Method for producing chimeric antigen receptor-expressing cells

Abstract

The present invention provides a method for producing immunoeffector cells that have been engineered to express chimeric antigen receptors (CARs), and a composition comprising such cells. [Solution] A method is provided for producing immunoeffector cells (e.g., T cells, NK cells) comprising (i) a nucleic acid molecule encoding a controllable chimeric antigen receptor (CCAR), or (ii) a nucleic acid molecule encoding a CAR and a regulatory molecule, and a composition produced by such a method.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 982,698, filed on 27 February 2020, which is incorporated herein by reference in its entirety.

[0002] Sequence List This application has been filed electronically in ASCII format and includes a sequence listing which is incorporated herein by reference in its entirety. The ASCII copy was created on 19 February 2021, named N2067-7169WO_SL.txt, and has a size of 386,964 bytes.

[0003] This disclosure relates, in general terms, to methods for producing immunoeffector cells (e.g., T cells or NK cells) that have been engineered to express chimeric antigen receptors (CARs), and to compositions comprising such cells. [Background technology]

[0004] Adoptive cell transfer (ACT) therapy using T cells, particularly T cells transduced with chimeric antigen receptors (CARs), has shown promise in trials for several hematological cancers. Currently, the production of genetically modified T cells is a complex process. There is a need for methods and processes to improve the production of CAR-expressing cell therapy products, enhance product quality, and maximize their therapeutic efficacy. [Overview of the project] [Means for solving the problem]

[0005] This disclosure relates to methods for producing immunoeffector cells (e.g., T cells or NK cells) engineered to express CARs, and compositions produced using such methods. Methods for using such compositions to treat a target disease, such as cancer, are also disclosed.

[0006] In some embodiments, the disclosure features a method for producing a population of cells (e.g., T cells) comprising a first nucleic acid molecule encoding a controllable chimeric antigen receptor (CCAR) or a second nucleic acid molecule encoding a chimeric antigen receptor (CAR) and a regulatory molecule. In some embodiments, the disclosure features a method for producing a population of cells (e.g., T cells) comprising a first nucleic acid molecule encoding a controllable chimeric antigen receptor (CCAR). In some embodiments, the disclosure features a method for producing a population of cells (e.g., T cells) comprising a second nucleic acid molecule encoding a chimeric antigen receptor (CAR) and a regulatory molecule. In some embodiments, the second nucleic acid molecule comprises one or more nucleic acid molecules, for example, the second nucleic acid molecule comprises a third nucleic acid molecule and a fourth nucleic acid molecule, the third nucleic acid molecule comprising a nucleic acid sequence encoding a CAR, and the fourth nucleic acid molecule comprising a nucleic acid molecule encoding a regulatory molecule.

[0007] In some embodiments, the method includes (i) contacting (e.g., binding) a population of cells (e.g., T cells, e.g., T cells isolated from frozen or fresh leukocyte apheresis products) with a drug that stimulates the CD3 / TCR complex and / or a drug that stimulates costimulatory molecules on the surface of the cells; (ii) contacting a population of cells (e.g., T cells) with a first nucleic acid molecule encoding a CCAR (e.g., a DNA or RNA molecule) or a second nucleic acid molecule encoding a CAR and regulatory molecules (e.g., a DNA or RNA molecule), thereby providing a population of cells (e.g., T cells) containing the first or second nucleic acid molecule; and (iii) collecting a population of cells (e.g., T cells) for storage (e.g., re-formulating the population of cells in cryopreservation medium) or administration. In some embodiments, step (ii) is performed together with step (i) or within 20 hours after the start of step (i), for example, within 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), for example, within 18 hours after the start of step (i), and step (iii) is performed within 30 (e.g., 26) hours after the start of step (i), for example, within 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (i), for example, within 24 hours after the start of step (i). In some embodiments, step (ii) is performed together with step (i) or within 20 hours after the start of step (i), for example, within 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), for example, within 18 hours after the start of step (i), and step (iii) is performed within 30 hours after the start of step (ii), for example, within 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (ii). In some embodiments, the population of cells from step (iii) is not proliferated, or is proliferated by 5, 10, 15, 20, 25, 30, 35, or 40%, for example, 10%, compared to the population of cells at the start of step (i). In some embodiments, the first or second nucleic acid molecule in step (ii) is on a viral vector.In some embodiments, the first or second nucleic acid molecule in step (ii) is an RNA molecule on a viral vector. In some embodiments, step (ii) includes transducing a population of cells (e.g., T cells) with a viral vector containing the first or second nucleic acid molecule.

[0008] In some embodiments, the agent stimulating the CD3 / TCR complex is an agent stimulating CD3 (e.g., an anti-CD antibody). In some embodiments, the agent stimulating the co-stimulator is an agent stimulating CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, the agent stimulating the CD3 / TCR complex or the agent stimulating the co-stimulator is selected from antibodies (e.g., single-domain antibodies (e.g., heavy-chain variable-domain antibodies), peptide bodies, Fab fragments, or scFv), small molecules, or ligands (e.g., naturally occurring ligands, recombinant ligands, or chimeric ligands). In some embodiments, the agent stimulating the CD3 / TCR complex or the agent stimulating the co-stimulator does not contain beads. In some embodiments, the agent stimulating the CD3 / TCR complex includes an anti-CD3 antibody, and the agent stimulating the co-stimulator includes an anti-CD28 antibody. In some embodiments, the agent stimulating the CD3 / TCR complex includes an anti-CD3 antibody covalently bound to a colloidal polymer nanomatrix, and the agent stimulating the costimulatory molecule includes an anti-CD28 antibody covalently bound to a colloidal polymer nanomatrix. In some embodiments, the agent stimulating the CD3 / TCR complex and the agent stimulating the costimulatory molecule include T Cell TransAct®.

[0009] In some embodiments, step (i) increases the percentage of cells containing the first or second nucleic acid molecule in the population of cells from step (iii). In some embodiments, the population of cells from step (iii) shows a higher percentage (e.g., at least 10, 20, 30, 40, 50, or 60%) of cells containing the first or second nucleic acid molecule compared to cells produced by a similar method except without step (i).

[0010] In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells in the cell population from step (iii) is the same as the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells in the cell population at the start of step (i), or differs by only 5 or 10% or less. In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells in the cell population from step (iii) is increased by at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3 times compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells in the cell population at the start of step (i). In some embodiments, the percentage of naive T cells containing a first or second nucleic acid molecule, e.g., CD45RA+CD45RO-CCR7+ T cells containing a first or second nucleic acid molecule, in the cell population increases during the period of step (ii), for example, by at least 30, 35, 40, 45, 50, 55, or 60% within 18-24 hours after the start of step (ii). In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (iii) does not decrease, or decreases by 5 or 10% or less, compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (i).

[0011] In some embodiments, the cell population from step (iii) exhibits a higher percentage (e.g., at least 10, 20, 30, or 40%) of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, compared to cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the population of cells from step (iii) is higher (e.g., at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3 times higher) than the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the percentage of naive T cells containing the first or second nucleic acid molecule, e.g., CD45RA+CD45RO-CCR7+ T cells containing the first or second nucleic acid molecule, in the population of cells from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 times higher) than the percentage of naive T cells containing the first or second nucleic acid molecule, e.g., CD45RA+CD45RO-CCR7+ T cells containing the first or second nucleic acid molecule, in cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i).In some embodiments, the cell population from step (iii) exhibits a higher percentage (e.g., at least 10, 20, 30, or 40%) of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, compared to cells produced by a similar method, except that the method further includes a step of growing the above cell population (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the population of cells from step (iii) is higher (e.g., at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3 times higher) in cells produced by a similar method, except that the method further includes a step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii). In some embodiments, the percentage of naive T cells containing a first or second nucleic acid molecule, e.g., CD45RA+CD45RO-CCR7+ T cells containing a first or second nucleic acid molecule, in the population of cells from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 times higher) than the percentage of naive T cells containing a first or second nucleic acid molecule, e.g., CD45RA+CD45RO-CCR7+ T cells containing a first or second nucleic acid molecule, in cells produced by the same method, except that the further step includes growing the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, e.g., 5, 6, 7, 8, or 9 days, e.g., CD45RA+CD45RO-CCR7+ T cells containing a first or second nucleic acid molecule, in the population of cells from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 times higher).

[0012] In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population from step (iii) is the same as the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population at the start of step (i), or differs by only 5 or 10% or less. In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the cell population from step (iii) is reduced by at least 20, 25, 30, 35, 40, 45, or 50% compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the cell population at the start of step (i). In some embodiments, the percentage of central memory T cells containing a first or second nucleic acid molecule, e.g., CCR7+CD45RO+ cells containing a first or second nucleic acid molecule, decreases during the period of step (ii), for example, by at least 8, 10, 12, 14, 16, 18, or 20% within 18-24 hours after the start of step (ii). In some embodiments, the percentage of central memory cells in the cell population from step (iii), e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, does not increase, or increases by 5 or 10% or less, compared to the percentage of central memory cells in the cell population at the start of step (i).

[0013] In some embodiments, the population of cells from step (iii) exhibits a lower percentage (e.g., at least 10, 20, 30, or 40%) of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, compared to cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 20, 30, 40, or 50%) than the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the percentage of central memory T cells containing the first or second nucleic acid molecule, e.g., CCR7+CD45RO+ T cells containing the first or second nucleic acid molecule, in the population of cells from step (iii) is lower (e.g., at least 10, 20, 30, or 40%) than the percentage of central memory T cells containing the first or second nucleic acid molecule, e.g., CCR7+CD45RO+ T cells containing the first or second nucleic acid molecule, in cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i).In some embodiments, the population of cells from step (iii) exhibits a lower percentage (e.g., at least 10, 20, 30, or 40%) of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, compared to cells produced by a similar method, except that the step further includes growing the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 20, 30, 40, or 50%) than the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in cells produced by a similar method, except that the step further includes growing the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the percentage of central memory T cells containing a first or second nucleic acid molecule, e.g., CCR7+CD45RO+ T cells containing a first or second nucleic acid molecule, in the population of cells from step (iii) is lower (e.g., at least 10, 20, 30, or 40%) than the percentage of central memory T cells containing a first or second nucleic acid molecule, e.g., CCR7+CD45RO+ T cells containing a first or second nucleic acid molecule, in cells produced by the same method, except that the method further includes a step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 CCR7+CD45RO+ T cells containing a first or second nucleic acid molecule, in the population of cells from step (iii) is lower (e.g., at least 10, 20, 30, or 40%).

[0014] In some embodiments, the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population from step (iii) is increased compared to the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population at the start of step (i). In some embodiments, the percentage of stem memory T cells containing a first or second nucleic acid molecule, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population from step (iii) is increased compared to the percentage of stem memory T cells containing a first or second nucleic acid molecule, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population at the start of step (i). In some embodiments, the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i). In some embodiments, the percentage of stem memory T cells containing a first or second nucleic acid molecule, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells containing a first or second nucleic acid molecule, in the population of cells from step (iii) is higher than the percentage of stem memory T cells containing a first or second nucleic acid molecule, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells containing a first or second nucleic acid molecule, in cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i).In some embodiments, the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the population of cells from step (iii) is higher than the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in cells produced by a similar method, except that the method further includes a step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the percentage of stem memory T cells containing a first or second nucleic acid molecule, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells containing a first or second nucleic acid molecule, in the population of cells from step (iii) is higher than the percentage of stem memory T cells containing a first or second nucleic acid molecule, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells containing a first or second nucleic acid molecule, in cells produced by the same method, except that the step further includes growing the population of cells (e.g., T cells) in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

[0015] In some embodiments, the median GeneSetScore (Up TEM vs. Down TSCM) of the cell population from step (iii) is approximately the same as the median GeneSetScore (Up TEM vs. Down TSCM) of the cell population at the start of step (i), or differs by only about 25, 50, 75, 100, or 125% or less (for example, increased by only a small amount). In some embodiments, the median GeneSetScore (Up TEM vs. Down TSCM) of the cell population from step (iii) is lower (e.g., at least about 100, 150, 200, 250, or 300%) than the median GeneSetScore (Up TEM vs. Down TSCM) of cells produced by the same method except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), or that the median GeneSetScore (Up TEM vs. Down TSCM) of cells produced by the same method except that step (iii) further includes a step of growing the cell population (e.g., T cells) in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the median GeneSetScore (Up Treg vs. Down Teff) of the cell population from step (iii) is approximately the same as the median GeneSetScore (Up Treg vs. Down Teff) of the cell population at the start of step (i), or differs by only about 25, 50, 100, 150, or 200% or less (for example, increased by only a small amount).In some embodiments, the median GeneSetScore (Up Treg vs. Down Teff) of the cell population from step (iii) is lower (e.g., at least about 50, 100, 125, 150, or 175%) than the median GeneSetScore (Up Treg vs. Down Teff) of cells produced by the same method except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), or that the cell population (e.g., T cells) is further grown in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the median GeneSetScore (Down stemness) of the cell population from step (iii) is approximately the same as the median GeneSetScore (Down stemness) of the cell population at the start of step (i), or differs by only about 25, 50, 100, 150, 200, or 250% or less (for example, increased by only a small amount). In some embodiments, the median GeneSetScore (Downstemness) of the cell population from step (iii) is lower (e.g., at least about 50, 100, or 125%) than the median GeneSetScore (Downstemness) of cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), or that the cell population (e.g., T cells) is further grown in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).In some embodiments, the median GeneSetScore (Up hypoxia) of the cell population from step (iii) is approximately the same as the median GeneSetScore (Up hypoxia) of the cell population at the start of step (i), or differs by only about 125, 150, 175, or 200% or less (for example, increased by only a small amount). In some embodiments, the median GeneSetScore (Up hypoxia) of the cell population from step (iii) is lower (e.g., at least about 40, 50, 60, 70, or 80%) than the median GeneSetScore (Up hypoxia) of cells produced by the same method except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), or that the cell population (e.g., T cells) is further grown in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the median GeneSetScore (Up autophagy) of the cell population from step (iii) is approximately the same as the median GeneSetScore (Up autophagy) of the cell population at the start of step (i), or differs by only about 180, 190, 200, or 210% or less (for example, increased by only that amount or less). In some embodiments, the median GeneSetScore (Up autophagy) of the cell population from step (iii) is lower (e.g., at least about 20, 30, or 40%) than the median GeneSetScore (Up autophagy) of cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), or that the cell population (e.g., T cells) is further grown in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

[0016] In some embodiments, the population of cells from step (iii) is incubated with cells expressing an antigen recognized by CCAR or CAR, and then secretes IL-2 at a higher level (e.g., at least 2, 4, 6, 8, 10, 12, or 14 times higher) than cells produced by the same method except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), or cells produced by the same method except that step (iii) further includes a step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

[0017] In some embodiments, the population of cells from step (iii), after being administered in vivo, persists longer or proliferates at a higher level compared to cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), or compared to cells produced by a similar method, except that the step further includes a step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii) (for example, as evaluated using the method described in Example 1 with respect to Figure 4C).

[0018] In some embodiments, the population of cells from step (iii) is administered in vivo, and step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), except that cells produced by the same method are administered in vivo, or the population of cells (e.g., T cells) is further proliferated in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii), except that the step further includes growing the population of cells (e.g., T cells) in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days, except that cells produced by the same method have more potent antitumor activity (e.g., low dose, e.g., 0.15 × 10⁻¹⁵) 6 , 0.2 × 10 6 , 0.25 × 10 6 or 0.3 × 10 6 It exhibits more potent antitumor activity in viable cells containing the first or second nucleic acid molecule at doses of one or fewer units.

[0019] In some embodiments, the population of cells from step (iii) is not proliferated compared to the population of cells at the start of step (i), for example, by the number of surviving cells, or is proliferated by 5, 10, 15, 20, 25, 30, 35, or 40% or less, and optionally, the number of surviving cells in the population of cells from step (iii) is reduced from the number of surviving cells in the population of cells at the start of step (i).

[0020] In some embodiments, the population of cells from step (iii) does not grow compared to the population of cells at the start of step (i), or grows for less than 2 hours, for example, less than 1 or 1.5 hours.

