and / or one or more constant regions, e.g., an Ig-hinge-containing spacer, a transmembrane domain containing all or a portion of a transmembrane domain from CD28, an intracellular signaling domain from CD28, and a CD3ζ signaling domain. In some embodiments, the CAR comprises an antibody or fragment, e.g., an scFv, a spacer, e.g., an Ig-hinge-containing spacer, a transmembrane domain from CD28, an intracellular signaling domain from 4-1BB, and a signaling domain from CD3ζ.

[0294] In some embodiments, the nucleic acid molecule encoding such a CAR construct further comprises a sequence encoding a T2A ribosomal skipping element and / or a tEGFR sequence, e.g., downstream of the sequence encoding the CAR. In some embodiments, the sequence encodes a T2A ribosomal skipping element as set forth in SEQ ID NO:6 or 17, or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:6 or 17. In some embodiments, T cells expressing an antigen receptor (e.g., CAR) can also be generated to express truncated EGFR (EGFRt) as a non-immunogenic selective epitope (e.g., by introduction of a construct encoding CAR and EGFRt separated by a T2A ribosomal switch to allow the two proteins to be expressed from the same construct), which can then be used as a marker to detect such cells (see, e.g., U.S. Pat. No. 8,802,374). In some embodiments, the sequence encodes a tEGFR sequence as set forth 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 more sequence identity to SEQ ID NO:7 or 16. In some cases, peptides such as T2A can cause the ribosome to skip synthesis of the peptide bond at the C-terminus of the 2A element (ribosomal skipping), resulting in a separation between the end of the 2A sequence and the next peptide downstream (see, e.g., 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 foot and mouth disease virus (F2A, e.g., SEQ ID NO:22), equine rhinitis A virus (E2A, e.g., SEQ ID NO:21), Thosea asigna virus (T2A, e.g., SEQ ID NO:6 or 17), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO:19 or 20), as described in U.S. Patent Application Publication No. 20070116690.

[0295] The recombinant receptor such as CAR expressed by the cell administered to the subject generally recognizes or specifically binds to the molecule that is expressed in, associated with, and / or specific to the disease or condition or cell to be treated.When specifically bound to a molecule, such as an antigen, the receptor generally delivers an immune stimulatory signal, such as ITAM transduction signal, into the cell, thereby promoting the immune response that targets the disease or condition.For example, in some embodiments, the cell expresses CAR that specifically binds to the antigen expressed by the cell or tissue of disease or condition or the cell or tissue associated with disease or condition.

[0296] b. Chimeric Autoantibody Receptor (CAAR) In some embodiments, among the recombinant receptors expressed by engineered cells used in connection with the provided methods, uses, articles of manufacture, and compositions are chimeric autoantibody receptors (CAARs). In some embodiments, CAARs are specific for autoantibodies. In some embodiments, cells expressing CAARs, such as T cells engineered to express CAARs, can be used to specifically bind and kill autoantibody-expressing cells, but not to specifically bind and kill normal antibody-expressing cells. In some embodiments, CAAR-expressing cells can be used to treat autoimmune diseases associated with the expression of autoantigens, such as autoimmune diseases. In some embodiments, CAAR-expressing cells can target B cells that ultimately produce autoantibodies and present autoantibodies on their cell surface, marking these B cells as disease-specific targets for therapeutic intervention. In some embodiments, CAAR-expressing cells can be used to efficiently target and kill pathogenic B cells in autoimmune diseases by targeting disease-causing B cells with antigen-specific chimeric autoantibody receptors. In some embodiments, the recombinant receptor is a CAAR, such as any described in U.S. Patent Application Publication No. 2017 / 0051035.

[0297] In some embodiments, CAAR comprises an autoantibody binding domain, a transmembrane domain, and an intracellular signaling region. In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain capable of inducing a primary activation signal in T cells, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling region comprises a secondary or costimulatory signaling region (secondary intracellular signaling region).

[0298] In some embodiments, the autoantibody binding domain comprises an autoantigen or a fragment thereof. The selection of the autoantigen can depend on the type of autoantibody to be targeted. For example, the autoantigen may be selected because it recognizes an autoantibody on a target cell, such as a B cell, that is associated with a particular disease state, for example, an autoimmune disease, such as an autoantibody-mediated autoimmune disease. In some embodiments, the autoimmune disease comprises pemphigus vulgaris (PV). Exemplary autoantigens include desmoglein 1 (Dsg1) and Dsg3.

[0299] c. Multi-targeting In some embodiments, the cells used in connection with the provided methods, uses, articles of manufacture, and compositions include cells that use a multi-targeting strategy. In some embodiments, the cells express a multi-chain chimeric antigen receptor (CAR) or express two or more engineered receptors on the cell, each recognizing the same of a different antigen, and typically each containing a different intracellular signaling component. Such multi-targeting strategies are described, for example, in International Patent Application Publication No. WO 2014055668 A1 (describes, for example, the combination of an activating CAR and a costimulatory CAR that target off-targets, e.g., two different antigens that are present individually on normal cells, but together only on cells of the disease or condition to be treated), and in Fedorov et al., Sci. Transl. Medicine, 5(215)(2013) (describes cells expressing an activating CAR and an inhibitory CAR, e.g., where the activating CAR binds to one antigen expressed on both normal or non-diseased cells, and cells of the disease or condition to be treated, and the inhibitory CAR binds to another antigen expressed only on normal cells or cells not desired to be treated).

[0300] For example, in some embodiments, the cell generally comprises a receptor expressing a first engineered antigen receptor (e.g., CAR or TCR), which can induce an activation or stimulatory signal to the cell when specifically binding to an antigen, such as the first antigen, generally recognized by the first receptor. In some embodiments, the cell further comprises a second engineered antigen receptor (e.g., CAR or TCR), such as a chimeric costimulatory receptor, which can induce a costimulatory signal to the immune cell when specifically binding to a second antigen, generally recognized by the second receptor. In some embodiments, the first antigen and the second antigen are the same. In some embodiments, the first antigen and the second antigen are different.

[0301] In some embodiments, the first and / or second engineered antigen receptor (e.g., CAR or TCR) can induce activation signals to cells. In some embodiments, the receptor contains intracellular signaling components that contain ITAM or ITAM-like motifs. In some embodiments, the activation induced by the first receptor includes signaling or protein expression changes in cells, resulting in the initiation of immune response, such as ITAM phosphorylation and / or the initiation of ITAM-mediated signaling cascades, the formation of immunological synapses and / or the clustering of molecules (e.g., CD4 or CD8) near the bound receptor, the activation of one or more transcription factors, such as NF-κB and / or AP-1, and / or the induction of gene expression of factors such as cytokines, proliferation, and / or survival.

[0302] In some embodiments, the first and / or second receptor comprises an intracellular signaling domain or region of a costimulatory receptor such as CD28, CD137 (4-1BB), OX40, and / or ICOS. In some embodiments, the first and second receptors comprise intracellular signaling domains of different costimulatory receptors. In one embodiment, the first receptor contains a CD28 costimulatory signaling region and the second receptor contains a 4-1BB costimulatory signaling region, or vice versa.