[0021] In some embodiments, steps (i) and / or (ii) are carried out in a cell medium (e.g., serum-free medium) containing IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-7, IL-21, IL-6 (e.g., IL-6 / sIL-6Ra), an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof.

[0022] In some embodiments, steps (i) and / or (ii) are carried out in serum-free cell medium containing a serum substitute. In some embodiments, the serum substitute is CTS® Immune Cell Serum Replacement (ICSR).

[0023] In some embodiments, the method further includes, before step (i), (iv) (optionally) receiving fresh leukocyte apheresis product (or an alternative source of hematopoietic tissue such as fresh whole blood product, fresh bone marrow product, or fresh tumor or organ biopsy or excision (e.g., fresh product from thymectomy)) from an entity, e.g., a laboratory, hospital, or healthcare provider, and (v) isolating a population of the cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) that were contacted in step (i) from the fresh leukocyte apheresis product (or an alternative source of hematopoietic tissue such as fresh whole blood product, fresh bone marrow product, or fresh tumor or organ biopsy or excision (e.g., fresh product from thymectomy)). In some embodiments, step (iii) is carried out within 35 hours after the start of step (v), for example, within 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of step (v), for example, within 30 hours after the start of step (v). In some embodiments, the population of cells from step (iii) is not proliferated compared to the population of cells at the end of step (v), as evaluated by the number of viable cells, or is proliferated by 5, 10, 15, 20, 25, 30, 35, or 40%, for example, 10% or less.

[0024] In some embodiments, the method further includes, prior to step (i), receiving cryopreserved T cells isolated from leukocyte apheresis products (or alternative sources of hematopoietic tissue such as cryopreserved T cells isolated from whole blood, bone marrow, or tumor or organ biopsy or resection (e.g., thymectomy)) from an entity, such as a laboratory, hospital, or healthcare provider.

[0025] In some embodiments, the method further comprises (iv) (optionally) receiving cryopreserved leukocyte apheresis products (or alternative sources of hematopoietic tissue such as cryopreserved whole blood products, cryopreserved bone marrow products, or cryopreserved tumor or organ biopsy or excision (e.g., cryopreserved products from thymectomy)) prior to step (i), and (v) isolating a population of the cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) that were contacted in step (i) from the cryopreserved leukocyte apheresis products (or alternative sources of hematopoietic tissue such as cryopreserved whole blood products, cryopreserved bone marrow products, or cryopreserved tumor or organ biopsy or excision (e.g., cryopreserved products from thymectomy)). In some embodiments, step (iii) is performed within 35 hours after the start of step (v), for example, within 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of step (v), for example, within 30 hours after the start of step (v). In some embodiments, the population of cells from step (iii) is not proliferated compared to the population of cells at the end of step (v), as judged by the number of viable cells, or is proliferated by 5, 10, 15, 20, 25, 30, 35, or 40%, for example, 10% or less.

[0026] In some embodiments, the method further includes step (vi): culturing a portion of the cell population from step (iii) for at least 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 6, 6.5, or 7 days, for example, at least 2 days and no more than 7 days, and measuring the CAR expression level in the portion (for example, measuring the percentage of viable CAR-expressing cells in the portion). In some embodiments, step (iii) includes collecting and freezing a population of cells (e.g., T cells), and step (vi) includes thawing a portion of the cell population from step (iii), culturing the portion for at least 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 6, 6.5, or 7 days, for example, at least 2 days and no more than 7 days, and measuring the CAR expression level in the portion (for example, measuring the percentage of viable CAR-expressing cells in the portion).

[0027] In some embodiments, methods for producing a population of cells (e.g., T cells) comprising a first nucleic acid molecule encoding a controllable chimeric antigen receptor (CCAR) or a second nucleic acid molecule encoding a chimeric antigen receptor (CAR) and a regulatory molecule are provided herein. In some embodiments, the disclosure is characterized by a method for producing a population of cells (e.g., T cells) comprising a first nucleic acid molecule encoding a controllable chimeric antigen receptor (CCAR). In some embodiments, the disclosure is characterized by a method for producing a population of cells (e.g., T cells) comprising a second nucleic acid molecule encoding a chimeric antigen receptor (CAR) and a regulatory molecule. In some embodiments, the second nucleic acid molecule comprises one or more nucleic acid molecules, for example, the second nucleic acid molecule comprises a third nucleic acid molecule and a fourth nucleic acid molecule, the third nucleic acid molecule comprising a nucleic acid sequence encoding a CAR, and the fourth nucleic acid molecule comprising a nucleic acid molecule encoding a regulatory molecule.

[0028] In some embodiments, the method includes (1) contacting a population of cells (e.g., T cells, e.g., T cells isolated from frozen leukocyte apheresis products) with a cytokine selected from IL-2, IL-7, IL-15, IL-21, IL-6 or a combination thereof; (2) contacting the population of cells (e.g., T cells) with a first nucleic acid molecule encoding a CCAR (e.g., a DNA or RNA molecule) or a second nucleic acid molecule encoding a CAR and regulatory molecules (e.g., a DNA or RNA molecule), thereby providing a population of cells (e.g., T cells) containing the first or second nucleic acid molecule; and (3) collecting the population of cells (e.g., T cells) for storage (e.g., re-formulating the population of cells in cryopreservation medium) or administration. In some embodiments, step (2) is performed together with step (1) or within 5 hours after the start of step (1), for example within 1, 2, 3, 4, or 5 hours after the start of step (1), and step (3) is performed within 26 hours after the start of step (1), for example within 22, 23, or 24 hours after the start of step (1), for example within 24 hours after the start of step (1). In some embodiments, the population of cells from step (3) is not proliferated, or is proliferated by 5, 10, 15, 20, 25, 30, 35, or 40%, for example, 10%, compared to the population of cells at the start of step (1), as evaluated by the number of viable cells. In some embodiments, the first or second nucleic acid molecule in step (2) is on a viral vector. In some embodiments, the first or second nucleic acid molecule in step (ii) is an RNA molecule on a viral vector. In some embodiments, step (ii) includes transducing the population of cells (e.g., T cells) with a viral vector containing a first or second nucleic acid molecule.

[0029] In some embodiments, step (1) includes contacting the population of cells (e.g., T cells) with IL-2. In some embodiments, step (1) includes contacting the population of cells (e.g., T cells) with IL-7. In some embodiments, step (1) includes contacting the population of cells (e.g., T cells) with IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)). In some embodiments, step (1) includes contacting the population of cells (e.g., T cells) with IL-21. In some embodiments, step (1) includes contacting the population of cells (e.g., T cells) with IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, step (1) includes contacting the population of cells (e.g., T cells) with IL-7 and IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)). In some embodiments, step (1) includes contacting the population of cells (e.g., T cells) with IL-7 and IL-21. In some embodiments, step (1) includes contacting the population of cells (e.g., T cells) with IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)) and IL-21. In some embodiments, step (1) includes contacting the population of cells (e.g., T cells) with IL-7, IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)) and IL-21. In some embodiments, step (1) includes contacting the population of cells (e.g., T cells) with IL-6 (e.g., IL-6 / sIL-6Ra) and IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)). In some embodiments, step (1) includes bringing the population of cells (e.g., T cells) into contact with IL-2 and IL-6 (e.g., IL-6 / sIL-6Ra).

[0030] In some embodiments, the population of cells from step (3) exhibits a higher percentage (e.g., at least 10, 15, 20, 25, 30, 35, or 40%) of naive cells among cells containing the first or second nucleic acid molecule compared to cells produced by a similar method, except that the step further includes contacting the population of cells with, for example, an anti-CD3 antibody.

[0031] In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells in the cell population from step (3) is (a) the same as the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells in the cell population at the start of step (1), or differs by 5 or 10% or less, or (b) is increased compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells in the cell population at the start of step (1), for example, increased by at least 10 or 20%.

[0032] In some embodiments, the population of cells from step (3) exhibits a higher percentage (e.g., at least 10, 20, 30, or 40%) of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, compared to cells produced by a similar method, except that step (3) is performed more than 26 hours after the start of step (1), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (1).

[0033] In some embodiments, the population of cells from step (3) exhibits a higher percentage (e.g., at least 10, 20, 30, or 40%) of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, compared to cells produced by a similar method, except that the method further includes a step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (2) and before step (3).

[0034] In some embodiments, the population of cells from step (3), after in vivo administration, persists longer or proliferates at a higher level compared to cells produced by a similar method, except that step (3) is performed more than 26 hours after the start of step (1), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (1) (as evaluated, for example, using the method described in Example 1 with respect to Figure 4C).

[0035] In some embodiments, the population of cells from step (3) persists longer or proliferates at a higher level than cells produced by a similar method, except that the step further includes a step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, for example 5, 6, 7, 8, or 9 days, after administration in vivo, after step (2) and before step (3) (for example, as evaluated using the method described in Example 1 with respect to Figure 4C).

[0036] In some embodiments, the population of cells from step (3) is not proliferated compared to the population of cells at the start of step (1), as evaluated by, for example, the number of surviving cells, or is proliferated by 5, 10, 15, 20, 25, 30, 35, or 40%, for example, 10% or less, and optionally, the number of surviving cells in the population of cells from step (3) is reduced from the number of surviving cells in the population of cells at the start of step (1).

[0037] In some embodiments, the population of cells from step (3) does not grow compared to the population of cells at the start of step (1), or grows for less than 2 hours, for example, less than 1 or 1.5 hours.

[0038] In some embodiments, the population of cells is not exposed in vitro to an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a co-stimulatory molecule on the surface of the cells, or, if exposed, the exposure step is less than 2 hours, for example, 1 or 1.5 hours or less. In some embodiments, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3 (e.g., an anti-CD3 antibody), and the agent that stimulates the co-stimulatory molecule is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. Optionally, the agent that stimulates the CD3 / TCR complex or the agent that stimulates the co-stimulatory molecule is selected from antibodies (e.g., single-domain antibodies (e.g., heavy-chain variable-domain antibodies), peptide bodies, Fab fragments, or scFv), small molecules, or ligands (e.g., naturally occurring ligands, recombinant ligands, or chimeric ligands).

[0039] In some embodiments, steps (1) and / or (2) are performed in a cell medium containing serum or an LSD1 inhibitor or MALT1 inhibitor, with serum being 5, 4, 3, 2, 1 or 0% or less, and optionally, steps (1) and / or (2) are performed in a cell medium containing serum or an LSD1 inhibitor, with serum being 5, 4, 3, 2, 1 or 0% or less.

[0040] In some embodiments, the method further includes the step of receiving cryopreserved leukocyte apheresis products (or alternative sources of hematopoietic tissue such as cryopreserved whole blood products, cryopreserved bone marrow products, or cryopreserved tumor or organ biopsy or excision products (e.g., cryopreserved products from thymectomy)) from an entity, such as a laboratory, hospital, or healthcare provider.

[0041] In some embodiments, the cell population at the start of step (i) or step (1) is enriched with IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ). In some embodiments, the cell population at the start of step (i) or step (1) contains 50, 60, or 70% or more IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ). In some embodiments, steps (i) and (ii) or steps (1) and (2) are carried out in a cell medium containing IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, IL-15 increases the ability of the cell population to proliferate, for example, after 10, 15, 20, or 25 days. In some embodiments, IL-15 increases the percentage of IL6Rβ-expressing cells in the cell population.

[0042] In some embodiments, the CCAR or CAR includes an antigen-binding domain, a transmembrane domain, and / or an intracellular signaling domain. In some embodiments, the antigen-binding domain is CD19, CD20, CD22, BCMA, mesothelin, EGFRvIII, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptide, sTn-O-glycopeptide, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, Regman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, Lewis Y, CD24, PDGFR-β, SSEA-4, folate receptor α, ERBB (e.g., ERBB2), Her2 / neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, FAP, Regman, HPV It binds to an antigen selected from the peptides of any of the following antigens presented on the MHC: E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, β-human chorionic gonadotropin, AFP, thyroglobulin, PLAC1, globo H, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, mut hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4, or MHC.In some embodiments, the antigen-binding domain comprises a CDR, VH, VL, or scFv sequence disclosed herein, and optionally (a) the antigen-binding domain binds to BCMA and comprises a CDR, VH, VL, scFv, or CAR sequence disclosed in Tables 3-15 or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto; (b) the antigen-binding domain binds to CD19 and comprises a CDR, VH, VL, scFv, or CAR sequence disclosed in Table 2 or a sequence having at least 80%, 85%, or 99% identity thereto. (c) The antigen-binding domain includes a sequence having 0%, 95%, or 99% identity; (d) The antigen-binding domain includes a sequence that binds to CD20 and is a CDR, VH, VL, scFv, or CAR sequence disclosed herein or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto; or (d) The antigen-binding domain includes a sequence that binds to CD22 and is a CDR, VH, VL, scFv, or CAR sequence disclosed herein or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the antigen-binding domain includes VH and VL, which are linked by a linker, which optionally includes the amino acid sequence of SEQ ID NO: 63 or 104. In some embodiments, (a) the transmembrane domain includes a transmembrane domain of a protein selected from the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154; (b) the transmembrane domain includes a transmembrane domain of CD8; (c) the transmembrane domain includes the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (d) the first or second nucleic acid molecule includes a nucleic acid sequence encoding the transmembrane domain, wherein the nucleic acid sequence includes the nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.In some embodiments, the antigen-binding domain is linked to the transmembrane domain by a hinge region, and optionally, (a) the hinge region includes the amino acid sequence of SEQ ID NO: 2, 3, or 4 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or (b) the first or second nucleic acid molecule includes a nucleic acid sequence encoding the hinge region, and the nucleic acid sequence includes the nucleic acid sequence of SEQ ID NO: 13, 14, or 15 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the intracellular signaling domain includes a primary signaling domain, which optionally includes a functional signaling domain derived from CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278(ICOS), FcεRI, DAP10, DAP12, or CD66d, which optionally includes (a) a functional signaling domain derived from CD3ζ, (b) an amino acid sequence of SEQ ID NO: 9 or 10 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or (c) a first or second nucleic acid molecule that includes a nucleic acid sequence encoding the primary signaling domain, which includes a nucleic acid sequence of SEQ ID NO: 20 or 21 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.In some embodiments, the intracellular signaling domain includes a co-stimulatory signaling domain, which optionally comprises an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activator molecule (SLAM protein), an activated NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, or CD4. 0, CDS, ICAM-1, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), N Kp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49 f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD1 It contains a functional signaling domain selected from ligands that specifically bind to 60(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB, or CD83.In some embodiments, (a) the co-stimulus signaling domain includes a functional signaling domain derived from 4-1BB, (b) the co-stimulus signaling domain includes the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or (c) the first or second nucleic acid molecule includes a nucleic acid sequence encoding the co-stimulus signaling domain, wherein the nucleic acid sequence includes the nucleic acid sequence of SEQ ID NO: 18 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the intracellular signaling domain includes a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3ζ, and optionally, the intracellular signaling domain includes the amino acid sequence of SEQ ID NO: 7 (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and the amino acid sequence of SEQ ID NO: 9 or 10 (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto), and optionally, the intracellular signaling domain includes the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or 10. In some embodiments, the CCAR or CAR further includes a leader sequence containing the amino acid sequence of SEQ ID NO: 1.

[0043] In some embodiments, a population of cells containing a first or second nucleic acid molecule (e.g., autologous or allogeneic T cells or NK cells containing a first or second nucleic acid molecule) prepared by the method described above is provided herein.

[0044] In some embodiments, populations of cells engineered to contain a first nucleic acid molecule encoding a CCAR or a second nucleic acid molecule encoding a CAR and a regulatory molecule are provided herein. In some embodiments, populations of cells engineered to contain a first nucleic acid molecule encoding a CCAR are provided herein. In some embodiments, populations of cells engineered to contain a second nucleic acid molecule encoding a CAR and a regulatory molecule are provided herein. In some embodiments, the second nucleic acid molecule comprises one or more nucleic acid molecules, for example, the second nucleic acid molecule comprises a third nucleic acid molecule and a fourth nucleic acid molecule, the third nucleic acid molecule comprises a nucleic acid sequence encoding a CAR, and the fourth nucleic acid molecule comprises a nucleic acid sequence encoding a regulatory molecule.

[0045] In some embodiments, the population comprises (a) approximately the same percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells as the same percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells as the same population of cells before being manipulated to include the first or second nucleic acid molecule; and (b) approximately the same percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ cells as the same population of cells before being manipulated to include the first or second nucleic acid molecule. (c) a change of within approximately 5% to approximately 10% of naive T cells, e.g., CD45RO-CCR7+ T cells; (d) a percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells that has been increased by, for example, at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8 or 3 times compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells in the same cell population before being manipulated to contain the first or second nucleic acid molecule; (d) the first or second nucleic acid molecule (e) (f) A change of approximately 5% to approximately 10% in cells, for example, central memory T cells, for example, CCR7+CD45RO+ T cells; (f) A percentage of central memory cells, for example, central memory T cells, for example, CCR7+CD45RO+ T cells that is reduced, for example, by at least 20, 25, 30, 35, 40, 45, or 50%, compared to the percentage of central memory cells, for example, central memory T cells, for example, CCR7+CD45RO+ T cells in the same population of cells before being manipulated to contain a first or second nucleic acid molecule;(g) Approximately the same percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, as the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the same population of cells before they are manipulated to contain a first or second nucleic acid molecule; (h) Stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the same population of cells before they are manipulated to contain a first or second nucleic acid molecule (i) A change of approximately 5% to 10% compared to the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells; or (i) an increased percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, compared to the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the same cell population before being manipulated to contain a first or second nucleic acid molecule.

[0046] In some embodiments, a population of cells is provided that has been manipulated to contain a first nucleic acid molecule encoding a CCAR or a second nucleic acid molecule encoding a CAR and a regulatory molecule, wherein (a) the median GeneSetScore (Up TEM vs. Down TSCM) of the cell population is approximately the same as the median GeneSetScore (Up TEM vs. Down TSCM) of the same cell population before manipulation to contain the first or second nucleic acid molecule, or differs by only about 25, 50, 75, 100, or 125% or less (e.g., increased by only less than or equal to that); (b) the median GeneSetScore (Up Treg vs. Down Teff) of the cell population is the same as the median GeneSetScore (Up Treg vs. Down TSCM) of the cell population before manipulation to contain the first or second nucleic acid molecule (c) The median GeneSetScore (Down stemness) of the above cell population is approximately the same as the median GeneSetScore (Down stemness) of the above cell population before it is manipulated to contain the first or second nucleic acid molecule, or differs by approximately 25, 50, 100, 150, 200, or 250% or less (for example, increased by only a small amount); (d) The median GeneSetScore (Up hypoxia) of the above cell population is approximately the same as the median GeneSetScore (Up hypoxia) of the above cell population before it is manipulated to contain the first or second nucleic acid molecule, or differs by approximately 125, 150, 175, or 200% or less (for example, increased by only a small amount); or (e) the median GeneSetScore (Up hypoxia) of the above cell population The autophagy is approximately the same as the median GeneSetScore (Up autophagy) of the aforementioned cell population before it is manipulated to contain the first or second nucleic acid molecule, or differs by only about 180, 190, 200, or 210% or less (e.g., increased by only a small amount).

[0047] In some embodiments, the population of cells includes a first nucleic acid molecule encoding a CCAR.

[0048] In some embodiments, the CCAR is a fusion polypeptide comprising a degraded polypeptide (e.g., a degraded polypeptide disclosed herein) and a CAR polypeptide (e.g., a CAR polypeptide disclosed herein). In some embodiments, (i) the degraded polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 310-315, 320-324, 337-339, 360-361, 367-369 and 374 (or sequences having at least 85, 87, 90, 95, 97, 98, 99 or 100% identity therewith), and optionally, the degraded polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 312. (ii) The degrading polypeptide comprises a β-turn of IKZF1 or IKZF3 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto), and optionally, the degrading polypeptide comprises a β-hairpin or β-chain of IKZF1 or IKZF3 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto); (iii) The degrading polypeptide (iv) The pharmacoplasmic peptide contains an α-helix of IKZF1 or IKZF3 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity therewith); (iv) The degraded polypeptide contains, from the N-terminus to the C-terminus, a first β-chain, a β-hairpin, a second β-chain and a first α-helix of IKZF1 or IKZF3 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity therewith; (v) The degraded polypeptide comprises, from the N-terminus to the C-terminus, a first β-chain, a β-hairpin, a second β-chain, a first α-helix, and a second α-helix of IKZF1 or IKZF3 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto), wherein optionally, the β-hairpin and the second α-helix are separated by 60, 50, 40, or 30 or fewer amino acid residues;(vi) The degraded polypeptide contains approximately 10 to approximately 95 amino acid residues, approximately 15 to approximately 90 amino acid residues, approximately 20 to approximately 85 amino acid residues, approximately 25 to approximately 80 amino acid residues, approximately 30 to approximately 75 amino acid residues, approximately 35 to approximately 70 amino acid residues, approximately 40 to approximately 65 amino acid residues, approximately 45 to approximately 65 amino acid residues, approximately 50 to approximately 65 amino acid residues, or approximately 55 to approximately 65 amino acid residues of IKZF1 or IKZF3 (or sequences having at least 85, 87, 90, 95, 97, 98, 99 or 100% identity therewith); (vii) The decomposed polypeptide consists of at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, at least 65 amino acids, at least 70 amino acids of IKZF1 or IKZF3 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99 or 100% identity thereto), (viii) The association of a fusion polypeptide with cereblon (CRBN) in the absence of COF1 or COF2, e.g., an immunomodulatory imid (IMiD), e.g., lenalidomide, pomalidomide, or thalidomide; the association of a fusion polypeptide with cereblon (CRBN) in the presence of COF1 or COF2, e.g., an IMiD, e.g., lenalidomide, pomalidomide, or thalidomide; (ix) Ubiquitination of the fusion polypeptide in the absence of COF1 or COF2, e.g., IMiD, e.g., lenalidomide, pomalidomide, or thalidomide, is less than or equal to 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the ubiquitination of the fusion polypeptide in the presence of COF1 or COF2, e.g., IMiD, e.g., lenalidomide, pomalidomide, or thalidomide;(x) The degradation of the fusion polypeptide in the absence of COF1 or COF2, e.g., IMiD, e.g., lenalidomide, pomalidomide, or thalidomide, is less than or equal to, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the degradation of the fusion polypeptide in the presence of COF1 or COF2, e.g., IMiD, e.g., lenalidomide, pomalidomide, or thalidomide; and / or (xi) The expression level of the fusion polypeptide in the presence of COF1 or COF2, e.g., IMiD, e.g., lenalidomide, pomalidomide, or thalidomide, is reduced by, for example, at least 40%, 50%, 60%, 70%, 80%, 90%, or 99% compared to the expression level of the fusion polypeptide in the absence of COF1 or COF2, e.g., IMiD, e.g., lenalidomide, pomalidomide, or thalidomide. ;

[0049] In some embodiments, the degraded polypeptide includes or comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 375 to 377 (or sequences having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity therewith), and optionally, the degraded polypeptide includes or comprises the amino acid sequence of SEQ ID NO. 375. In some embodiments, the degraded polypeptide includes a β-turn of IKZF2 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity therewith), and optionally, the degraded polypeptide includes a β-hairpin or β-chain of IKZF2 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity therewith). In some embodiments, the degraded polypeptide includes an α-helix of IKZF2 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto). In some embodiments, the degraded polypeptide includes a first β-chain, a β-hairpin, a second β-chain, and a first α-helix of IKZF2 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto) from the N-terminus to the C-terminus. In some embodiments, the degraded polypeptide comprises, from the N-terminus to the C-terminus, a first β-chain, a β-hairpin, a second β-chain, a first α-helix, and a second α-helix of IKZF2 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto), wherein the β-hairpin and the second α-helix are optionally separated by 60, 50, 40, or 30 or fewer amino acid residues. In some embodiments, the degraded polypeptide comprises approximately 10 to approximately 95 amino acid residues, approximately 15 to approximately 90 amino acid residues, approximately 20 to approximately 85 amino acid residues, approximately 25 to approximately 80 amino acid residues, approximately 30 to approximately 75 amino acid residues, approximately 35 to approximately 70 amino acid residues, approximately 40 to approximately 65 amino acid residues, approximately 45 to approximately 65 amino acid residues, approximately 50 to approximately 65 amino acid residues, or approximately 55 to approximately 65 amino acid residues of IKZF2 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity therewith).In some embodiments, the degraded polypeptide comprises at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, at least 65 amino acids, at least 70 amino acids, at least 75 amino acids, at least 80 amino acids, at least 85 amino acids, at least 90 amino acids, at least 90 amino acids, or at least 95 amino acids of IKZF2 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity therewith). In some embodiments, the association of the fusion polypeptide with cereblon (CRBN) in the absence of COF3, e.g., compound I-112 disclosed in Table 29, is less than or equal to, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15%, or 20%, compared to the association of the fusion polypeptide with CRBN in the presence of COF3, e.g., compound I-112 disclosed in Table 29. In some embodiments, the ubiquitination of the fusion polypeptide in the absence of COF3, for example, compound I-112 disclosed in Table 29, is less than or equal to, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, or 70%, of the ubiquitination of the fusion polypeptide in the presence of COF3, for example, compound I-112 disclosed in Table 29. In some embodiments, the degradation of the fusion polypeptide in the absence of COF3, for example, compound I-112 disclosed in Table 29, is less than or equal to, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, or 70%, of the degradation of the fusion polypeptide in the presence of COF3, for example, compound I-112 disclosed in Table 29. In some embodiments, the expression level of the fusion polypeptide in the presence of COF3, for example compound I-112 disclosed in Table 29, is reduced by, for example, at least 40, 50, 60, 70, 80, 90, or 99% compared to the expression level of the fusion polypeptide in the absence of COF3, for example compound I-112 disclosed in Table 29.

[0050] In some embodiments, (i) the degraded polypeptide is fused to the CAR polypeptide; (ii) the degraded polypeptide and the CAR polypeptide are linked by a peptide bond; (iii) the degraded polypeptide and the CAR polypeptide are linked by a bond other than a peptide bond; (iv) the degraded polypeptide is directly linked to the CAR polypeptide; (v) the degraded polypeptide is indirectly linked to the CAR polypeptide; (vi) the degraded polypeptide and the CAR polypeptide are operably linked via a linker, e.g., a glycine-serine linker, e.g., a linker containing the amino acid sequence GGGGSGGGGTGGGGSG (SEQ ID NO: 335); (vii) the degraded polypeptide is linked to the C-terminus or N-terminus of the CAR polypeptide; or (viii) the degraded polypeptide is located in the center of the CAR polypeptide.

[0051] In some embodiments, the CCAR is a fusion polypeptide comprising a degradation domain (e.g., a degradation domain disclosed herein) and a CAR polypeptide (e.g., a CAR polypeptide disclosed herein), wherein optionally, the degradation domain is separated from the CAR polypeptide by a heteroprotease cleavage site, and optionally, the CCAR comprises the degradation domain, the heteroprotease cleavage site, and the CAR polypeptide from the N-terminus to the C-terminus.

[0052] In some embodiments, the degradation domain has a first state associated with the expression of a first level of the fusion polypeptide and a second state associated with the expression of a second level of the fusion polypeptide, the second level being increased by, for example, at least 2, 3, 4, 5, 10, 20, or 30 times compared to the first level in the presence of the stabilizing compound, and optionally, (a) in the absence of the stabilizing compound, the fusion polypeptide is degraded by a cytodegradation pathway, for example, by at least 50%, 60%, 70%, 80%, 90%, or more of the fusion polypeptide; (b) in the presence of the stabilizing compound, the degradation domain adopts a conformation that is more resistant to cytodegradation compared to the conformation in the absence of the stabilizing compound; and / or (c) in the presence of the stabilizing compound, the conformation of the fusion polypeptide is more tolerant of cleavage at heteroprotease sites compared to the conformation in the absence of the stabilizing compound.

[0053] In some embodiments, the degradation domain is selected from an estrogen receptor (ER) domain, an FKB protein (FKBP) domain, or a dihydrofolate reductase (DHFR) domain, and optionally, (a) the degradation domain is an estrogen receptor (ER) domain, for example, the degradation domain comprises the amino acid sequence of SEQ ID NO: 342 or 344 or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto, and optionally, the stabilizing compound is bazedoxifene or 4-hydroxytamoxifene (4-OHT) or a pharmaceutically acceptable salt thereof; (b) the degradation domain is an FKB protein (c) The degradation domain is a nitrate (FKBP) domain, for example, the degradation domain comprises the amino acid sequence of SEQ ID NO: 346 or a sequence having at least 85, 87, 90, 95, 97, 98, 99 or 100% identity thereto, and optionally the stabilizing compound is Shield-1 or a pharmaceutically acceptable salt thereof; or (c) the degradation domain is a dihydrofolate reductase (DHFR) domain, for example, the degradation domain comprises the amino acid sequence of SEQ ID NO: 347 or a sequence having at least 85, 87, 90, 95, 97, 98, 99 or 100% identity thereto, and optionally the stabilizing compound is trimethoprim or a pharmaceutically acceptable salt thereof.

[0054] In some embodiments, the heterologous protease cleavage site is cleaved by a mammalian intracellular protease, and optionally, (a) the heterologous protease cleavage site is cleaved by a protease selected from the group consisting of furin, PCSK1, PCSK5, PCSK6, PCSK7, cathepsin B, granzyme B, factor XA, enterokinase, genenase, saltase, precision protease, thrombin, TEV protease, and elastase 1; (b) the heterologous protease cleavage site is cleaved by a protease selected from the group consisting of furin, PCSK1, PCSK5, PCSK6, PCSK7, cathepsin B, granzyme B, factor XA, enterokinase, genenase, saltase, precision protease, thrombin, TEV protease, and elastase 1; or (b) the heterologous protease cleavage site is cleaved by a mammalian intracellular protease. The cross-sectional areas are the RX(K / R)R consensus motif (X can be any amino acid; SEQ ID NO: 348), RXXX[KR]R consensus motif (X can be any amino acid; SEQ ID NO: 349), RRX consensus motif (SEQ ID NO: 350), IEPDX consensus motif (SEQ ID NO: 351), Ile-Glu / Asp-Gly-Arg (SEQ ID NO: 352), Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 353), and Pro-Gly-Ala-Ala-His-Tyr. (SEQ ID NO: 354), LPXTG / A consensus motif (SEQ ID NO: 355), Leu-Glu-Val-Phe-Gln-Gly-Pro (SEQ ID NO: 356), Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO: 357), ENLYFQG (SEQ ID NO: 358), and [AGSV]-X (where X can be any amino acid; SEQ ID NO: 359) contain a sequence having a cleavage motif selected from the group consisting of (SEQ ID NO: 354), LPXTG / A consensus motif (SEQ ID NO: 355), Leu-Glu-Val-Phe-Gln-Gly-Pro (SEQ ID NO: 356), Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO: 357), ENLYFQG (SEQ ID NO: 358), and [AGSV]-X (where X can be any amino acid; SEQ ID NO: 359); or (c) the heterologous protease cleavage site is RTKR (SEQ ID NO: 378); GT It includes a furin cleavage site selected from the group consisting of GAEDPRPSRKRRSLGDVG (sequence number 379);GTGAEDPRPSRKRR (sequence number 381);LQWLEQQVAKRRTKR (sequence number 383);GTGAEDPRPSRKRRSLGG (sequence number 385);GTGAEDPRPSRKRRSLG (sequence number 387);SLNLTESHNSRKKR (sequence number 389);CKINGYPKRGRKRR (sequence number 391); and SARNRQKR (sequence number 336).In some embodiments, the heterologous protease cleavage site is cleaved by a mammalian extracellular protease, optionally (a) the heterologous protease cleavage site is cleaved by a protease selected from the group consisting of factor XA, enterokinase, genenase, saltase, precision protease, thrombin, TEV protease, and elastase 1; or (b) the heterologous protease cleavage site is cleaved by Ile-Glu / Asp-Gly-Arg (SEQ ID NO: 352), Asp-Asp-A The amino acid sequence includes an amino acid sequence selected from the group consisting of sp-Asp-Lys (SEQ ID NO: 353), Pro-Gly-Ala-Ala-His-Tyr (SEQ ID NO: 354), LPXTG / A consensus motif (SEQ ID NO: 355), Leu-Glu-Val-Phe-Gln-Gly-Pro (SEQ ID NO: 356), Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO: 357), ENLYFQG (SEQ ID NO: 358), and [AGSV]-X (where X can be any amino acid; SEQ ID NO: 359).