[0303] In some embodiments, the first and / or second receptor comprises both an intracellular signaling domain containing an ITAM or ITAM-like motif and an intracellular signaling domain of a costimulatory receptor.

[0304] In some embodiments, the first receptor contains the intracellular signaling domain that contains ITAM or ITAM-like motif, and the second receptor contains the intracellular signaling domain of costimulatory receptor.The costimulatory signal combined with the activation signal induced in the same cell results in a robust and sustained immune response, such as T cell-mediated effector function, such as increased gene expression, secretion of cytokines and other factors, and cell death.

[0305] In some embodiments, the ligation of the first receptor alone or the ligation of the second receptor alone does not induce a robust immune response.In some aspects, when only one receptor is ligated, cells become tolerant or unresponsive to antigen, or are inhibited, and / or are not induced to proliferate, secrete factors, or perform effector functions.However, in some such embodiments, when multiple receptors are ligated, such as when the cells expressing the first and second antigens meet, desired responses are achieved, such as complete immune activation or stimulation, as shown by, for example, secretion of one or more cytokines, proliferation, persistence, and / or performing immune effector functions, such as cytotoxic killing of target cells.

[0306] In some embodiments, the two receptors induce activation and inhibitory signals to cells, respectively, so that the binding of one of the receptors to the antigen activates the cell or induces a response, while the binding of the second inhibitory receptor to the antigen induces a signal that suppresses or attenuates the response.An example is the combination of activating CAR and inhibitory CAR or iCAR.For example, such a strategy can be used that activating CAR binds to an antigen that is expressed in disease or condition but also expressed on normal cells, and inhibitory receptor binds to a separate antigen that is expressed on normal cells but not expressed on disease or condition cells.

[0307] In some embodiments, multi-targeting strategy is used when the antigen associated with a particular disease or condition is expressed on non-diseased cells and / or on the engineered cells themselves, either transiently (e.g., upon stimulation associated with genetic engineering) or permanently.In such cases, specificity, selectivity, and / or efficacy can be improved by requiring the ligation of two separate and individually specific antigen receptors.

[0308] In some embodiments, multiple antigens, for example, the first and second antigens, are expressed in the targeted cell, tissue, or disease or condition, such as cancer cells.In some aspects, the cell, tissue, disease, or condition is multiple myeloma or multiple myeloma cells.In some embodiments, one or more of the multiple antigens are generally also expressed in cells that are not desired to be targeted by cell therapy, such as normal or non-diseased cells or tissues, and / or in the engineered cells themselves.In such embodiments, specificity and / or efficacy are achieved by requiring the ligation of multiple receptors to achieve a cellular response.

[0309] d. T cell receptor (TCR) In some embodiments, engineered cells, e.g., T cells, are provided that express a T cell receptor (TCR) or an antigen-binding portion thereof that recognizes a peptide epitope or T cell epitope of a target polypeptide, such as an antigen of a tumor, a virus, or an autoimmune protein.

[0310] In some embodiments, a "T cell receptor" or "TCR" is a molecule that contains variable alpha and beta chains (also known as TCRα and TCRβ, respectively) or variable gamma and delta chains (also known as TCRα and TCRβ, respectively), or an antigen-binding portion thereof, and can specifically bind to peptides bound to MHC molecules. In some embodiments, the TCR is in αβ form. Typically, TCRs that exist in αβ and γδ forms are generally structurally similar, but the T cells that express them may have distinct anatomical locations or functions. TCRs can be found on the surface of cells or in soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes), where they are generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0311] Unless otherwise specified, the term "TCR" should be understood to include the complete TCR and its antigen-binding portion or antigen-binding fragment. In some embodiments, the TCR is an intact or full-length TCR, including the αβ or γδ forms of the TCR. In some embodiments, the TCR is less than full-length, but is an antigen-binding portion that binds to a specific peptide bound to an MHC molecule, e.g., binds to an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of the TCR can contain only a portion of the structural domain of the full-length or intact TCR, but can still bind to a peptide epitope, such as an MHC-peptide complex, to which the complete TCR binds. In some cases, the antigen-binding portion contains sufficient variable domains of the TCR, e.g., the variable α and variable β chains of the TCR, to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of the TCR contain the complementarity determining regions involved in the recognition of peptides, MHC, and / or MHC-peptide complexes.

[0312] In some embodiments, the variable domain of TCR contains hypervariable loops or complementarity determining regions (CDRs), which are generally the main contributors to antigen recognition and binding capacity and specificity. In some embodiments, the CDRs of TCR or a combination thereof form all or substantially all of the antigen binding site of a given TCR molecule. The various CDRs in the variable region of TCR chain are generally separated by framework regions (FRs), which generally show lower variability between TCR molecules compared to CDRs (see, for example, Jores et al., Proc. Nat'l Acad. Sci. USA 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; also see Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the main CDR responsible for antigen binding or specificity, or is the most important of the three CDRs on a given TCR variable region for antigen recognition and / or for interaction with the processed peptide portion of a peptide-MHC complex. In some circumstances, CDR1 of the α chain can interact with the N-terminal portion of a certain antigen peptide. In some circumstances, CDR1 of the β chain can interact with the C-terminal portion of a peptide. In some circumstances, CDR2 is the main CDR that contributes most strongly to or is responsible for interaction with or recognition of the MHC portion of an MHC-peptide complex. In some embodiments, the variable region of the β chain can contain an additional hypervariable region (CDR4 or HVR4) that is generally involved in superantigen binding and not involved in antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

[0313] In some embodiments, the TCR may also contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997). In some aspects, each chain of the TCR may possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with the invariant protein of the CD3 complex, which is involved in mediating signal transduction.

[0314] In some embodiments, a TCR chain contains one or more constant domains. For example, the extracellular portion of a given TCR chain (e.g., an α chain or a β chain) contains two immunoglobulin-like domains, such as a variable domain (e.g., Vα or Vβ; typically amino acids 1-116 according to the Kabat numbering, Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.) and a constant domain adjacent to the cell membrane (e.g., an α chain constant domain or Cα, typically positions 117-259 of the chain according to the Kabat numbering, or a β chain constant domain or Cβ). β, typically positions 117-295 of the chain according to Kabat). For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains, and two membrane-distal variable domains, each of which contains a CDR. The constant domain of the TCR may contain a short connecting sequence in which cysteine ​​residues form disulfide bonds, thereby linking the two chains of the TCR. In some embodiments, the TCR may have additional cysteine ​​residues in each of the α and β chains, such that the TCR contains two disulfide bonds in the constant domain.

[0315] In some embodiments, the TCR chain contains a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules, such as CD3 and its subunits. For example, a TCR containing a constant domain with a transmembrane region can anchor the protein to the cell membrane and associate with the invariant subunit of the CD3 signaling apparatus or complex. The intracellular tail of the CD3 signaling subunit (e.g., CD3γ, CD3δ, CD3ε, and CD3ζ chains) contains one or more immunoreceptor tyrosine-based activation motifs or ITAMs that are involved in the signaling ability of the TCR complex.