[0055] In some embodiments, the CCAR is a tunable CAR (RCAR) (e.g., an RCAR disclosed herein). In some embodiments, the RCAR includes (i) an intracellular signaling member comprising an intracellular signaling domain, e.g., a primary intracellular signaling domain and a first switch domain; (ii) an antigen-binding member comprising an antigen-binding domain and a second switch domain; and (iii) a transmembrane domain which may optionally be located on the intracellular signaling member and / or the antigen-binding member. In some embodiments, the RCAR includes (i) an intracellular signaling member comprising an intracellular signaling domain, e.g., a primary intracellular signaling domain and a first switch domain; (ii) an inhibitory extracellular domain member comprising an inhibitory extracellular domain (e.g., an inhibitory extracellular domain comprising an extracellular domain of B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM, LAG3, TIGIT, CTLA-4, BTLA, LAIR1, or a TGF-β receptor, or an inhibitory extracellular domain comprising a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereof) and a second switch domain; and (iii) a transmembrane domain which can optionally be located on the intracellular signaling member and / or the inhibitory extracellular domain member. In some embodiments, the RCAR includes (i) an intracellular signaling member comprising an intracellular signaling domain, e.g., a primary intracellular signaling domain and a first switch domain; (ii) a costimulatory extracellular domain member comprising a costimulatory extracellular domain (e.g., an extracellular domain of ICOS, CD28, VEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226, or a costimulatory extracellular domain comprising a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity therewith) and a second switch domain; and (iii) a transmembrane domain which can optionally be located on the intracellular signaling member and / or the costimulatory extracellular domain member.

[0056] In some embodiments, the first and second switch domains can form a dimerizing switch in the presence of a dimerizing molecule, optionally, (i) the dimerizing switch is an intracellular dimerizing switch or an extracellular dimerizing switch; (ii) the dimerizing switch is a homodimerizing switch or a heterodimerizing switch; (iii) the dimerizing switch comprises an FKBP-FRB based switch, for example, the dimerizing switch comprising a switch domain containing an FRB-binding fragment or an analogue of FKBP and an FKBP-binding fragment The invention comprises a switch domain containing a ment or an analog of FRB, and optionally, the FKBP-binding fragment or FRB analog comprises one or more mutations disclosed herein (e.g., the F2032 mutation, the T2098 mutation, or one or more mutations selected from the E2032 and T2098 mutations), and optionally, the dimerizing molecule is an mTOR inhibitor, e.g., a rapamycin analog, e.g., RAD001; and / or (iv) the antigen-binding domain binds to a target antigen but does not promote the T cell immune effector response until the dimerizing molecule is present.

[0057] In some embodiments, (i) the intracellular signaling member comprises a primary intracellular signaling domain, e.g., a primary intracellular signaling domain disclosed herein, e.g., a CD3ζ domain; (ii) the intracellular signaling member comprises a co-stimulatory signaling domain, e.g., a co-stimulatory signaling domain disclosed herein, e.g., a 4-1BB domain or a CD28 domain; (iii) the antigen-binding member does not comprise a primary intracellular signaling domain, e.g., an antigen-binding member comprises a co-stimulatory signaling domain and does not comprise a primary intracellular signaling domain; (iv) the inhibitory extracellular domain member does not comprise a primary intracellular signaling domain, e.g., an inhibitory extracellular domain member comprises a co-stimulatory signaling domain and does not comprise a primary intracellular signaling domain; and / or (v) the co-stimulatory extracellular domain member does not comprise a primary intracellular signaling domain, e.g., a co-stimulatory extracellular domain member comprises a co-stimulatory signaling domain and does not comprise a primary intracellular signaling domain.

[0058] In some embodiments, the population of cells includes a second nucleic acid molecule encoding a CAR and a regulatory molecule.

[0059] In some embodiments, the second nucleic acid molecule comprises a nucleic acid sequence encoding a CAR and a nucleic acid sequence encoding a regulatory molecule, and optionally, the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the regulatory molecule are (i) located on a single nucleic acid molecule, for example, separated by a nucleic acid sequence encoding a self-cleavage site; or (ii) located on separate nucleic acid molecules.

[0060] In some embodiments, the regulatory molecule comprises a chimeric protein including (i) a multimeric ligand-binding region and (ii) a caspase 9 molecule. In some embodiments, the caspase 9 molecule is a cleavage-type caspase 9, and optionally, the caspase 9 molecule lacks a caspase recruitment domain. In some embodiments, the multimeric ligand-binding region is selected from the group consisting of FKBP, cyclophylline receptors, steroid receptors, tetracycline receptors, heavy-chain antibody subunits, light-chain antibody subunits, single-chain antibodies composed of tandem heavy-chain and light-chain variable regions separated by a flexible linker domain, and their variant sequences, and optionally, the multimeric ligand-binding region is an FKBP12 region.

[0061] In some embodiments, the regulatory molecule comprises a cleaved epidermal growth factor receptor (EGFRt). In some embodiments, the EGFRt has one, two, three, four, or all of the following properties: (i) the EGFRt contains one or both of EGFR domain III and EGFR domain IV; (ii) the EGFRt does not contain one, two, three, or all of EGFR domain I, EGFR domain II, EGFR perimembrane domain and EGFR tyrosine kinase domain; (iii) the EGFRt does not mediate signaling or trafficking; (iv) the EGFRt does not bind to endogenous EGFR ligands, such as epidermal growth factor (EGF); and (v) the EGFRt binds to anti-EGFR antibody molecules (e.g., cetuximab, matsuzumab, necitumumab, and panitumumab), EGFR-specific siRNA, or EGFR-targeting small molecules.

[0062] In some embodiments, pharmaceutical compositions comprising a population of cells disclosed herein and a pharmaceutically acceptable carrier are provided herein.

[0063] In some embodiments, a method for increasing the immune response of a target is provided herein, comprising the step of administering a population of cells disclosed herein or a pharmaceutical composition disclosed herein to the target, thereby increasing the immune response of the target. In some embodiments, a method for treating a cancer of a target is provided herein, comprising the step of administering a population of cells disclosed herein or a pharmaceutical composition disclosed herein to the target, thereby treating the cancer of the target. In some embodiments, the cancer is a solid tumor or its metastasis selected from, for example, one or more of the following: mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, skin cancer, melanoma, kidney cancer, liver cancer, brain tumor, thymoma, sarcoma, carcinoma, uterine cancer, kidney cancer, gastrointestinal cancer, urothelial carcinoma, pharyngeal cancer, head and neck cancer, rectal cancer, esophageal cancer, or bladder cancer.In some embodiments, cancers include, for example, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoblastic leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prelymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, Diffuse large B-cell lymphoma (DLBCL), DLBCL with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasm, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (mucosa-associated lymphoid tissue type extranodal follicular marginal zone lymphoma), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, diffuse red medullary small cell B-cell lymphoma of the spleen, hairy cell leukemia - variant, lymphoplasmacytic lymphoma, H chain disease, plasma cell myeloma, solitary osteoplasmacytoma, extraskeletal plasmacytoma, nodular marginal zone lymphoma, pediatric nodular marginal zone lymphoma The lymphoma is a humoral cancer selected from lymphoma, primary cutaneous follicular lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma occurring in HHV8-associated multicentric Castleman disease, primary exudative lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or lymphoma that cannot be classified. In some embodiments, the method further includes a step of administering a second therapeutic agent to the target.

[0064] In some embodiments, the method further includes administering an effective amount of IMiD (e.g., thalidomide and its derivatives, e.g., lenalidomide, pomalidomide, and thalidomide) or compound I-112 to a subject after administration of a population of cells or a pharmaceutical composition. In some embodiments, the subject has developed, is developing, or is expected to develop an adverse reaction after administration of the population of cells or the pharmaceutical composition. In some embodiments, the administration of IMiD or compound I-112 is in response to the occurrence or expectation of an adverse reaction in the subject. In some embodiments, the administration of IMiD or compound I-112 reduces or prevents adverse effects. In some embodiments, the population of cells comprises a nucleic acid molecule encoding a CCAR, where the CCAR is a fusion polypeptide comprising a degraded polypeptide (e.g., a degraded polypeptide disclosed herein) and a CAR polypeptide (e.g., a CAR polypeptide disclosed herein).

[0065] In some embodiments, methods for treating a target cancer are provided herein, which include, i) a step of ex vivo contacting a population of cells with IMiD (e.g., thalidomide and its derivatives, e.g., lenalidomide, pomalidomide and thalidomide) or compound I-112, wherein the population of cells comprises a nucleic acid molecule encoding a CCAR, and the CCAR is a fusion polypeptide comprising a degradation polypeptide (e.g., a degradation polypeptide disclosed herein) and a CAR polypeptide (e.g., a CAR polypeptide disclosed herein), and ii) The step of administering an effective amount of a population of cells to the subject. This includes and thereby treats the above-mentioned cancer.

[0066] In some embodiments, in the presence of IMiD or compound I-112, the expression level of CCAR is reduced by, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to the expression level of CCAR before the cell population is ex vivo contacted with IMiD or compound I-112. In some embodiments, the method further includes the step of reducing the amount of IMiD or compound I-112 that comes into contact with the cell population, for example, inside and / or around the cell population, after step i) and before step ii).

[0067] In some embodiments, this method proceeds after step ii), iii) further comprising the step of administering an effective amount of IMiD or compound I-112 to the subject. In some embodiments, administration of IMiD or compound I-112 reduces the expression level of CCAR by, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent compared to the expression level of CCAR after step ii) and before step iii). In some embodiments, the subject has developed, is developing, or is expected to develop an adverse reaction. In some embodiments, administration of IMiD or compound I-112 is in response to the occurrence of an adverse reaction in the subject or the expectation of the occurrence of an adverse reaction in the subject. In some embodiments, administration of IMiD or compound I-112 reduces or prevents an adverse effect.

[0068] In some embodiments, after step iii), iv) Step of discontinuing administration of IMiD or compound I-112 The following further includes: In some embodiments, discontinuation of IMiD or compound I-112 administration increases the CCAR expression level by, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times compared to the CCAR expression level after step iii) and before step iv). In some embodiments, discontinuation of IMiD or compound I-112 administration restores the CCAR expression level to the expression level after step ii) and before step iii). In some embodiments, the subject has relapsed, is relapsed, or is expected to relapse. In some embodiments, discontinuation of IMiD or compound I-112 administration is in response to tumor relapse in the subject or the expectation of relapse in the subject. In some embodiments, discontinuation of IMiD or compound I-112 administration treats or prevents tumor relapse.

[0069] In some embodiments, after step iv), v) A step of repeating steps iii) and / or iv). It further includes and thereby treats cancer.

[0070] In some embodiments, a method for treating a target cancer is provided herein, comprising the steps of i) administering an effective amount of a population of cells to the target, wherein the population of cells comprises a nucleic acid molecule encoding a CCAR, and the CCAR is a fusion polypeptide comprising a degraded polypeptide (e.g., a degraded polypeptide disclosed herein) and a CAR polypeptide (e.g., a CAR polypeptide disclosed herein), thereby treating the cancer. In some embodiments, the population of cells is ex vivo contacted with an IMiD (e.g., thalidomide and its derivatives, e.g., lenalidomide, pomalidomide and thalidomide) or compound I-112 prior to administration. In some embodiments, in the presence of IMiD or compound I-112, the expression level of CCAR is reduced by, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent compared to the expression level of CCAR before the cell population is ex vivo contacted with IMiD or compound I-112. In some embodiments, after the cell population is ex vivo contacted with IMiD or compound I-112 and before the cell population is administered to the subject, for example, the amount of IMiD or compound I-112 in contact with the cell population inside and / or around the cell population is reduced.

[0071] In some embodiments, the cell population is not ex vivo contacted with IMiD or compound I-112 prior to administration.

[0072] In some embodiments, after step i), ii) A step of administering an effective dose of IMiD or compound I-112 to the target. The following further includes: In some embodiments, administration of IMiD or compound I-112 reduces the expression level of CCAR by, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to the expression level of CCAR after step i) and before step ii). In some embodiments, the subject has developed, is developing, or is expected to develop an adverse reaction. In some embodiments, administration of IMiD or compound I-112 is in response to the occurrence of an adverse reaction in the subject or the expectation of the occurrence of an adverse reaction in the subject. In some embodiments, administration of IMiD or compound I-112 reduces or prevents an adverse effect.

[0073] In some embodiments, this method proceeds after step ii), iii) Step of discontinuing administration of IMiD or compound I-112 The following further include: In some embodiments, discontinuation of IMiD or compound I-112 administration increases the CCAR expression level by, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times compared to the CCAR expression level after step ii) and before step iii). In some embodiments, discontinuation of IMiD or compound I-112 administration restores the CCAR expression level to the expression level after step i) and before step ii). In some embodiments, the subject has relapsed, is relapsed, or is expected to relapse. In some embodiments, discontinuation of IMiD or compound I-112 administration is in response to tumor relapse in the subject or the expectation of relapse in the subject. In some embodiments, discontinuation of IMiD or compound I-112 administration treats or prevents tumor relapse.

[0074] In some embodiments, after step iii), iv) A step that repeats step ii) and / or iii). It further includes, thereby treating the above-mentioned cancer.

[0075] In some embodiments, a method for treating a target cancer is provided herein, comprising the steps of i) administering an effective amount of IMiD (e.g., thalidomide and its derivatives, e.g., lenalidomide, pomalidomide and thalidomide) or compound I-112 to the target, wherein the target comprises a population of cells, the population of cells comprising a nucleic acid molecule encoding a CCAR, and the CCAR is a fusion polypeptide comprising a degraded polypeptide (e.g., a degraded polypeptide disclosed herein) and a CAR polypeptide (e.g., a CAR polypeptide disclosed herein), thereby treating the cancer. In some embodiments, administration of IMiD or compound I-112 reduces the expression level of CCAR by, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 percent compared to the expression level of CCAR before administration of IMiD or compound I-112. In some embodiments, the subject has developed, is developing, or is expected to develop an adverse reaction. In some embodiments, administration of IMiD or compound I-112 is in response to the occurrence or expected occurrence of an adverse reaction in the subject. In some embodiments, administration of IMiD or compound I-112 reduces or prevents adverse effects.

[0076] In some embodiments, after step i), ii) Step of discontinuing administration of IMiD or compound I-112 The following further includes: In some embodiments, discontinuation of IMiD or compound I-112 administration increases the CCAR expression level by, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times compared to the CCAR expression level after step i) and before step ii). In some embodiments, discontinuation of IMiD or compound I-112 administration restores the CCAR expression level to the level before administration of IMiD or compound I-112. In some embodiments, the subject has relapsed, is relapsed, or is expected to relapse. In some embodiments, discontinuation of IMiD or compound I-112 administration is in response to tumor relapse in the subject or the expectation of relapse in the subject. In some embodiments, discontinuation of IMiD or compound I-112 administration treats or prevents tumor relapse.

[0077] In some embodiments, this method proceeds after step ii), iii) A step that repeats step i) and / or ii). It further includes, thereby treating the above-mentioned cancer.

[0078] In some embodiments, a method for treating a target cancer is provided herein, comprising the steps of i) administering to the target (1) a stabilizing compound and (2) an effective amount of a population of cells, thereby treating the cancer, wherein the population of cells comprises a nucleic acid molecule encoding a CCAR, the CCAR being a fusion polypeptide comprising a degradation domain (e.g., a degradation domain disclosed herein) and a CAR polypeptide (e.g., a CAR polypeptide disclosed herein), wherein optionally, the degradation domain is separated from the CAR polypeptide by a heterologous protease cleavage site. In some embodiments, the expression level of the CCAR in the presence of the stabilizing compound is, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times higher than the expression level of the CCAR in the absence of the stabilizing compound.

[0079] In some embodiments, after step i), ii) Step of discontinuing administration of the stabilizing compound. The following further includes: In some embodiments, discontinuation of administration of the stabilizing compound reduces the expression level of CCAR by, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times compared to the expression level of CCAR after step i) and before step ii). In some embodiments, the subject has responded to the treatment in step i) (e.g., the subject has a complete response to the treatment in step i), the subject shows a reduction in tumor mass, the subject shows a decrease in tumor cells, or the treatment in step i) is effective in the subject). In some embodiments, discontinuation of administration of the stabilizing compound is in response to the subject's response to the treatment in step i) (e.g., the subject has a complete response to the treatment in step i), the subject shows a reduction in tumor mass, the subject shows a decrease in tumor cells, or the treatment in step i) is effective in the subject).

[0080] In some embodiments, after step i), iii) Step of discontinuing administration of the stabilizing compound. The following further include: In some embodiments, discontinuation of administration of the stabilizing compound reduces the expression level of the CCAR by, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times compared to the expression of the CCAR after step i) and before step ii). In some embodiments, the subject has developed, is developing, or is expected to develop an adverse reaction. In some embodiments, discontinuation of administration of the stabilizing compound is in response to the occurrence of an adverse reaction in the subject or the expectation of the occurrence of an adverse reaction in the subject. In some embodiments, discontinuation of administration of the stabilizing compound reduces or prevents an adverse effect.

[0081] In some embodiments, after step ii) or iii), iv) Step of administering an effective amount of the stabilizing compound. The following further include: In some embodiments, administration of the stabilizing compound increases the expression level of the CCAR by, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times compared to the expression level of the CCAR after step ii) or iii) and before step iv). In some embodiments, the subject has relapsed, is relapsed, or is expected to relapse. In some embodiments, administration of the stabilizing compound is in response to tumor relapse in the subject or the expectation of relapse in the subject. In some embodiments, administration of the stabilizing compound treats or prevents tumor relapse.

[0082] In some embodiments, after step iv), v) A step that repeats step ii), iii), or iv). It further includes and thereby treats cancer.

[0083] In some embodiments, this method, before step i), vi) Step of ex vivo contacting the above cell population with a stabilizing compound. The following further includes: In some embodiments, the expression level of CCAR in the presence of the stabilizing compound is, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times higher than the expression level of CCAR in the absence of the stabilizing compound.

[0084] In some embodiments, the cell population is not ex vivo contacted with the stabilizing compound before administration.

[0085] In some embodiments, a population of cells or a pharmaceutical composition disclosed herein is provided for use in a method for increasing the immune response of a target, the method comprising the step of administering an effective amount of the population of cells or an effective amount of the pharmaceutical composition to the target. In some embodiments, a population of cells or a pharmaceutical composition disclosed herein is provided for use in a method for treating a cancer of a target, the method comprising the step of administering an effective amount of the population of cells or a pharmaceutical composition to the target.

[0086] In some embodiments, the disclosure features a method for producing a population of cells (e.g., T cells) expressing a chimeric antigen receptor (CAR), for example, a CAR disclosed herein, for example, a CCAR disclosed herein. In some embodiments, the cell population further expresses a regulatory molecule. In some embodiments, the cell population expresses a CCAR disclosed herein. In some embodiments, the cell population expresses a CAR disclosed herein and a regulatory molecule disclosed herein.In some embodiments, the method comprises the steps of (i) contacting (e.g., binding) a population of cells (e.g., T cells, e.g., T cells isolated from frozen or fresh leukocyte apheresis products) with a drug that stimulates the CD3 / TCR complex and / or a drug that stimulates costimulatory molecules on the cell surface; (ii) contacting a population of cells (e.g., T cells) with a nucleic acid molecule encoding CAR (e.g., DNA or RNA molecule) thereby providing a population of cells (e.g., T cells) containing the nucleic acid molecule; and (iii) collecting a population of cells (e.g., T cells) for storage (e.g., re-formulating a population of cells in cryopreservation medium) or administration, wherein (a) step (ii) is carried out together with step (i) or within 20 hours after the start of step (i), for example, within 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), for example, within 18 hours after the start of step (i), and step (iii) is carried out together with step (i) (b) Step (ii) is carried out together with Step (i), or within 20 hours after the start of Step (i), for example, within 22, 23, 24 or 25 hours after the start of Step (i), for example, within 24 hours after the start of Step (i); (b) Step (ii) is carried out together with Step (i), or within 20 hours after the start of Step (i), for example, within 12, 13, 14, 15, 16, 17 or 18 hours after the start of Step (i), for example, within 18 hours after the start of Step (i); and Step (iii) is carried out after the start of Step (ii) (c) The procedure is carried out within 30 hours, for example, within 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours after the start of step (ii); or (c) the population of cells from step (iii) is not proliferated, as assessed by the number of viable cells, compared to the population of cells at the start of step (i), for example, or is proliferated by 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40%, for example, 10% or less. In some embodiments, the nucleic acid molecule in step (ii) is a DNA molecule. In some embodiments, the nucleic acid molecule in step (ii) is an RNA molecule.In some embodiments, the nucleic acid molecule in step (ii) is on a viral vector, such as a lentiviral vector, adenovirus vector, or retroviral vector. In some embodiments, the nucleic acid molecule in step (ii) is on a non-viral vector. In some embodiments, the nucleic acid molecule in step (ii) is on a plasmid. In some embodiments, the nucleic acid molecule in step (ii) is not on any vector. In some embodiments, step (ii) includes transducing a population of cells (e.g., T cells) with a viral vector containing a nucleic acid molecule encoding a CAR. In some embodiments, step (ii) is carried out in conjunction with step (i). In some embodiments, step (ii) is carried out within 20 hours after the start of step (i). In some embodiments, step (ii) is carried out within 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i). In some embodiments, step (ii) is carried out within 18 hours after the start of step (i). In some embodiments, step (iii) is carried out within 26 hours after the start of step (i). In some embodiments, step (iii) is performed within 22, 23, 24, or 25 hours after the start of step (i). In some embodiments, step (iii) is performed within 24 hours after the start of step (i). In some embodiments, step (iii) is performed within 30 hours after the start of step (ii). In some embodiments, step (iii) is performed within 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (ii).

[0087] In some embodiments, the cell population from step (iii) is not proliferated. In some embodiments, the cell population from step (iii) is proliferated by only 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% or less compared to the cell population at the start of step (i), as evaluated by the number of surviving cells. In some embodiments, the cell population from step (iii) is proliferated by only 10% or less compared to the cell population at the start of step (i), as evaluated by the number of surviving cells.

[0088] In some embodiments, the agent stimulating the CD3 / TCR complex is an agent that stimulates CD3. In some embodiments, the agent stimulating the co-stimulator is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, the agent stimulating the co-stimulator is an agent that stimulates CD28. In some embodiments, the agent stimulating the CD3 / TCR complex is selected from antibodies (e.g., single-domain antibodies (e.g., heavy-chain variable-domain antibodies), peptide bodies, Fab fragments, or scFv), small molecules, or ligands (e.g., naturally occurring ligands, recombinant ligands, or chimeric ligands). In some embodiments, the agent stimulating the co-stimulatory molecule is selected from antibodies (e.g., single-domain antibodies (e.g., heavy-chain variable-domain antibodies), peptide bodies, Fab fragments, or scFv), small molecules, or ligands (e.g., naturally occurring ligands, recombinant ligands, or chimeric ligands). In some embodiments, the agent stimulating the CD3 / TCR complex does not contain beads. In some embodiments, the agent stimulating the co-stimulatory molecule does not contain beads. In some embodiments, the agent stimulating the CD3 / TCR complex includes an anti-CD3 antibody. In some embodiments, the agent stimulating the co-stimulatory molecule includes an anti-CD28 antibody. In some embodiments, the agent stimulating the CD3 / TCR complex includes an anti-CD3 antibody covalently bound to a colloidal polymer nanomatrix. In some embodiments, the agent stimulating the co-stimulatory molecule includes an anti-CD28 antibody covalently bound to a colloidal polymer nanomatrix. In some embodiments, the agent stimulating the CD3 / TCR complex and the agent stimulating the co-stimulatory molecule include T Cell TransAct®.

[0089] In some embodiments, the agent stimulating the CD3 / TCR complex does not contain a hydrogel. In some embodiments, the agent stimulating the costimulatory molecule does not contain a hydrogel. In some embodiments, the agent stimulating the CD3 / TCR complex does not contain alginate. In some embodiments, the agent stimulating the costimulatory molecule does not contain alginate.

[0090] In some embodiments, the agent stimulating the CD3 / TCR complex includes a hydrogel. In some embodiments, the agent stimulating the co-stimulatory molecule includes a hydrogel. In some embodiments, the agent stimulating the CD3 / TCR complex includes an alginate. In some embodiments, the agent stimulating the co-stimulatory molecule includes an alginate. In some embodiments, the agent stimulating the CD3 / TCR complex or the agent stimulating the co-stimulatory molecule includes MagCloudz® manufactured by Quad Technologies.

[0091] In some embodiments, step (i) increases the percentage of CAR-expressing cells in the cell population from step (iii), for example, the cell population from step (iii) exhibits a higher percentage of CAR-expressing cells (e.g., at least 10, 20, 30, 40, 50, or 60%) compared to cells produced by a similar method except without step (i).

[0092] In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (iii) is the same as the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (i). In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (iii) is different from the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (i) by only 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12% or less. In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (iii) differs from the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (i) by only 5 or 10% or less.

[0093] In some embodiments, the population of cells from step (iii) exhibits a higher percentage (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%) of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, compared to cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the cell population from step (iii) exhibits a higher percentage (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%) of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, compared to cells produced by a similar method, except that the further step includes growing a cell population (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, e.g., 5, 6, 7, 8, or 9 days, e.g., 5, 6, 7, 8, or 9 days) of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%) compared to cells produced by a similar method, except that the further step includes growing a cell population (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, e.g., 5, 6, 7, 8, or 9 days.

[0094] In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population from step (iii) is the same as the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population at the start of step (i). In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population from step (iii) is different from the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population at the start of step (i) by only 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12% or less. In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population from step (iii) is different from the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population at the start of step (i) by only 5 or 10% or less.

[0095] In some embodiments, the population of cells from step (iii) exhibits a lower percentage (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%) of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, compared to cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the cell population from step (iii) exhibits a lower percentage (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%) of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, compared to cells produced by a similar method, except that the step further includes growing a cell population (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9) of central memory cells, such as central memory T cells, such as CD95+ central memory T cells.

[0096] In some embodiments, the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the cell population from step (iii) is increased compared to the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the cell population at the start of step (i). In some embodiments, the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the cell population from step (iii) is increased compared to the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the cell population at the start of step (i). In some embodiments, the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the cell population from step (iii) is higher than the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in cell populations produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i). In some embodiments, the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the cell population from step (iii) is higher than the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i).In some embodiments, the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the cell population from step (iii) is higher than the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in cell populations produced by a similar method, except that the method further includes a step of growing the cell population (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in a population of cells from step (iii) is higher than the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in a population of cells produced by a similar method, except that the method further includes a step of growing a population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

[0097] In some embodiments, the median GeneSetScore (Up TEM vs. Down TSCM) of the cell population from step (iii) is approximately the same as the median GeneSetScore (Up TEM vs. Down TSCM) of the cell population at the start of step (i), or differs by only about 25, 50, 75, 100, or 125% or less (e.g., increased by only about that amount). In some embodiments, the median GeneSetScore (Up TEM vs. Down TSCM) of the cell population from step (iii) is lower than the median GeneSetScore (Up TEM vs. Down TSCM) of a cell population produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i) (e.g., at least about 100, 150, 200, 250, or 300% lower). In some embodiments, the median GeneSetScore (Up TEM vs. Down TSCM) of the cell population from step (iii) is lower (e.g., at least about 100, 150, 200, 250, or 300%) than the median GeneSetScore (Up TEM vs. Down TSCM) of a cell population produced by a similar method, except that the method further includes a step of growing the cell population (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the median GeneSetScore (Up Treg vs. Down Teff) of the cell population from step (iii) is approximately the same as the median GeneSetScore (Up Treg vs. Down Teff) of the cell population at the start of step (i), or differs by only about 25, 50, 100, 150, or 200% or less (e.g., increased by only less than or equal to that).In some embodiments, the median GeneSetScore (Up Treg vs. Down Teff) of the cell population from step (iii) is lower (e.g., at least about 50, 100, 125, 150, or 175%) than the median GeneSetScore (Up Treg vs. Down Teff) of a cell population produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the median GeneSetScore (Up Treg vs. Down Teff) of the cell population from step (iii) is lower (e.g., at least about 50, 100, 150, or 175%) than the median GeneSetScore (Up Treg vs. Down Teff) of a cell population produced by a similar method, except that the method further includes a step of growing the cell population (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the median GeneSetScore (Down stemness) of the cell population from step (iii) is approximately the same as the median GeneSetScore (Down stemness) of the cell population at the start of step (i), or differs by only about 25, 50, 100, 150, 200, or 250% or less (e.g., increased by only less than or equal to that). In some embodiments, the median GeneSetScore (Downstemness) of the cell population from step (iii) is lower (e.g., at least about 50, 100, or 125%) than the median GeneSetScore (Downstemness) of a cell population produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i).In some embodiments, the median GeneSetScore (Down stemness) of the cell population from step (iii) is lower (e.g., at least about 50, 100, or 125%) than the median GeneSetScore (Down stemness) of a cell population produced by a similar method, except that the method further includes a step of growing the cell population (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the median GeneSetScore (Up hypoxia) of the cell population from step (iii) is approximately the same as the median GeneSetScore (Up hypoxia) of the cell population at the start of step (i), or differs by only about 125, 150, 175, or 200% or less (e.g., increased by only less than or equal to that). In some embodiments, the median GeneSetScore (Up hypoxia) of the cell population from step (iii) is lower (e.g., at least about 40, 50, 60, 70, or 80%) than the median GeneSetScore (Up hypoxia) of a cell population produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the median GeneSetScore (Up hypoxia) of the cell population from step (iii) is lower (e.g., at least about 40, 50, 60, 70, or 80%) than the median GeneSetScore (Up hypoxia) of a cell population produced by a similar method, except that step (ii) and before step (iii) further includes a step of growing a cell population (e.g., T cells) in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days. In some embodiments, the median GeneSetScore (Up autophagy) of the cell population from step (iii) is approximately the same as the median GeneSetScore (Up autophagy) of the cell population at the start of step (i), or differs by only about 180, 190, 200, or 210% or less (for example, increased by only that amount or less).In some embodiments, the median GeneSetScore (Up autophagy) of the cell population from step (iii) is lower (e.g., at least about 20, 30, or 40%) than the median GeneSetScore (Up autophagy) of a cell population produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the median GeneSetScore (Up autophagy) of the cell population from step (iii) is lower (e.g., at least about 20, 30, or 40%) than the median GeneSetScore (Up autophagy) of a cell population produced by a similar method, except that the step further includes growing a cell population (e.g., T cells) in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

[0098] In some embodiments, the population of cells from step (iii) is incubated with cells expressing an antigen recognized by CAR, and then secretes IL-2 at a higher level (e.g., at least 2, 4, 6, 8, 10, 12, or 14 times higher) than cells produced by the same method except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), or cells produced by the same method except that step (iii) further includes a step of growing a population of cells (e.g., T cells) in vitro for more than 3 days, for example 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

[0099] In some embodiments, the cell population from step (iii), after in vivo administration, proliferates at a longer duration or higher (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher) than cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i) (e.g., as evaluated using the method described in Example 1 with reference to Figure 4C). In some embodiments, the cell population from step (iii) persists longer or grows at a higher level (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90%) than cells produced by a similar method, except that the step further includes a step of growing the cell population (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after in vivo administration (e.g., as evaluated using the method described in Example 1 with reference to Figure 4C).