[0316] In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), or may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β, or γ and δ), linked, for example, by one or more disulfide bonds.

[0317] In some embodiments, TCRs can be generated from known TCR sequences, such as sequences of Vα, β chains, for which substantially full-length coding sequences are readily available.Methods for obtaining full-length TCR sequences, including V chain sequences, from cell sources are well known.In some embodiments, nucleic acids encoding TCRs can be obtained from various sources, for example, by polymerase chain reaction (PCR) amplification of TCR-encoding nucleic acids in or isolated from a given cell or cells, or by synthesis of publicly available TCR DNA sequences.

[0318] In some embodiments, the TCR is obtained from a biological source, e.g., from a cell, e.g., a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other publicly available source. In some embodiments, the T cell can be obtained from an in vivo isolated cell. In some embodiments, the TCR is a thymically selected TCR. In some embodiments, the TCR is a neoepitope-restricted TCR. In some embodiments, the T cell can be a cultured T cell hybridoma or clone. In some embodiments, the TCR or antigen-binding portion thereof or antigen-binding fragment thereof can be synthetically generated from knowledge of the sequence of the TCR.

[0319] In some embodiments, the TCR is generated from a TCR identified or selected from screening a library of candidate TCRs against a target polypeptide antigen or its target T cell epitope. The TCR library can be generated by amplifying the repertoire of Va and Vβ from T cells isolated from a subject, including cells present in PBMC, 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 a T cell source of a normal healthy subject, i.e., a normal TCR library. In some embodiments, the TCR can be amplified from a T cell source of a diseased subject, i.e., a diseased TCR library. In some embodiments, degenerate primers are used to amplify the gene repertoire of Va and Vβ by, for example, RT-PCR in a sample, such as T cells obtained from a human. In some embodiments, the scTv library can be assembled from naive Vα and Vβ libraries, where the amplification products are cloned or assembled to be separated by a linker. Depending on the subject and the source of the cells, the library can be HLA allele-specific. Alternatively, in some embodiments, the TCR library can be generated by mutagenesis or diversification of parent or backbone TCR molecules. In some aspects, the TCR is subjected to directed evolution, such as by mutagenesis of the α or β chain. In some aspects, specific residues within the CDRs of the TCR are altered. In some embodiments, the selected TCR can be modified by affinity maturation. In some embodiments, the antigen-specific T cells can be selected, such as by screening to evaluate CTL activity against the peptide. In some aspects, for example, the TCR present on the antigen-specific T cells can be selected by avidity, such as a specific affinity or avidity for the antigen.

[0320] In some embodiments, the TCR or antigen-binding portion thereof is modified or engineered. In some embodiments, directed evolution methods are used to generate TCRs with altered properties, such as higher affinity for specific MHC-peptide complexes. In some embodiments, directed evolution is achieved by display methods, including but not limited to yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), or T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84). In some embodiments, the display approach involves engineering or modifying a known, parent or reference TCR. For example, in some cases, a wild-type TCR can be used as a template to create mutagenized TCRs in which one or more residues in the CDRs are mutated, and mutants with desired altered properties, such as higher affinity for a desired target antigen, are selected.

[0321] In some embodiments, the peptide of the target polypeptide for use in the creation or generation of the TCR of interest is known or can be easily identified. In some embodiments, the peptide suitable for use in the generation of the TCR or antigen-binding portion can be determined based on the presence of HLA-restricted motifs in the target polypeptide of interest, such as the target polypeptide described below. In some embodiments, the peptide is identified using available computer prediction models. In some embodiments, such models 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) for predicting MHC class I binding sites. In some embodiments, the MHC-restricted epitope is HLA-A0201, which is expressed in approximately 39-46% of all Caucasians and is therefore a suitable choice of MHC antigen for use in preparing TCRs or other MHC-peptide binding molecules.

[0322] HLA-A0201 binding motifs and cleavage sites for proteasomes and immunoproteasomes are known using computer prediction models. For predicting MHC class I binding sites, such models include, but are not limited to, ProPred1 (described in detail in Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17 (12): 1236-1237 2001), and SYFPEITHI (refer to Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction. in Immunoinformatics Methods in Molecular Biology, vol 409 (1): 75-93 2007).

[0323] In some embodiments, the TCR or its antigen-binding portion may be a recombinantly produced natural protein or a variant thereof, with one or more characteristics, such as binding characteristics, altered. In some embodiments, the TCR may be derived from one of a variety of animal species, such as human, mouse, rat, or other mammals. The TCR may be cell-bound or soluble. In some embodiments, for the purposes of the provided method, the TCR is cell-bound, expressed on the surface of a cell.

[0324] In some embodiments, the TCR is a full-length TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR (sc-TCR). In some embodiments, the dTCR or scTCR has the structure described in WO 03 / 020763, WO 04 / 033685, WO2011 / 044186.

[0325] In some embodiments, the TCR contains a sequence corresponding to a transmembrane sequence. In some embodiments, the TCR contains a sequence corresponding to a cytoplasmic sequence. In some embodiments, the TCR can form a complex with CD3. In some embodiments, either a dTCR or an scTCR-containing TCR can be linked to a signaling domain, resulting in an active TCR on the surface of a T cell. In some embodiments, the TCR is expressed on the surface of a cell.

[0326] In some embodiments, the dTCR contains a first polypeptide in which a sequence corresponding to the variable region sequence of the TCR α-chain is fused to the N-terminus of a sequence corresponding to the extracellular sequence of the constant region of the TCR α-chain, and a second polypeptide in which a sequence corresponding to the variable region sequence of the TCR β-chain is fused to the N-terminus of a sequence corresponding to the extracellular sequence of the constant region of the TCR β-chain, and the first polypeptide and the second polypeptide are linked by a disulfide bond. In some embodiments, the bond can correspond to the native interchain disulfide bond present in the native dimeric αβ TCR. In some embodiments, the interchain disulfide bond is not present in the native TCR. For example, in some embodiments, one or more cysteines can be incorporated into the extracellular sequence of the constant region of the dTCR polypeptide pair. In some cases, both native and non-native disulfide bonds may be desirable. In some embodiments, the TCR contains a transmembrane sequence for membrane anchoring.

[0327] In some embodiments, the dTCR contains a TCR α-chain containing a variable α domain, a constant α domain, and a first dimerization motif attached to the C-terminus of the constant α domain, and a TCR β-chain containing a variable β domain, a constant β domain, and a first dimerization motif attached to the C-terminus of the constant β domain, wherein the first and second dimerization motifs readily interact to form a covalent bond between the amino acids in the first dimerization motif and the amino acids in the second dimerization motif, linking the TCR α-chain and the TCR β-chain together.