[0100] In some embodiments, the population of cells from step (iii) is administered in vivo, and step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), except that cells produced by the same method are administered in vivo, or the population of cells (e.g., T cells) after step (ii) and before step (iii) is grown in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days, except that the step further includes growing the population of cells (e.g., T cells) in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days, except that cells produced by the same method have more potent antitumor activity (e.g., low dose, e.g., 0.15 × 10⁻¹⁵) 6 , 0.2 × 10 6 , 0.25 × 10 6 or 0.3 × 10 6 It exhibits stronger antitumor activity in individual surviving CAR-expressing cells.

[0101] In some embodiments, the population of cells from step (iii) does not proliferate compared to the population of cells at the start of step (i), as assessed by, for example, the number of viable cells. In some embodiments, the population of cells from step (iii) is reduced from the number of viable cells in the population of cells at the start of step (i), as assessed by, for example, the number of viable cells. In some embodiments, the population of cells from step (iii) proliferates by 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% or less compared to the population of cells at the start of step (i), as assessed by, for example, the number of viable cells. In some embodiments, the population of cells from step (iii) does not proliferate compared to the population of cells at the start of step (i), or proliferates for less than 0.5, 1, 1.5, or 2 hours, for example, less than 1 or 1.5 hours.

[0102] In some embodiments, steps (i) and (ii) are carried out in a cell medium containing IL-2, IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)), IL-6 (e.g., IL-6 / sIL-6Ra), an LSD1 inhibitor, or a MALT1 inhibitor. In some embodiments, steps (i) and (ii) are carried out in a cell medium (e.g., serum-free medium) containing IL-7, IL-21, or a combination thereof. In some embodiments, steps (i) and (ii) are carried out in a cell medium containing IL-2, IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)), IL-21, IL-7, IL-6 (e.g., IL-6 / sIL-6Ra), an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof. In some embodiments, step (i) is carried out in a cell medium containing IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-6 (e.g., IL-6 / sIL-6Ra), an LSD1 inhibitor, or a MALT1 inhibitor. In some embodiments, step (ii) is carried out in a cell medium containing IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-6 (e.g., IL-6 / sIL-6Ra), an LSD1 inhibitor, or a MALT1 inhibitor. In some embodiments, step (i) is carried out in a cell medium (e.g., serum-free medium) containing IL-7, IL-21, or a combination thereof. In some embodiments, step (ii) is carried out in a cell medium (e.g., serum-free medium) containing IL-7, IL-21, or a combination thereof. In some embodiments, step (i) is carried out in a cell medium containing IL-2, IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)), IL-21, IL-7, IL-6 (e.g., IL-6 / sIL-6Ra), an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof. In some embodiments, step (ii) is carried out in a cell medium containing IL-2, IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)), IL-21, IL-7, IL-6 (e.g., IL-6 / sIL-6Ra), an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof. In some embodiments, the cell medium is a serum-free medium containing a serum substitute.In some embodiments, the serum substitute is CTS® Immune Cell Serum Replacement (ICSR).

[0103] In some embodiments, the method described above further includes the step of (iv) receiving a fresh leukocyte apheresis product (or an alternative source of hematopoietic tissue such as a fresh whole blood product, a fresh bone marrow product, or a fresh tumor or organ biopsy or excision (e.g., a fresh product from a thymectomy)) from an entity prior to step (i).

[0104] In some embodiments, the method described above further includes, prior to step (i), the step of isolating a population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) that were contacted in step (i) from (v) a fresh leukocyte apheresis product (or an alternative source such as a fresh whole blood product, a fresh bone marrow product, or a fresh tumor or organ biopsy or excision (e.g., a fresh product from a thymectomy)). In some embodiments, step (iii) is carried out within 35 hours after the start of step (v), for example, within 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours after the start of step (v), for example, within 30 hours after the start of step (v). In some embodiments, the population of cells from step (iii) is not proliferated, as assessed by the number of viable cells, compared to the population of cells at the end of step (v), or is proliferated by 5, 10, 15, 20, 25, 30, 35 or 40%, e.g., 10% or less.

[0105] In some embodiments, the method described above further includes, prior to step (i), receiving cryopreserved T cells isolated from leukocyte apheresis products (or alternative sources of hematopoietic tissue such as cryopreserved T cells isolated from whole blood, bone marrow, or tumor or organ biopsy or excision (e.g., thymectomy)) from an entity, such as a laboratory, hospital, or healthcare provider.

[0106] In some embodiments, the method described above further includes the step of (iv) receiving a cryopreserved leukocyte apheresis product (or an alternative source of hematopoietic tissue such as a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved tumor or organ biopsy or excision (e.g., a cryopreserved product from a thymectomy)) prior to step (i).

[0107] In some embodiments, the method described above further includes, before step (i), the step of isolating a population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) that were contacted in step (i) from (v) a cryopreserved leukocyte apheresis product (or an alternative source of hematopoietic tissue such as a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved tumor or organ biopsy or excision (e.g., a cryopreserved product from thymectomy)). In some embodiments, step (iii) is carried out within 35 hours after the start of step (v), for example, within 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours after the start of step (v), for example, within 30 hours after the start of step (v). In some embodiments, the population of cells from step (iii) is not proliferated, as assessed by the number of viable cells, compared to the population of cells at the end of step (v), or is proliferated by 5, 10, 15, 20, 25, 30, 35 or 40%, e.g., 10% or less.

[0108] In some embodiments, the disclosure features a method for producing a population of cells (e.g., T cells) expressing a chimeric antigen receptor (CAR), for example, a CAR disclosed herein, for example, a CCAR disclosed herein. In some embodiments, the cell population further expresses a regulatory molecule. In some embodiments, the cell population expresses a CCAR disclosed herein. In some embodiments, the cell population expresses a CAR disclosed herein and a regulatory molecule disclosed herein. In some embodiments, the method includes (1) contacting a population of cells (e.g., T cells, e.g., T cells isolated from frozen leukocyte apheresis products) with a cytokine selected from IL-2, IL-7, IL-15, IL-21, IL-6 or a combination thereof; (2) contacting a population of cells (e.g., T cells) with a nucleic acid molecule encoding CAR (e.g., a DNA or RNA molecule), thereby providing a population of cells (e.g., T cells) containing the nucleic acid molecule; and (3) collecting a population of cells (e.g., T cells) for preservation (e.g., re-formulating a population of cells in a cryopreservation medium) or administration, (a) Step (2) is performed together with Step (1) or within 5 hours after the start of Step (1), for example, within 1, 2, 3, 4, or 5 hours after the start of Step (1); and Step (3) is performed within 26 hours after the start of Step (1), for example, within 22, 23, 24, or 25 hours after the start of Step (1), for example, within 24 hours after the start of Step (1); or (b) the population of cells from Step (3) is not proliferated, as assessed by the number of viable cells, compared to the population of cells at the start of Step (1), for example, or is proliferated by 5, 10, 15, 20, 25, 30, 35, or 40%, for example, 10% or less. In some embodiments, the nucleic acid molecule in Step (2) is a DNA molecule. In some embodiments, the nucleic acid molecule in Step (2) is an RNA molecule. In some embodiments, the nucleic acid molecule in Step (2) is on a viral vector, for example, a viral vector selected from a lentiviral vector, an adenovirus vector, or a retroviral vector. In some embodiments, the nucleic acid molecule in step (2) is located on a non-viral vector.In some embodiments, the nucleic acid molecule in step (2) is on a plasmid. In some embodiments, the nucleic acid molecule in step (2) is not on any vector. In some embodiments, step (2) includes transducing a population of cells (e.g., T cells) with a viral vector containing a nucleic acid molecule encoding a CAR.

[0109] In some embodiments, step (2) is performed together with step (1). In some embodiments, step (2) is performed within 5 hours after the start of step (1). In some embodiments, step (2) is performed within 1, 2, 3, 4, or 5 hours after the start of step (1). In some embodiments, step (3) is performed within 26 hours after the start of step (1). In some embodiments, step (3) is performed within 22, 23, 24, or 25 hours after the start of step (1). In some embodiments, step (3) is performed within 24 hours after the start of step (1).

[0110] In some embodiments, the population of cells from step (3) does not proliferate, as assessed by the number of surviving cells, compared to, for example, the population of cells at the start of step (1). In some embodiments, the population of cells from step (3) proliferates by only 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% or less, as assessed by the number of surviving cells, compared to, for example, the population of cells at the start of step (1). In some embodiments, the population of cells from step (3) proliferates by only 10% or less, as assessed by the number of surviving cells, compared to, for example, the population of cells at the start of step (1).

[0111] In some embodiments, step (1) includes contacting a population of cells (e.g., T cells) with IL-2. In some embodiments, step (1) includes contacting a population of cells (e.g., T cells) with IL-7. In some embodiments, step (1) includes contacting a population of cells (e.g., T cells) with IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, step (1) includes contacting a population of cells (e.g., T cells) with IL-21. In some embodiments, step (1) includes contacting a population of cells (e.g., T cells) with IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, step (1) includes contacting a population of cells (e.g., T cells) with IL-2 and IL-7. In some embodiments, step (1) includes contacting a population of cells (e.g., T cells) with IL-2 and IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)). In some embodiments, step (1) includes contacting a population of cells (e.g., T cells) with IL-2 and IL-21. In some embodiments, step (1) includes contacting a population of cells (e.g., T cells) with IL-2 and IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, step (1) includes contacting a population of cells (e.g., T cells) with IL-7 and IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)). In some embodiments, step (1) includes contacting a population of cells (e.g., T cells) with IL-7 and IL-21. In some embodiments, step (1) includes contacting a population of cells (e.g., T cells) with IL-7 and IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, step (1) includes contacting a population of cells (e.g., T cells) with IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)) and IL-21. In some embodiments, step (1) includes contacting a population of cells (e.g., T cells) with IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)) and IL-6 (e.g., IL-6 / sIL-6Ra).In some embodiments, step (1) includes contacting a population of cells (e.g., T cells) with IL-21 and IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, step (1) includes contacting a population of cells (e.g., T cells) with IL-7, IL-15 (e.g., hetIL-15 (IL-15 / sIL-15Ra)) and IL-21.

[0112] In some embodiments, the cell population from step (3) exhibits a higher percentage of naive cells among CAR-expressing cells (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%) compared to cells produced by a similar method, except that the step further includes contacting the cell population with, for example, an anti-CD3 antibody.

[0113] In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells in the cell population from step (3) is the same as the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells in the cell population at the start of step (1). In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells in the cell population from step (3) is different from the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells in the cell population at the start of step (1) by only 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12% or less. In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (3) differs from the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (1) by only 5 or 10% or less. In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (3) is increased compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (1). In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells in the cell population from step (3) is increased by at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells in the cell population at the start of step (1).In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (3) is increased by at least 10 or 20% compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (1).

[0114] In some embodiments, the population of cells from step (3) exhibits a higher percentage (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%) of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, compared to cells produced by a similar method, except that step (3) is performed more than 26 hours after the start of step (1), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (1). In some embodiments, the cell population from step (3) exhibits a higher percentage (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%) of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, compared to cells produced by a similar method, except that the further step includes growing a population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9) of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells.

[0115] In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population from step (3) is the same as the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population at the start of step (i). In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population from step (3) is different from the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population at the start of step (i) by only 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12% or less. In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population from step (3) is different from the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population at the start of step (i) by only 5 or 10% or less. In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population from step (3) is reduced compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population at the start of step (1). In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population from step (3) is reduced by at least 10 or 20% compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population at the start of step (1).In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population from step (3) is reduced by at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population at the start of step (1).

[0116] In some embodiments, the population of cells from step (3) exhibits a lower percentage (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%) of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, compared to cells produced by a similar method, except that step (3) is performed more than 26 hours after the start of step (1), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (1). In some embodiments, the cell population from step (3) exhibits a lower percentage (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%) of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, compared to cells produced by a similar method, except that the step further includes growing a population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (2) and before step (3).

[0117] In some embodiments, the population of cells from step (3), after being administered in vivo, proliferates at a longer duration or higher level (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher) compared to cells produced by a similar method, except that step (3) is performed more than 26 hours after the start of step (1), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (1) (e.g., as evaluated using the method described in Example 1 with respect to Figure 4C). In some embodiments, the cell population from step (3) persists longer or grows at a higher level (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90%) than cells produced by a similar method, except that the step further includes growing the cell population (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after administration in vivo (e.g., as evaluated using the method described in Example 1 with respect to Figure 4C).

[0118] In some embodiments, the population of cells from step (3) does not proliferate compared to the population of cells at the start of step (1), as evaluated by, for example, the number of viable cells. In some embodiments, the population of cells from step (3) proliferates by only 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% or less compared to the population of cells at the start of step (1), as evaluated by, for example, the number of viable cells. In some embodiments, the population of cells from step (3) proliferates by only 10% or less compared to the population of cells at the start of step (1), as evaluated by, for example, the number of viable cells. In some embodiments, the number of viable cells in the population of cells from step (3) decreases from the number of viable cells in the cells at the start of step (1), as evaluated by, for example, the number of viable cells.

[0119] In some embodiments, the population of cells from step (3) does not proliferate compared to the population of cells at the start of step (1), as assessed by, for example, the number of viable cells. In some embodiments, the population of cells from step (3) proliferates for less than 0.5, 1, 1.5, or 2 hours, for example, less than 1 or 1.5 hours, compared to the population of cells at the start of step (1).

[0120] In some embodiments, a population of cells is not in vitro contacted with a drug that stimulates the CD3 / TCR complex and / or a drug that stimulates a co-stimulatory molecule on the surface of those cells, or, if contacted, the contact step is less than 2 hours, for example, 1 or 1.5 hours or less. In some embodiments, the drug that stimulates the CD3 / TCR complex is a drug that stimulates CD3 (e.g., an anti-CD3 antibody). In some embodiments, the drug that stimulates the co-stimulatory molecule is a drug that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, the drug that stimulates the co-stimulatory molecule is a drug that stimulates CD28. In some embodiments, the agent that stimulates the CD3 / TCR complex is selected from antibodies (e.g., single-domain antibodies (e.g., heavy-chain variable-domain antibodies), peptide bodies, Fab fragments, or scFvs), small molecules, or ligands (e.g., naturally occurring ligands, recombinant ligands, or chimeric ligands).

[0121] In some embodiments, steps (1) and / or (2) are carried out in a cell medium containing 5, 4, 3, 2, 1, or 0% or less of serum. In some embodiments, steps (1) and / or (2) are carried out in a cell medium containing 2% or less of serum. In some embodiments, steps (1) and / or (2) are carried out in a cell medium containing about 2% of serum. In some embodiments, steps (1) and / or (2) are carried out in a cell medium containing an LSD1 inhibitor or a MALT1 inhibitor. In some embodiments, step (1) is carried out in a cell medium containing 5, 4, 3, 2, 1, or 0% or less of serum. In some embodiments, step (1) is carried out in a cell medium containing 2% or less of serum. In some embodiments, step (1) is carried out in a cell medium containing about 2% of serum. In some embodiments, step (2) is carried out in a cell medium containing 5, 4, 3, 2, 1, or 0% or less of serum. In some embodiments, step (2) is carried out in a cell medium containing 2% or less of serum. In some embodiments, step (2) is carried out in a cell medium containing approximately 2% serum. In some embodiments, step (1) is carried out in a cell medium containing an LSD1 inhibitor or a MALT1 inhibitor. In some embodiments, step (2) is carried out in a cell medium containing an LSD1 inhibitor or a MALT1 inhibitor.

[0122] In some embodiments, the method described above further includes the step of (iv) receiving a fresh leukocyte apheresis product (or an alternative source of hematopoietic tissue such as a fresh whole blood product, a fresh bone marrow product, or a fresh tumor or organ biopsy or excision (e.g., a fresh product from a thymectomy)) from an entity prior to step (i).