[0328] In some embodiments, TCR is scTCR.Typically, scTCR can be generated by known methods.See, for example, Soo Hoo, WF et al. PNAS (USA) 89, 4759 (1992);Wulfing, C. and Pluckthun, A., J. Mol. Biol. 242, 655 (1994);Kurucz, I. et al. PNAS (USA) 90 3830 (1993);International Publication PCT Nos. WO 96 / 13593, WO 96 / 18105, WO99 / 60120, WO99 / 18129, WO 03 / 020763, WO2011 / 044186;And Schlueter, CJ et al. J. Mol. Biol. 256, 859 (1996). In some embodiments, the scTCR contains a non-native disulfide interchain bond introduced to facilitate TCR chain association (see, e.g., International Publication No. WO 03 / 020763). In some embodiments, the scTCR is a non-disulfide truncated TCR in which a heterologous leucine zipper fused to its C-terminus facilitates chain association (see, e.g., International Publication No. WO 99 / 60120). In some embodiments, the scTCR contains a TCR alpha variable domain covalently linked to a TCR beta variable domain via a peptide linker (see, e.g., International Publication No. WO 99 / 18129).

[0329] In some embodiments, the scTCR contains a first segment composed of an amino acid sequence corresponding to a TCR alpha chain variable region, a second segment composed of an amino acid sequence corresponding to a TCR beta chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to a TCR beta chain constant domain extracellular sequence, and a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment.

[0330] In some embodiments, the scTCR contains a first segment composed of an alpha chain variable region sequence fused to the N-terminus of an alpha chain extracellular constant domain sequence, and a second segment composed of a beta chain variable region sequence fused to the N-terminus of a beta chain extracellular constant sequence and transmembrane sequence sequence, and optionally a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment.

[0331] In some embodiments, the scTCR contains a first segment comprised of a TCR β chain variable region sequence fused to the N-terminus of a β chain extracellular constant domain sequence, and a second segment comprised of an α chain variable region sequence fused to the N-terminus of an α chain extracellular constant sequence and transmembrane sequence sequence, and optionally a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment.

[0332] In some embodiments, the linker of the scTCR linking the first and second TCR segments can be any linker capable of forming a single polypeptide chain while retaining the binding specificity of the TCR. In some embodiments, the linker sequence may have, for example, the formula -P-AA-P-, where P is proline and AA represents an amino acid sequence, where the amino acids are glycine and serine. In some embodiments, the first and second segments are paired such that their variable region sequences are oriented for such binding. Thus, in some cases, the linker has a length sufficient to bridge the distance between the C-terminus of the first segment and the N-terminus of the second segment, or vice versa, but is not so long as to block or reduce binding of the scTCR to the target ligand. In some embodiments, the linker can contain 10-45 amino acids or about 10-45 amino acids, such as 10-30 amino acids or 26-41 amino acid residues, such as 29, 30, 31, or 32 amino acids. In some embodiments, the linker has a length of about 10-45 amino acids, such as 10-30 amino acids or 26-41 amino acid residues, such as 29, 30, 31, or 32 amino acids. TIFF2025063189000002.tif4128, where P is proline, G is glycine, and S is serine (SEQ ID NO:32). In some embodiments, the linker has the sequence I have TIFF2025063189000003.tif5128.

[0333] In some embodiments, the scTCR contains a covalent disulfide bond linking the residues of the immunoglobulin region of the constant domain of the α chain to the residues of the immunoglobulin region of the constant domain of the β chain. In some embodiments, there is no interchain disulfide bond in the native TCR. For example, in some embodiments, one or more cysteines can be incorporated into the constant region extracellular sequence of the first and second segments of the scTCR polypeptide. In some cases, both natural and non-natural disulfide bonds may be desirable.

[0334] In some embodiments of dTCR or scTCR containing introduced interchain disulfide bond, there is no natural disulfide bond.In some embodiments, one or more of the natural cysteines that form natural interchain disulfide bond are replaced with another residue, such as serine or alanine.In some embodiments, the introduced disulfide bond can be formed by mutating the non-cysteine ​​residues on the first and second segments to cysteine.Exemplary non-natural disulfide bonds of TCR are described in International Publication No. WO2006 / 000830.

[0335] In some embodiments, the TCR or antigen-binding fragment thereof exhibits an affinity for the target antigen with an equilibrium binding constant of, or about, 10-5 to 10-12 M and all individual values ​​and ranges therein. In some embodiments, the target antigen is an MHC-peptide complex or ligand.

[0336] In some embodiments, one or more nucleic acids encoding TCRs, such as α and β chains, can be amplified by PCR, cloning, or other suitable means and cloned into one or more suitable expression vectors. The expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification, or for expression, or both, such as plasmids and viruses.

[0337] In some embodiments, the vector can be a pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), or pEX series (Clontech, Palo Alto, Calif.) vector. In some cases, bacteriophage vectors such as λG10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. In some embodiments, plant expression vectors can be used, including pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, viral vectors such as retroviral vectors are used.

[0338] In some embodiments, recombinant expression vectors can be prepared using standard recombinant DNA techniques. In some embodiments, vectors can contain control sequences, such as transcription and translation initiation and termination codons, that are specific to the type of host (e.g., bacteria, fungi, plants, or animals) into which the vector will be introduced, as appropriate and taking into account whether the vector is DNA-based or RNA-based. In some embodiments, vectors can contain non-natural promoters that are operably linked to the nucleotide sequence that codes for TCR or antigen-binding portion (or other MHC-peptide binding molecules). In some embodiments, the promoter can be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter, and the promoter found in the long terminal repeat of murine stem cell virus. Other known promoters are also contemplated.

[0339] In some embodiments, to generate vectors encoding TCR, α chain and β chain are PCR amplified from the total cDNA isolated from T cell clone expressing TCR of interest and cloned into expression vector.In some embodiments, α chain and β chain are cloned into the same vector.In some embodiments, α chain and β chain are cloned into different vectors.In some embodiments, generated α chain and β chain are integrated into retroviral vector, for example lentiviral vector.

[0340] D. Characterization of output cell composition In certain embodiments, the method provided herein creates or produces a cell composition, such as an output cell composition, that contains genetically engineered cells. In certain embodiments, the output cell composition is a cell composition that results from some or all of the steps for genetically engineering cells. In certain embodiments, the output cell composition results from the process of genetically engineering the cells of the input composition. In certain embodiments, the process contains one or more steps for activating, transducing or transfecting, expanding, and / or harvesting cells, such as cells obtained from the input cell composition. In certain embodiments, the output cell composition contains cells that have been genetically engineered. In certain embodiments, the cells of the output cell composition have undergone all of the steps for the process of genetic engineering.

[0341] In some embodiments, the output cell composition contains one or more nucleic acids introduced via genetic engineering, thereby containing cells that express recombinant or engineered products of such nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or sample obtained from the cell, e.g., obtained from another organism or cell that is not normally found in the cell being engineered and / or the organism from which such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, e.g., a nucleic acid not found in nature, including those that contain chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

[0342] In some embodiments, the output cell composition contains genetically engineered cells. In certain embodiments, the output cell composition contains engineered T cells. In some embodiments, the engineered T cells include engineered CD4+ T cells and engineered CD8+ T cells. In certain embodiments, the output cell composition contains or comprises 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% engineered T cells. In certain embodiments, the engineered cells express a recombinant receptor. In certain embodiments, the output cell composition contains or comprises 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 recombinant receptor. In some embodiments, the recombinant receptor is a TCR or a CAR. In certain embodiments, the recombinant receptor is a CAR.

[0343] In certain embodiments, the output cell composition has a ratio of engineered CD4+ T cells to CD8+ T cells of 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 particular embodiments, the output cell composition has a ratio of engineered CD4+ T cells to CD8+ T cells of between 2:1 and 0.5:1. In some embodiments, the output cell composition has a ratio of engineered CD4+ T cells to CD8+ T cel...

Claims

1. 1. A method for producing a cell composition, comprising: combining a first cell composition comprising naive-like CD4+ T cells with a second cell composition comprising naive-like CD8+ T cells to produce an input cell composition having a ratio of naive-like CD4+ T cells to naive-like CD8+ T cells that is between or about 0.8:1 and 2.2:1, inclusive. A method comprising:

2. 2. The method of claim 1, wherein 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 the biological sample obtained from the subject.

3. Before combining, Determining the number, number per volume, number per weight and / or percentage of naive-like CD4+ T cells in the first cell composition and / or the number, number per volume, number per weight and / or percentage of naive-like CD8+ T cells in the second cell composition.

3. The method of claim 1 or claim 2, comprising:

4. Before combining, Determining the number, volumetric number, weight number and / or percentage of naive-like CD4+ T cells and / or the number, volumetric number, weight number and / or percentage of naive-like CD8+ T cells in a biological sample from the subject.

3. The method of claim 2, comprising:

5. 5. The method of any one of claims 1 to 4, wherein the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells in the input cell composition is adjusted or altered compared to the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells in a biological sample from the subject.

6. 1. A method for producing a cell composition, comprising: determining the number, volumetric number, weight number and / or percentage of naive-like CD4+ T cells and naive-like CD8+ T cells in a biological sample obtained from the subject or one or more samples derived therefrom; producing an input cell composition comprising CD4+ T cells and CD8+ T cells, the input cell composition having a ratio of naive-like CD4+ T cells to naive-like CD8+ T cells that is between or about 2.2:1 and 0.8:1, inclusive, wherein the ratio in the input cell composition is adjusted or altered compared to the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells in a biological sample from the subject; A method comprising:

7. contacting the input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the input cell composition. The method of any one of claims 1 to 6, further comprising:

8. 1. A method for producing a cell composition, comprising: contacting an input cell composition comprising naive-like CD4+ T cells and naive-like CD8+ T cells from a biological sample from a subject with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the composition. Including, The method, wherein the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells present in the input cell composition is at or about 0.8:1 to 2.2:1, inclusive.

9. stimulating the cells before, during and / or after the contacting step, where stimulation comprises incubating the cells in the presence of one or more stimuli, where stimulation results in activation and / or proliferation of the cells.

9. The method of claim 7 or claim 8, further comprising:

10. 1. A method for producing a cell composition, comprising: combining a first cell composition comprising naive-like CD4+ T cells with a second cell composition comprising naive-like CD8+ T cells to produce an input cell composition having a ratio of naive-like CD4+ T cells to naive-like CD8+ T cells that is between or about 0.8:1 and 2.2:1, inclusive; contacting an input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the composition; stimulating the cells before, during and / or after said contacting step, where stimulating comprises incubating the cells in the presence of one or more stimuli, where stimulating results in activation and / or proliferation of the cells; A method comprising:

11. The naive-like CD4+ cells and / or the naive-like CD8+ cells are is surface positive for a marker selected from the group consisting of CD45RA, CD27, CD28 and CCR7; and / or is surface negative for a marker selected from the group consisting of CD25, CD45RO, CD56, CD62L and KLRG1; and / or have low expression of CD95, and / or negative for intracellular expression of cytokines selected from the group consisting of IL-2, IFN-γ, IL-4, and IL-10; The method of any one of claims 1 to 10.

12. The naive-like CD4+ cells and / or the naive-like CD8+ cells are and / or is surface positive for a T cell activation marker selected from the group consisting of CD45RA, CD27, CD28 and CCR7; is surface negative for a marker selected from the group consisting of CD45RO, CD56, CD62L and KLRG1; and / or have low expression of CD95, The method of any one of claims 1 to 11.

13. The method of any one of claims 1 to 12, wherein the naive-like CD4+ cells and / or the naive-like CD8+ cells are surface positive for CD45RA and CCR7.

14. The method of any one of claims 1 to 12, wherein the naive-like CD4+ cells and / or the naive-like CD8+ cells are surface positive for CD27 and CCR7.

15. The method of any one of claims 1 to 14, wherein the naive-like CD4+ cells and the naive-like CD8+ cells are surface positive for CD45RA, CD27 and CCR7, and surface negative for CD45RO.

16. The method of any one of claims 1 to 12, wherein the naive-like CD4+ cells and / or the naive-like CD8+ cells are surface positive for CCR7 and surface negative for CD62L.

17. 17. The method of any one of claims 3 to 16, wherein the number, number per volume, number per weight and / or percentage of naive-like CD4+ T cells and / or the number, number per volume, number per weight and / or percentage of naive-like CD8+ T cells are determined by flow cytometry.

18. The method of any one of claims 8 to 17, wherein the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells is adjusted compared to the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells in a biological sample from the subject.

19. 19. The method of any one of claims 1-18, wherein the input cell composition comprises a ratio of naive-like CD4+ cells to naive-like CD8+ cells that is at or about 0.8:1-2.0:1, about 0.8:1-1.6:1, about 0.8:1-1.4:1, about 0.8:1-1.2:1, about 1.5:1-2:1, about 1.6:1-1.8:1, or about 1.0:1-1.2:1, inclusive.

20. 20. The method of any one of claims 1-19, wherein the input cell composition comprises a ratio of naive-like CD4+ cells to naive-like CD8+ cells of or about 2:1, about 1.9:1, about 1.8:1, about 1.7:1, 1.69:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1 or 1.0:

1.

21. 21. The method of any one of claims 1-20, wherein the input cell composition comprises a ratio of naive-like CD4+ cells to naive-like CD8+ cells of 1.1:1 or about 1.1:

1.

22. 21. The method of any one of claims 1-20, wherein the input cell composition comprises a ratio of 1.1:1 or about 1.1:1 naive-like CD4+ cells to naive-like CD8+ cells that are surface positive for CD45RA and CCR7.

23. 21. The method of any one of claims 1-20, wherein the input cell composition comprises a ratio of naive-like CD4+ cells to naive-like CD8+ cells that are surface positive for CD45RA and CD27 of at or about 1.69:

1.

24. 21. The method of any one of claims 1-20, wherein the input cell composition comprises a ratio of naive-like CD4+ cells to naive-like CD8+ cells that are surface positive for CD27 and CCR7 of at or about 1.69:

1.