[0123] In some embodiments, the method described above further includes, prior to step (i), the step of isolating a population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) that were contacted in step (i) from (v) a fresh leukocyte apheresis product (or an alternative source of hematopoietic tissue such as a fresh whole blood product, a fresh bone marrow product, or a fresh tumor or organ biopsy or excision (e.g., a fresh product from thymectomy)). In some embodiments, step (iii) is carried out within 35 hours after the start of step (v), for example, within 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours after the start of step (v), for example, within 30 hours after the start of step (v). In some embodiments, the population of cells from step (iii) is not proliferated, as assessed by the number of viable cells, compared to the population of cells at the end of step (v), or is proliferated by 5, 10, 15, 20, 25, 30, 35 or 40%, e.g., 10% or less.

[0124] In some embodiments, the method described above further includes, prior to step (i), receiving cryopreserved T cells isolated from leukocyte apheresis products (or alternative sources such as cryopreserved T cells isolated from whole blood, bone marrow, or tumor or organ biopsy or excision (e.g., thymectomy)) from an entity, e.g., a laboratory, hospital, or healthcare provider.

[0125] In some embodiments, the method described above further includes the step of (iv) receiving a cryopreserved leukocyte apheresis product (or an alternative source of hematopoietic tissue such as a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved tumor or organ biopsy or excision (e.g., a cryopreserved product from a thymectomy)) prior to step (i).

[0126] In some embodiments, the method described above further includes, before step (i), the step of isolating a population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) that were contacted in step (i) from (v) a cryopreserved leukocyte apheresis product (or an alternative source of hematopoietic tissue such as a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved tumor or organ biopsy or excision (e.g., a cryopreserved product from thymectomy)). In some embodiments, step (iii) is carried out within 35 hours after the start of step (v), for example, within 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours after the start of step (v), for example, within 30 hours after the start of step (v). In some embodiments, the population of cells from step (iii) is not proliferated, as assessed by the number of viable cells, compared to the population of cells at the end of step (v), or is proliferated by 5, 10, 15, 20, 25, 30, 35 or 40%, e.g., 10% or less.

[0127] In some embodiments, the cell population at the start of step (i) or step (1) is enriched with IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ). In some embodiments, the cell population at the start of step (i) or step (1) contains 40, 45, 50, 55, 60, 65, or 70% or more of IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ).

[0128] In some embodiments, steps (i) and (ii) or steps (1) and (2) are carried out in a cell medium containing IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, IL-15 increases the ability of a cell population to proliferate, for example, after 10, 15, 20, or 25 days. In some embodiments, IL-15 increases the percentage of IL6Rβ-expressing cells in a cell population.

[0129] In some embodiments of the method described above, the method is carried out in a closed system. In some embodiments, T cell isolation, activation, transduction, incubation, and washing are all carried out in a closed system. In some embodiments of the method described above, the method is carried out in individual devices. In some embodiments, T cell isolation, activation, transduction, incubation, and washing are carried out in individual devices.

[0130] In some embodiments of the method described above, the method further includes the step of adding an adjuvant or transduction-promoting reagent to the cell culture medium to enhance transduction efficiency. In some embodiments, the adjuvant or transduction-promoting reagent comprises a cationic polymer. In some embodiments, the adjuvant or transduction-promoting reagent is selected from LentiBOOST® (Sirion Biotech), vectofusin-1, F108, hexadimethrin bromide (Polybrene), PEA, Pluronic F68, Pluronic F127, Synperonic, or LentiTrans®. In some embodiments, the adjuvant is LentiBOOST® (Sirion Biotech).

[0131] In some embodiments of the methods described above, the step of transducing a population of cells (e.g., T cells) with a viral vector includes subjecting the cell population and the viral vector to centrifugal force under conditions that enhance transduction efficiency. In one embodiment, the cells are transduced by spinoculation.

[0132] In some embodiments of the methods described above, cells (e.g., T cells) are activated and transduced in a cell culture flask containing a gas permeable membrane at the bottom, which supports a large volume of culture medium without substantially impairing gas exchange. In some embodiments, cell proliferation is achieved by convection-mediated access to nutrients, e.g., uninterrupted access.

[0133] In some embodiments of the methods described above, the CAR or CCAR includes an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0134] In some embodiments, the antigen-binding domain is CD19, CD20, CD22, BCMA, mesothelin, EGFRvIII, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptide, sTn-O-glycopeptide, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, Regman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, Lewis Y, CD24, PDGFR-β, SSEA-4, folate receptor α, ERBB (e.g., ERBB2), Her2 / neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, FAP, Regman, HPV It binds to an antigen selected from E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, β-human chorionic gonadotropin, AFP, thyroglobulin, PLAC1, globo H, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, mut hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4, or any of these antigen peptides presented on the MHC. In some embodiments, the antigen-binding domain includes a CDR, VH, VL, scFv, or CAR sequence as disclosed herein. In some embodiments, the antigen-binding domain comprises VH and VL, which are linked by a linker, which optionally comprises the amino acid sequence of SEQ ID NO: 63 or 104.

[0135] In some embodiments, the transmembrane domain includes the transmembrane domain of a protein selected from the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain includes the transmembrane domain of CD8. In some embodiments, the transmembrane domain includes the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding the transmembrane domain, and the nucleic acid sequence includes the nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0136] In some embodiments, the antigen-binding domain is linked to the transmembrane domain by a hinge region. In some embodiments, the hinge region includes the amino acid sequence of SEQ ID NOs: 2, 3, or 4, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding the hinge region, and the nucleic acid sequence includes the nucleic acid sequence of SEQ ID NOs: 13, 14, or 15, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0137] In some embodiments, the intracellular signaling domain includes a primary signaling domain. In some embodiments, the primary signaling domain includes a functional signaling domain derived from CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278(ICOS), FcεRI, DAP10, DAP12, or CD66d. In some embodiments, the primary signaling domain includes a functional signaling domain derived from CD3ζ. In some embodiments, the primary signaling domain includes the amino acid sequence of SEQ ID NO: 9 or 10 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding the primary signaling domain, and the nucleic acid sequence includes the nucleic acid sequence of SEQ ID NO: 20 or 21 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0138] In some embodiments, the intracellular signaling domain includes a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain includes MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activators (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, 4-1BB (CD137). , B7-H3, ICOS(CD278), GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, C D19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD 11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG 2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229) The ligands include CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB, or CD83. In some embodiments, the co-stimulatory signaling domain includes a functional signaling domain derived from 4-1BB. In some embodiments, the co-stimulatory signaling domain includes the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.In some embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding a co-stimulus signaling domain, and the nucleic acid sequence includes the nucleic acid sequence of Sequence ID No. 18 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0139] In some embodiments, the intracellular signaling domain includes a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3ζ. In some embodiments, the intracellular signaling domain includes the amino acid sequence of SEQ ID NO: 7 (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with it) and the amino acid sequence of SEQ ID NO: 9 or 10 (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with it). In some embodiments, the intracellular signaling domain includes the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or 10.

[0140] In some embodiments, the CAR or CCAR further includes a leader sequence comprising the amino acid sequence of SEQ ID NO: 1.

[0141] In some embodiments, the disclosure features a population of CAR-expressing cells (e.g., CCAR-expressing cells) (e.g., autologous or allogeneic CAR-expressing T cells or NK cells) prepared by any of the methods described herein or any other method disclosed herein. In some embodiments, a pharmaceutical composition comprising the population of CAR-expressing cells disclosed herein and a pharmaceutically acceptable carrier is provided herein.

[0142] In some embodiments, the total amount of beads (e.g., CD4 beads, CD8 beads, and / or TransACT beads) in the final CAR cell product produced using the method described herein is 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, or 0.5% or less of the total amount of beads added during the production process.

[0143] In some embodiments, the disclosure features a population of CAR-expressing cells (e.g., CCAR-expressing cells) (e.g., autologous or allogeneic CAR-expressing T cells or NK cells) having one or more of the following characteristics: (a) approximately the same percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells compared to the same population of cells before they were manipulated to express CAR; (b) a change of approximately 5% to approximately 10% of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells compared to the same population of cells before they were manipulated to express CAR; (c) a percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells compared to the same population of cells before they were manipulated to express CAR. (d) A percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells, increased by, for example, at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or three times compared to the past; (d) Approximately the same percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the same population of cells before being manipulated to express CAR; (e) A change of approximately 5% to approximately 10% of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the same population of cells before being manipulated to express CAR;(f) A percentage of central memory cells, e.g., CCR7+CD45RO+T cells, reduced by, for example, at least 20, 25, 30, 35, 40, 45, or 50%, compared to the percentage of central memory cells, e.g., CCR7+CD45RO+T cells, in the same population of cells before they were manipulated to express CAR; (g) Stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in approximately the same percentage as the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in the same population of cells before they were manipulated to express CAR; (h) A change of approximately 5% to 10% of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, compared to the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the same cell population before manipulation to express CAR; or (i) An increased percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, compared to the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the same cell population before manipulation to express CAR.

[0144] In some embodiments, the disclosure features a population of CAR-expressing cells (e.g., CCAR-expressing cells) (e.g., autologous or allogeneic CAR-expressing T cells or NK cells), where (a) the median GeneSetScore (Up TEM vs. Down TSCM) of the cell population is approximately the same as the median GeneSetScore (Up TEM vs. Down TSCM) of the same cell population before it was manipulated to express CAR, or differs by only about 25, 50, 75, 100, or 125% or less (e.g., increased by only a small amount); (b) the median GeneSetScore (Up Treg vs. Down Teff) of the cell population is the same as the median GeneSetScore (Up Treg vs. Down TSCM) of the cell population before it was manipulated to express CAR (c) The median GeneSetScore (Down stemness) of a cell population is approximately the same as the median GeneSetScore (Down stemness) of a cell population before it was manipulated to express CAR, or differs by approximately 25, 50, 100, 150, or 200% or less (e.g., increased by less than or equal to that); (d) The median GeneSetScore (Up hypoxia) of a cell population is approximately the same as the median GeneSetScore (Up hypoxia) of a cell population before it was manipulated to express CAR, or differs by approximately 125, 150, 175, or 200% or less (e.g., increased by less than or equal to that); or (e) the median GeneSetScore (Up hypoxia) of a cell population Autophagy is approximately the same as the median GeneSetScore (Up autophagy) of the cell population before it was manipulated to express CAR, or differs by only about 180, 190, 200, or 210% or less (e.g., increased by only a small amount).

[0145] In some embodiments, the present disclosure features a method for increasing the immune response of a target, which includes the step of administering to a population of CAR-expressing cells or a pharmaceutical composition disclosed herein to thereby increase the immune response of the target.

[0146] In some embodiments, a method of treating a subject's cancer is disclosed, which includes administering to the subject a population of CAR-expressing cells disclosed herein or a pharmaceutical composition disclosed herein, thereby treating the subject's cancer. In some embodiments, the cancer is, for example, mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, skin cancer, melanoma, renal cancer, liver cancer, brain tumor, thymoma, sarcoma, carcinoma, uterine cancer, renal cancer, gastrointestinal cancer, urothelial cancer, pharyngeal cancer, head and neck cancer, rectal cancer, esophageal cancer or bladder cancer, a solid cancer selected from one or more of these or its metastasis. In some embodiments, the cancer is, for example, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoblastic leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocyte leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), DLBCL with chronic inflammation, chronic myelogenous leukemia, myeloproliferative tumor, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphocyte proliferative disorder, MALT lymphoma (mucosa-associated lymphoid tissue type extranodal follicular marginal zone lymphoma), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small cell B-cell lymphoma, hairy cell leukemia-variant, lymphoplasmacytic lymphoma, H-chain disease, plasma cell myeloma, solitary bone plasmacytoma, extramedullary plasmacytoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma occurring in HHV8-related multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML) or an unclassifiable lymphoma.

[0147] In some embodiments, the method further comprises administering a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is an anti-cancer therapeutic agent, such as a chemotherapeutic agent, radiation therapy, or immunomodulatory therapy. In some embodiments, the second therapeutic agent is IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)).

[0148] Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, but suitable methods and materials are described below. All publications, patent applications, patents, and other references (e.g., sequence database reference numbers) cited herein are incorporated herein by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences mentioned herein (e.g., in any table herein) are incorporated by reference. When a gene or protein refers to multiple accession numbers, all sequence variants are included.