25. 1. A method for producing a cell composition, comprising: combining a first cell composition comprising CCR7+CD45RA+CD4+ T cells with a second cell composition comprising CCR7+CD45RA+CD8+ T cells to produce an input cell composition having a ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells that is between or about 0.8:1 and 2.2:1, inclusive. A method comprising:

26. 26. The method of claim 25, wherein 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 the biological sample obtained from the subject.

27. Before combining, Determining the number, number per volume, number per weight and / or percentage of CCR7+CD45RA+CD4+ T cells in the first cell composition and / or the number, number per volume, number per weight and / or percentage of CCR7+CD45RA+CD8+ T cells in the second cell composition.

27. The method of claim 25 or claim 26, comprising:

28. Before combining, Determining the number, volumetric number, weight number and / or percentage of CCR7+CD45RA+CD4+ T cells and / or the number, volumetric number, weight number and / or percentage of CCR7+CD45RA+CD8+ T cells in a biological sample from the subject. The method of any one of claims 25 to 27, comprising:

29. 29. The method of any one of claims 25 to 28, wherein the ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells in the input cell composition is adjusted or altered compared to the ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells in a biological sample from the subject.

30. 1. A method for producing a cell composition, comprising: determining the number, volumetric count, weight count and / or percentage of CCR7+CD45RA+CD4+ T cells and CCR7+CD45RA+CD8+ T cells in a biological sample obtained from the subject or in one or more samples derived therefrom; producing an input cell composition comprising CD4+ T cells and CD8+ T cells, the ratio of CCR7+CD45RA+CD4+ T cells to naive-like CD8+ T cells being between or about 2.2:1 and 0.8:1, inclusive, wherein the ratio in the input cell composition is adjusted or altered compared to the ratio of CCR7+CD45RA+CD4+ T cells to naive-like CD8+ T cells in a biological sample from the subject; A method comprising:

31. contacting the input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the input cell composition.

31. The method of claim 30, further comprising:

32. 1. A method for producing a cell composition, comprising: contacting an input cell composition comprising CCR7+CD45RA+CD4+ T cells and CCR7+CD45RA+CD8+ T cells from a biological sample from a subject with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the composition. wherein the ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells present in the input cell composition is at or about 0.8:1 to 2.2:1, inclusive.

33. stimulating the cells before, during and / or after the contacting step, where stimulation comprises incubating the cells in the presence of one or more stimuli, where stimulation results in activation and / or proliferation of the cells.

33. The method of claim 32, further comprising:

34. 1. A method for producing a cell composition, comprising: combining a first cell composition comprising CCR7+CD45RA+CD4+ T cells with a second cell composition comprising CCR7+CD45RA+CD8+ T cells to produce an input cell composition having a ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells that is between or about 0.8:1 and 2.2:1, inclusive; contacting an input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the composition; stimulating the cells before, during and / or after said contacting step, where stimulating comprises incubating the cells in the presence of one or more stimuli, where stimulating results in activation and / or proliferation of the cells; A method comprising:

35. The method of any one of claims 25 to 34, wherein 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.

36. The method of any one of claims 32 to 35, wherein the ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells is adjusted compared to the ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells in a biological sample from the subject.

37. 37. The method of any one of claims 25-36, wherein the input cell composition comprises a ratio of CCR7+CD45RA+CD4+ cells to CCR7+CD45RA+CD8+ cells of, or about, 0.8:1 to 2.0:1, 0.8:1 to 1.6:1, 0.8:1 to 1.4:1, 0.8:1 to 1.2:1, or 1.0:1 to 1.2:1, inclusive.

38. 38. The method of any one of claims 25-37, wherein the input cell composition comprises a ratio of CCR7+CD45RA+CD4+ cells to CCR7+CD45RA+CD8+ cells of, or about, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1 or 1.0:

1.

39. 39. The method of any one of claims 25-38, wherein the input cell composition comprises a ratio of CCR7+CD45RA+CD4+ cells to CCR7+CD45RA+CD8+ cells of at or about 1.1:

1.

40. 1. A method for producing 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 produce an input cell composition having a ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells that is between or about 1.2:1 and 2.4:1, inclusive. A method comprising:

41. The method of claim 40, wherein 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 the biological sample obtained from the subject.

42. Before combining, Determining the number, number per volume, number per weight and / or percentage of CD27+CCR7+CD4+ T cells in the first cell composition and / or the number, number per volume, number per weight and / or percentage of CD27+CCR7+CD8+ T cells in the second cell composition.

42. The method of claim 40 or claim 41, comprising:

43. Before combining, Determining the number, volumetric number, weight number and / or percentage of CD27+CCR7+CD4+ T cells and / or the number, volumetric number, weight number and / or percentage of CD27+CCR7+CD8+ T cells in a biological sample from the subject. The method of any one of claims 40 to 42, comprising:

44. 44. The method of any one of claims 40 to 43, wherein the ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells in the input cell 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 the subject.

45. 1. A method for producing a cell composition, comprising: determining the number, volumetric count, weight count and / or percentage of CD27+CCR7+CD4+ T cells and CD27+CCR7+CD8+ T cells in a biological sample obtained from the subject or one or more samples derived therefrom; producing an input cell composition comprising CD4+ T cells and CD8+ T cells, the ratio of CD27+CCR7+CD4+ T cells to naive-like CD8+ T cells being between or about 2.2:1 and 0.8:1, inclusive, wherein the ratio in the input cell composition is adjusted or altered compared to the ratio of CD27+CCR7+CD4+ T cells to naive-like CD8+ T cells in a biological sample from the subject; A method comprising:

46. contacting the input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the input cell composition. The method of any one of claims 40 to 45, further comprising:

47. 1. A method for producing a cell composition, comprising: contacting an input cell composition comprising CD27+CCR7+CD4+T cells and CD27+CCR7+CD8+T cells from a biological sample from a subject with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the composition. wherein the ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells present in the input cell composition is at or about 0.8:1 to 2.2:1, inclusive.

48. stimulating the cells before, during and / or after the contacting step, where stimulation comprises incubating the cells in the presence of one or more stimuli, where stimulation results in activation and / or proliferation of the cells.

48. The method of claim 47, further comprising:

49. 1. A method for producing 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 produce an input cell composition having a ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells that is between or about 0.8:1 and 2.2:1, inclusive; contacting an input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the composition; stimulating the cells before, during and / or after said contacting step, where stimulating comprises incubating the cells in the presence of one or more stimuli, where stimulating results in activation and / or proliferation of the cells; A method comprising:

50. The method of any one of claims 40 to 49, wherein 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.

51. The method of any one of claims 40 to 50, wherein the ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells is adjusted compared to the ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells in a biological sample from the subject.

52. 52. The method of any one of claims 40-51, wherein the input cell composition comprises a ratio of CD27+CCR7+CD4+ cells to CD27+CCR7+CD8+ cells of or about 0.8:1 to 2.0:1, about 0.8:1 to 1.6:1, about 0.8:1 to 1.4:1, about 0.8:1 to 1.2:1, or about 1.0:1 to 1.2:1, inclusive.

53. 53. The method of any one of claims 40-52, wherein the input cell composition comprises a ratio of CD27+CCR7+CD4+ cells to CD27+CCR7+CD8+ cells of, or about, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1 or 1.0:

1.

54. 54. The method of any one of claims 40-53, wherein the input cell composition comprises a ratio of CD27+CCR7+CD4+ cells to CD27+CCR7+CD8+ cells of at or about 1.1:

1.

55. 54. The method of any one of claims 40-53, wherein the input cell composition comprises a ratio of CD27+CCR7+CD4+ cells to CD27+CCR7+CD8+ cells of at or about 1.69:

1.

56. 1. A method for producing a cell composition, comprising: combining a first cell composition comprising CD62L-CCR7+CD4+ T cells with a second cell composition comprising CD62L-CCR7+CD8+ T cells to produce an input cell composition having a ratio of CD62L-CCR7+CD4+ T cells to CD62L-CCR7+CD8+ T cells that is between or about 0.5:1 and 2:1, inclusive. A method comprising:

57. 1. A method for producing a cell composition, comprising: determining the number, volumetric count, weight count and / or percentage of CD62L-CCR7+CD4+T cells and CD62L-CCR7+CD8+T cells in a biological sample obtained from the subject or one or more samples derived therefrom; producing an input cell composition comprising CD4+ T cells and CD8+ T cells, wherein the ratio of CD62L-CCR7+ CD4+ T cells to naive-like CD8+ T cells is between or about 0.5:1 and 2:1, inclusive, wherein the ratio in the input cell composition is adjusted or altered compared to the ratio of CD62L-CCR7+ CD4+ T cells to naive-like CD8+ T cells in a biological sample from the subject; A method comprising:

58. contacting the input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the input cell composition.

58. The method of claim 56 or claim 57, further comprising:

59. 1. A method for producing a cell composition, comprising: combining a first cell composition comprising CD62L-CCR7+CD4+ T cells with a second cell composition comprising CD62L-CCR7+CD8+ T cells to produce an input cell composition having a ratio of CD62L-CCR7+CD4+ T cells to CD62L-CCR7+CD8+ T cells that is between or about 0.5:1 and 2:1, inclusive; contacting an input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions for introducing the nucleic acid encoding the recombinant receptor into cells in the composition; stimulating the cells before, during and / or after said contacting step, where stimulating comprises incubating the cells in the presence of one or more stimuli, where stimulating results in activation and / or proliferation of the cells; A method comprising:

60. The method of claim 58 or claim 59, wherein 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.

61. The method of any one of claims 57 to 60, wherein the ratio of CD62L-CCR7+CD4+T cells to CD62L-CCR7+CD8+T cells is adjusted compared to the ratio of CD62L-CCR7+CD4+T cells to CD62L-CCR7+CD8+T cells in a biological sample from the subject.

62. 62. The method of any one of claims 57-61, wherein the input cell composition comprises a ratio of CD62L-CCR7+CD4+ cells to CD62L-CCR7+CD8+ cells of at or about 0.5:1 to 1.5:1, about 1:1 to 2:1, about 0.75:1 to 1.5:1, or about 0.8:1 to 1.2:1, inclusive.

63. 63. The method of claims 57-62, wherein the input cell composition comprises a ratio of CD62L-CCR7+CD4+ cells to CD62L-CCR7+CD8+ cells of, or about, 1.2:1, 1.1:1, 1.0:1, 0.9:1 or 0.8:

1.

64. The method of any one of claims 2-9, 18-24, 26-33, 35-39, 41-48, 51-54, 57-58 or 61-63, wherein the biological sample is or is obtained from a blood, plasma or serum sample.

65. 65. The method of any one of claims 2-9, 18-24, 26-33, 35-39, 41-48, 51-54, 57-58 or 61-64, wherein the biological sample is or comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product or a leukapheresis product.

66. The method of any one of claims 2-9, 18-24, 26-33, 35-39, 41-48, 51-54, 57-58 or 61-65, wherein the biological sample is or is obtained from an apheresis sample or a leukapheresis sample.

67. The method of any one of claims 2-9, 18-24, 26-33, 35-39, 41-48, 51-54, 57-58 or 61-66, wherein the subject is a human subject.

68. The input cell composition was 1 × 10 7 ~5×10 9 pcs or approx. 1×10 7 ~5×10 9 Total cells or total T cells, 5 x 10 7 ~1×10 9 Pieces or about 5 x 10 7 ~1×10 9 Total cells or total T cells, 1 x 10 8 ~5×10 8 pcs or approx. 1×10 8 ~5×10 8 total cells or total T cells, or 2 x 10 8 ~5×10 8 pcs or approx. 2×10 8 ~5×10 8 68. The method of any one of claims 1 to 67, comprising total cells or total T cells, or comprising a viable population of any of said.

69. The input cell composition is at least 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 Or 5 x 10 8 or at least about 1 x 10 8 , about 2×10 8 , about 3×10 8 , about 4×10 8 Or about 5 x 10 8 69. The method of any one of claims 1 to 68, comprising total cells or total T cells, or a viable population of any of said.

70. The method of any one of claims 9-24, 33-39, 48-54, 59-69, wherein the one or more stimulatory agents are capable of activating T cells, CD4+ T cells and / or CD8+ T cells, inducing a signal through the TCR complex, and / or inducing proliferation of T cells, CD4+ T cells and / or CD8+ T cells.

71. The method of any one of claims 9-24, 33-39, 48-54, 59-70, wherein the one or more stimulatory agents comprise a primary agent that binds to a member of the TCR complex, optionally a primary agent that specifically binds to CD3.

72. 72. The method of claim 71, wherein the one or more stimulatory agents further comprise a secondary agent that specifically binds to a T cell costimulatory molecule.

73. The method of claim 72, wherein the costimulatory molecule is selected from the group consisting of CD28, CD137 (4-1-BB), OX40 or ICOS.

74. 74. The method of claim 72 or claim 73, wherein the primary and secondary agents comprise antibodies, and optionally, the one or more stimulatory agents comprise incubation with an anti-CD3 antibody and an anti-CD28 antibody.

75. The method of any one of claims 9-24, 33-39, 48-54, 59-74, wherein the one or more stimuli are present on the surface of a solid support, optionally a bead.

76. 76. The method of claim 75, wherein the one or more stimuli are present on the surface of a bead, and the bead is a paramagnetic bead.

77. The method of any one of claims 9-24, 33-39, 48-54, 59-76, wherein the one or more stimulatory substances are selected from the group consisting of a CD3 binding molecule, a CD28 binding molecule, recombinant IL-2, recombinant IL-15, and recombinant IL-7, a vaccine comprising an antigen that is specifically recognized by an antigen receptor, and an anti-idiotypic antibody that specifically binds to an antigen receptor, or a combination thereof.

78. 78. The method of any one of claims 9-24, 33-39, 48-54, 59-77, wherein the incubation is carried out for 2-15 days, 2-12 days, 2-12 days, 2-8 days, 2-6 days, 2-4 days, 4-12 days, 4-10 days, 4-8 days, 4-6 days, 6-12 days, 6-10 days, 6-8 days, 8-12 days, 8-10 days, or 10-12 days.

79. The method of any one of claims 9-24, 33-39, 48-54, 59-78, wherein the incubation is carried out for at least or about 4, 6, 8, 10 or 12 days, or for at least or about 4, 6, 8, 10 or 12 days.

80. The method of any one of claims 7-24, 31-40, 46-55 or 59-79, wherein the agent comprising the nucleic acid molecule is a viral vector or a transposon.

81. 81. The method of claim 80, wherein the agent comprising the nucleic acid molecule is a viral vector, and the viral vector is a retroviral vector.

82. The method of claim 81, wherein the viral vector is a lentiviral vector or a gamma retroviral vector.

83. The method of any one of claims 7-24, 31-39, 46-54 or 58-82, wherein the recombinant receptor is capable of binding to a target antigen associated with, specific for and / or expressed on cells or tissue of a disease, disorder or condition.

84. 84. The method of claim 83, wherein the disease, disorder or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer.

85. 85. The method of claim 83 or claim 84, wherein the target antigen is a tumor antigen.

86. Target antigens include 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), ephrin receptor A2 (EPHa2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, epidermal growth factor receptor type III mutation (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 alpha (IL-22Rα), kinase insert domain receptor (kdr), kappa light chain, leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, preferentially expressed antigen in melanoma (PRAME), survivin, tumor-associated glycoprotein 72 (TAG7 2), 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, biantigen, cancer-testis antigen, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), natural killer group 2 member D (NKG2D) ligand, cancer / testis antigen 1B (CTAG;also known as NY-ESO-1 and LAGE-2), melan A (MART-1), glycoprotein 100 (gp100), glypican 3 (GPC3), G protein-coupled receptor 5D (GPRC5D), fetal tumor antigen, TAG72, tyrosinase-related protein 1 (TRP1; also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2; also known as dopachrome tautomerase, dopachrome delta-isomerase, or DCT), vascular endothelial growth factor receptor 2 (VEGF- 86. The method of any one of claims 83-85, wherein the antigen is selected from among antigens associated with a universal tag, such as CD123, CD133, CD138, CD138R2, carcinoembryonic antigen (CEA), estrogen receptor, progesterone receptor, prostate specific antigen, ephrin B2, CD123, CD133, c-Met, O-acetylated GD2 (OGD2), the CE7 epitope of L1-CAM, Wilms' tumor 1 (WT-1), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD138, a pathogen-specific or pathogen-expressed antigen, and a universal tag;

87. The method of any one of claims 7 to 24, 31 to 39, 46 to 55 or 58 to 86, wherein the recombinant receptor is or comprises a functional non-TCR antigen receptor or TCR or an antigen-binding fragment thereof.

88. The method of any one of claims 7-24, 31-39, 46-55 or 58-86, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

89. The method of claim 88, wherein the chimeric antigen receptor comprises an extracellular domain comprising an antigen-binding domain that specifically binds to an antigen, and an intracellular signaling domain comprising an ITAM.

90. 90. The method of claim 89, wherein the antigen-binding domain is or comprises an antibody or an antibody fragment thereof, optionally a single chain fragment.

91. The method of claim 89 or claim 90, wherein the intracellular signaling domain is or comprises the intracellular signaling domain of a CD3 chain, optionally a CD3-zeta (CD3ζ) chain, or a signaling portion thereof.

92. The method of any one of claims 89, 90 and 91, wherein the intracellular signaling region further comprises a costimulatory signaling region.

93. The method of claim 92, wherein the costimulatory signaling region comprises the intracellular signaling domain of a T cell costimulatory molecule, or a signaling portion thereof.

94. The method of claim 92 or claim 93, wherein the costimulatory signaling region comprises the intracellular signaling domain of CD28, 4-1BB or ICOS, or a signaling portion thereof.

95. the CAR comprises an antigen-specific scFv, a transmembrane domain, optionally a cytoplasmic signaling domain derived from a costimulatory molecule that is or comprises 4-1BB, and optionally a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule that is or comprises a CD3 zeta signaling domain, and optionally further comprises a spacer between the transmembrane domain and the scFv; the CAR comprises, in order, an scFv specific for an antigen, a transmembrane domain, optionally a cytoplasmic signaling domain derived from a costimulatory molecule that is or contains a 4-1BB signaling domain, and optionally a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule that is a CD3 zeta signaling domain; or the CAR comprises, in order, an scFv specific for an antigen, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule that is optionally a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule that is or includes a CD3 zeta signaling domain; The method of any one of claims 88 to 93.

96. The method of any one of claims 2-9, 11-24, 26-33, 35-39, 41-48, 51-55, 57-58 or 61-95, wherein the subject has a disease or condition, and optionally the recombinant receptor specifically recognizes or specifically binds to an antigen associated with the disease or condition, or an antigen expressed or present on cells of the disease or condition.

97. an output composition in which the ratio of recombinant receptor-expressing CD4+ T cells to recombinant receptor-expressing CD8+ T cells, and optionally the viable cell ratio thereof, varies by no more than 20%, or no more than 10%, or no more than 5% from the average of such ratios in a plurality of T cell compositions produced by said method, and / or varies by no more than one standard deviation from such average. The method of any one of claims 1 to 96,

98. an output composition in which the ratio of recombinant receptor-expressing CD4+ T cells to recombinant receptor-expressing CD8+ T cells, optionally viable cells thereof, is between 0.5:1 and 2:1, or between 0.8:1 and 1.6:1, or between 1:1 and 1.5:1, or about 0.5:1 and 2:1, or about 0.8:1 and 1.6:1, or about 1:1 and 1.5:1, inclusive; The method of any one of claims 1 to 97,

99. 99. The method of any one of claims 97 or 98, wherein the ratio of recombinant receptor-expressing CD4+ T cells to recombinant receptor-expressing CD8+ T cells, optionally viable cells thereof, in the output composition is or is about 1.2:1, 1.1:1, 1:1, 0.9:1, or 0.8:

1.

100. 100. The method of any one of claims 97-99, wherein the ratio of recombinant receptor-expressing CD4+ T cells to recombinant receptor-expressing CD8+ T cells, optionally viable cells thereof, in the output composition is 1:1 or is about 1:

1.

101. 101. An output composition produced by the method of any one of claims 1 to 100.

102. 102. A pharmaceutical composition comprising the output composition of claim 101.

103. 103. The pharmaceutical composition of claim 102, further comprising a pharmaceutical carrier.

104. Administering the output composition produced by the method of any one of claims 1 to 100 or the pharmaceutical composition of claim 102 or claim 103 to a mammalian subject. A method of treatment comprising:

105. The method of claim 104, wherein the cells are derived from the subject to which the cells are administered.

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