[0149] Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting. Headings, subheadings, or numbered or lettered elements, such as (a), (b), (i), etc., are provided merely for ease of reading. The use of headings or numbered or lettered elements herein does not require that steps or elements be performed in alphabetical order or that steps or elements be necessarily distinct from one another. Other features, objects, and advantages of the present disclosure will become apparent from the description, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0150] Figures 1A-1I: When purified T cells were incubated with cytokines, naive cells were the dominant transduced population. Figure 1A is a graph illustrating an exemplary cytokine process. Figure 1B is a set of graphs showing the percentage of CD3+CAR+ cells at each display time point after transduction. Figure 1C is a series of graphs showing transduction in the CD3+CCR7+CD45RO- population in the CD3 / CD28 bead-stimulated population (left) compared to the cytokine-only population (right) in two independent donors. In the case of the sample labeled "Short stim IL7+IL15" in Figure 1C, cells were stimulated with beads for two days and then removed in the presence of IL7 and IL15. Figures 1D, 1E, and 1F are a series of flow cytometry graphs showing transduction of T cell subsets cultured daily with IL2 (Figure 1D), IL15 (Figure 1E), and IL7+IL15 (Figure 1F) over three days. Figure 1G is a series of flow cytometry graphs showing T cell differentiation of CCR7 and CD45RO cells on day 0 (left) and day 1 (right) after stimulation with IL-2 (upper right panel) or IL-15 (lower right panel). Figures 1H and 1I are a series of graphs showing the percentage of CD3+CCR7+RO-, CD3+CCR7+RO+, CD3+CCR7-RO+, and CD3+CCR7-RO- cells on day 0 or after 24-hour incubation with the indicated cytokines. Figures 2A-2D: CART produced by cytokine stimulation for 1 day was functional. Figure 2A: Purified T cells were transduced at an MOI of 1 under all tested cytokine conditions, and the percentage of CAR-expressing cells observed on day 1 and day 10 was equivalent. CART was generated within 1 day, and after collection, it was grown for 9 days with CD3 / CD28 beads to mimic the in vivo setting. Figure 2A is a set of graphs showing the average percentage of CD3+CAR+ cells under each condition for day 1 CART (left) and day 10 CART (right). Figure 2B: The cytotoxicity of day 1 CART after growth was measured using Nalm6 as the target cell. Figure 2B is a graph showing the percentage of CD19-positive Nalm6 cells killed by CART under each condition. Day 10 CART grown with CD3 / CD28 beads is labeled "Day 10". All other samples were day 1 CART. Figure 2C: IFN-γ secretion by day 1 CART in response to Nalm6 target cells was tested. Figure 2C is a graph showing the amount of IFN-γ secretion by CART under each condition in the presence of CD19-positive or CD19-negative target cells. Figure 2D: The proliferative capacity of day 1 CART was tested by measuring EDU uptake. Figure 2D is a graph showing the average percentage of EDU-positive cells for each condition. Similar to Figure 2B, day 10 CART is labeled "Day 10," and all other samples were day 1 CART. Figures 3A-3B: Effects of MOI and culture medium composition on transduction on day 0. Figure 3A: Purified T cells were transduced at MOIs ranging from 1 to 10 in the presence of IL15, IL2+IL15, IL2+IL7, or IL7+IL15. A linear increase in transduction was observed regardless of the cytokine used. Figure 3A is a series of graphs plotting the percentage of CD3+CAR+ cells against the MOI of each tested condition. Figure 3B: Culture medium composition affected transduction in the cytokine process. Figure 3B is a pair of graphs showing the percentage of CD3+CAR+ cells on day 1 (left) and day 8 (right) for each tested condition. "2.50" indicates an MOI of 2.50. "5.00" indicates an MOI of 5.00. Figures 4A-4D: CAR T cells produced within 24 hours can eliminate tumors. Figure 4A: Purified T cells were transduced with CAR19 and harvested after 24 hours. Figure 4A is a series of flow cytometry plots showing transduction of T cells with CAR19 cultured with IL2, IL15, and IL7+IL15, showing transduction under each cytokine condition. Figure 4B: Graphs showing average survival rates of over 80% under all tested conditions. Figure 4C: Proliferation of day 1 CART in peripheral blood is increased in vivo compared to their day 10 counterparts. Percentage of viable CD45+CD11b-CD3+CAR+ cells at the time of display after injection for each tested condition. Day 10 CART is labeled "D10 1e6" or "D10 5e6", while all other samples were day 1 CART. Figure 4D: Day 1 CART, compared to Day 10 CART, was able to eliminate tumors in vivo, although with a pharmacokinetic delay. Figure 4D is a graph showing the total flux at the time indicated after tumor inoculation for each tested condition. CART was administered 4 days after tumor inoculation. Day 10 CART is indicated as "5e6 d.10", and all other samples were Day 1 CART. Figures 5A-5B: The cytokine process was scalable. Figure 5A: T cells were enriched with CliniMACS® Prodigy®, reducing the B cell compartment to less than 1%. Figure 5A is a series of flow cytometry plots showing cell staining with anti-CD3 antibody (left) or anti-CD19 and anti-CD14 antibodies (right) in Leucopack cells (top) or CD4+CD8+ enriched cells (bottom). Figure 5B: Purified T cells from freeze apheresis were transduced with CAR19 in either a 24-well plate or a enriched PL30 bag. CART was harvested after 24 hours. Figure 5B is a series of flow cytometry plots showing CD3 staining and CAR in cells produced in the presence of either IL2 or hetIL-15 (IL15 / sIL-15Ra). Figures 6A-6C: CART produced by the activation process showed superior antitumor efficacy in vivo. Figures 6A and 6B are graphs plotting tumor load against the indicated time point after tumor transplantation. "d.1" shows CART produced using the activation process. "d.9" shows CART produced using the traditional 9-day growth protocol used as a positive control in this study. Figure 6C is a series of representative images showing bioluminescence from mice. Figures 7A-7B: IL6Rα and IL6Rβ expressing cells were enriched in a poorly differentiated T cell population. Fresh T cells were stained for the surface antigens shown, and the expression levels of IL6Rα and IL6Rβ on CD4 (Figure 7A) and CD8 (Figure 7B) T cell subsets were tested. Figures 8A-8B: Both IL6Rα and IL6Rβ expressing cells were enriched in the poorly differentiated T cell population. Fresh T cells were stained for the indicated surface antigens, and the expression levels of the indicated surface antigens on CD4 (Figure 8A) and CD8 (Figure 8B) T cell subsets were tested. Figure 9: IL6Rα-expressing cells expressed surface markers for poorly differentiated T cells. Fresh T cells were stained for the surface antigens shown, and the expression levels of each surface antigen were examined in high, medium, and low IL6Rα-expressing cell subsets. Figure 10: IL6Rβ-expressing cells expressed surface markers for poorly differentiated T cells. Fresh T cells were stained for the surface antigens shown, and the expression levels of each surface antigen were examined in high, medium, and low IL6Rβ-expressing cell subsets. Figure 11: IL6Rα expression was downregulated after TCR engagement, but IL6Rβ expression was not. After activating T cells with αCD3αCD28 beads on day 0, the expression levels of IL6Rα and IL6Rβ were tested at the time shown. Figure 12: Proliferation rate of cytokine-treated T cells after TCR engagement. On day 0, T cells were activated with αCD3αCD28 beads in the presence of the indicated cytokines, and the cell count was monitored at the indicated time. Figures 13A-13B: Treatment with IL2, IL7, and IL15 did not affect cell size and viability after TCR engagement. On day 0, T cells were activated with αCD3αCD28 beads in the presence of the indicated cytokines, and cell size (Figure 13A) and viability (Figure 13B) were monitored at the indicated time. Figure 14: Expression dynamics of various surface molecules in CD4 T cells after cytokine treatment. On day 0, T cells were activated with αCD3αCD28 beads in the presence of the indicated cytokines, and the expression of various surface molecules was examined by flow cytometry at the indicated time. Figure 15: Expression dynamics of various surface molecules in CD8 T cells after cytokine treatment. On day 0, T cells were activated with αCD3αCD28 beads in the presence of the indicated cytokines, and the expression of various surface molecules was examined by flow cytometry at the indicated time. Figure 16: After TCR engagement, IL6Rβ expression was restricted primarily in the CD27-expressing T cell subset. On day 0, T cells were activated with αCD3αCD28 beads in the presence of the indicated cytokines, and on day 15, IL6Rβ expression was assessed by flow cytometry. Figure 17: After TCR engagement, IL6Rβ expression was suppressed primarily in the CD57-nonexpressing T cell subset. On day 0, T cells were activated with αCD3αCD28 beads in the presence of the indicated cytokines, and on day 25, IL6Rβ expression was assessed by flow cytometry. Figure 18: Common γ-chain cytokine-treated T cells produced functional cytokines on day 25. On day 0, T cells were activated with αCD3αCD28 beads in the presence of the indicated cytokines, and on day 25, the percentage of IL2, IFN-γ, and TNFα-producing T cells was examined by flow cytometry. Figures 19A-19B: BCMA CAR expression on day 1 using an ARM with MOI=2.5 in T cells from two healthy donors. Figure 19A is a panel of histograms showing BCMA CAR expression as measured by flow cytometry. Figure 19B is a table listing the reagents / conditions used for flow cytometry analysis. Figures 20A-20C: In vitro CAR expression dynamics from day 1 to day 4 in cells produced using the ARM process. CAR was stably expressed on day 3. Figure 20A is a panel of histograms showing CAR expression at the indicated time point, measured by flow cytometry. Figures 20B and 20C are graphs showing CAR+% and MFI values ​​over time, respectively. Figures 21A and 21B: In vivo triage in a KMS-11-luc xenograft mouse model of multiple myeloma. Each mouse received 1.5E6 of the day 1 CART product. Figure 21A is a panel of histograms showing day 1 and day 7 CAR expression in CART cells. Figure 21B is a graph showing tumor dynamics (BLI levels) after CART treatment. Figures 22A, 22B, and 22C: In vivo triage of BCMA CARs by dose titration in a KMS-11-luc multiple myeloma xenograft mouse model. Figure 22A is a panel of histograms showing CAR expression on day 1 and day 3. Figure 22B is a graph showing tumor uptake dynamics after CART treatment using two different doses: one dose of 1.5e5 CAR+ T cells and one dose of 5e4 CAR+ T cells. The dose of CAR+ cells was normalized based on day 3 CAR expression. Figure 22C is a graph showing body weight dynamics over time during this study. Figures 23A, 23B, and 23C: Figures 23A and 23B are graphs showing the percentage of T cells expressing CAR on the cell surface (Figure 23A) and the mean fluorescence intensity (MFI) of CD3+CAR+ cells observed over time (Figure 23B) (repeat efficiency is averaged from the two flow panels shown in Figure 23C). Figure 23C is a panel of flow cytometry plots showing the gating strategy of surface CAR expression on viable CD3+ cells based on UTD samples. The numbers in the plots represent the CAR positivity rate (%). Figures 24A and 24B: Figure 24A is a graph showing the end-to-end composition of the starting material (Prodigy® product) and at various time points from the start of culture to harvesting. Native (n), central memory (cm), effector memory (em), and effector (eff) subsets were determined by the surface expression or absence of CD4, CD8, CCR7, and CD45RO. CD4 composition is shown. At each time point, the bars on the left show the cellular composition of the entire CD3+ population (bulk), and the bars on the right show the cellular composition of the CAR+ fraction. Figure 24B is a panel of flow cytometry plots showing the gating strategies applied to the viable CD3+ events to determine the overall transduction efficiency (top row), CD4 / CD8 composition (middle row), and memory subset (bottom row) within the entire CD3+ population (bulk) and CAR+ fraction. Figure 25: Dynamics of T cell subsets expressing surface CARs over time, expressed as the number of viable cells in each subset. Figure 26: Viable cells collected 12-24 hours after the start of culture, as determined from the pre-wash count (number of viable cells collected at the time of collection relative to the number of viable cells inoculated). Figure 27: Survival rate of fast CARTs collected 12-24 hours after the start of culture, as determined before and after washing at the time of collection. Figures 28A, 28B, 28C, and 28D: Figure 28A is a graph showing the composition of the starting material (healthy donor leukopak: LKPK) and the T cell enriched product analyzed by flow cytometry. The numerical values indicate the percentage of the parent (viable, single cells). T: T cells; mono: monocytes; B: B cells; CD56 (NK): NK cells. Figure 28B is a panel of flow cytometry plots showing the gating strategy for viable CD3+ events used to determine transduction efficiency (forward scatter light FSC vs CAR) and T cell subsets (CD4 vs CD8 and CCR7 vs CD45RO). For ARM CD19CAR (CD19 CAR T cells manufactured using the Activated Rapid Manufacturing (ARM) process) and TM-CD19CAR (CD19 CAR T cells manufactured using the Traditional Manufacturing (TM) process), the lower left panel represents the bulk culture and the right panel represents the CAR+ T cells. "ARM-UTD" and "TM-UTD" refer to non-transduced T cells (UTD) manufactured according to the ARM and TM processes, respectively. The numerical values within the quadrants indicate the percentage of the parent population. The boxes within the TM-UTD and TM-CD19CAR plots indicate the CM skew towards the phenotype. The boxes within the ARM-UTD and ARM-CD19CAR plots indicate the maintenance of naive-like cells by the ARM process. NA: not applicable. Figure 28C is a graph showing the end-to-end T cell composition of ARM-CD19CAR and TM-CD19CAR. The composition is shown for the "bulk" and "CAR+" populations where applicable. The percentage of each population refers to the percentage of the parent, either CD3+ or CAR+CD3+ as applicable. The percentage of CD4 cells in a representative bulk or CAR+ population is shown. LKPK: leukopak starting material; 4 and 8: CD4+ and CD8+, respectively; eff: effector; em: effector memory; cm: central memory; n: naive-like. The data are representative of three full-scale tests using three different healthy donors (n = 3) and several small-scale tests used to optimize the process. Figure 28D is a table showing the percentages shown in Figure 28C. Figures 29A, 29B, 29C, and 29D: Cytokine concentrations in cell culture supernatant. IFN-γ (Figures 29A and 29B) and IL-2 (Figures 29C and 29D). Figures 29A and 29C: TM-CD19CAR, ARM-CD19CAR, and representative UTDs were co-cultured with NALM6-WT (ALL) and TMD-8 (DLBCL) or cultured without cancer cells (T cells alone). Supernatant was collected after 48 hours. Figures 29B and 29D: ARM-CD19CAR was co-cultured with NALM6-WT and NALM6-19KO (CD19 negative) or cultured alone. Supernatant was collected after 24 or 48 hours. To further evaluate antigen-specific cytokine secretion, ARM-CD19CAR was cultured alone for 24 hours, washed, and then co-cultured with target cells for 24 hours. The data presented is representative of two experiments using a total of three donors, obtained from two healthy donor T cells. Figures 30A, 30B, and 30C: Figure 30A is a graph illustrating the schematic of a xenograft mouse model for testing the antitumor activity of ARM-CD19CAR. Figure 30B is a panel of flow cytometry plots showing the determination of CAR expression in ARM-CD19CAR cells from sentinel vials. Prior to flow cytometry analysis, ARM-CD19CAR cells were cultured for the periods indicated in the figure. Gating of CAR expression was performed based on isotype control (Iso) staining. Figure 30C is a graph showing the in vivo efficacy of ARM-CD19CAR in a xenograft mouse model. Pre-B ALL strain NALM6 expressing the luciferase reporter gene was injected into NGS mice; tumor load was expressed as total body luminescence (p / s) and shown as the mean tumor load with a 95% confidence interval. Mice were treated with each dose (number of viable CARs + T cells) of ARM-CD19CAR or TM-CD19CAR on day 7 after tumor inoculation. The high-dose ARM-CD19CAR group was discontinued on day 33 due to the development of X-GVHD. Vehicle (PBS) and untransduced T cells (UTDs) were used as negative controls. ARM-UTD 1×10 6Except for the dose and all TM-CD19CAR dose groups, which had n=4, all groups had n=5 mice. Five xenotransplant studies were conducted using CAR+ T cells generated from five different healthy donors, three of which included comparisons with TM-CD19CAR. Figures 31A, 31B, 31C, and 31D: Plasma cytokine levels in NALM6 tumor-bearing mice treated with ARM-CD19CAR or TM-CD19CAR at respective CAR-T cell doses. Blood samples were collected from the mice and plasma cytokines were measured by the MSD assay. IFN-γ (Figures 31A and 31B) and IL-2 (Figures 31C and 31D) are shown for mice treated with CAR-T cells (Figures 31A and 31C) or ARM- and TM-UTD cells (Figures 31B and 31D). Bars within each dose represent the mean cytokine levels within the group at various time points (from left to right: days 4, 7, 10, 12, 16, 19, 23, and 26). Horizontal bars and numbers are represented by letters (ARM-CD19CAR (1 × 10)). 6 Dose group) and TM-CD19CAR (0.5 × 10 6 Comparison of fold changes between dose groups: IFN-γ showed a 3-fold increase; and IL-2 showed a 10-fold increase. Groups withdrawn due to tumor burden or weight loss do not show the last time point. Plasma cytokine levels were measured for both trials. no tum: no tumor. Figure 32: Time course of total and CAR+ T cell concentrations in NALM6 tumor-bearing mice treated with PBS vehicle, UTD, TM-CD19CAR, or ARM-CD19CAR. Blood samples were collected 4, 7, 14, 21, and 28 days after CAR-T cell injection. Total T cell (CD3+, top) and CAR+ T cell (CD3+CAR+, bottom) concentrations were analyzed by flow cytometry at planned time points and are shown as mean cells with a 95% confidence interval. Figures 33A and 33B: IL-6 levels (pg / mL) in the supernatant of three-party co-cultures. Subsequently, ARM-CD19CAR / K562 co-cultured cells (Figure 33A) or TM-CD19CAR / K562 co-cultured cells (Figure 33B), incubated for 6 or 24 hours at different ratios (1:1 and 1:2.5), were added to PMA-differentiated THP-1 cells for a further 24 hours. Results from CAR-T cells co-cultured with K562-CD19 cells, CAR-T cells co-cultured with K562-mesothelin cells, and CAR-T cells alone are shown. For clarity, the 1:5 ratio is not shown. The bars labeled ARM-CD19CAR only and TM-CD19CAR only represent CAR-T cell cultures (6h, 24h) that do not contain target cells. Mean + SEM, repeated twice: n=1 (TM-CD19CAR) and n=3 (ARM-CD19CAR). Figures 34A, 34B, and 34C: The ARM process preserves the stem cell properties of BCMA CAR+ T cells. CAR expression was evaluated for RI61, R1G5, and BCMA10 CART cells produced using the ARM process at thawing (Figure 34A) and 48 hours after thawing (Figure 34B). The CCR7 / CD45RO marker was also evaluated for the 48-hour post-thawed product (Figure 34C). The data shown are representative from two experiments conducted using two donor T cells. Figures 35A and 35B: The TM process primarily resulted in central memory T cells (TCMs) (CD45RO+ / CCR7+), while the native-like T cell population was almost completely eliminated in CAR+ T cells produced by the TM process. PI61, R1G5, and BCMA10 CART cells produced using the TM process were evaluated for CAR expression on day 9 (Figure 35A). The CCR7 / CD45RO marker was also evaluated in the day 9 product after thawing (Figure 35B). The data shown are representative from two experiments conducted using two donor T cells. Figures 36A, 36B, 36C, and 36D: ARM-treated BCMA CAR-T cells exhibit BCMA-specific activation and secrete higher levels of IL-2 and IFN-γ. IL-2 and IFN-γ concentrations in the cell supernatant. RI61, R1G5, and BCMA10 CART cells, produced using the ARM or TM process, and their respective UTDs were co-cultured with KMS-11 in a 2.5:1 ratio. Supernatants were collected after 20 hours. For ARM products, IFN-γ concentrations are shown in Figure 36A and IL-2 concentrations in Figure 36B. For TM products, IFN-γ concentrations are shown in Figure 36C and IL-2 concentrations in Figure 36D. The data shown are representative from two experiments conducted using two donor T cells. Figures 37A, 37B, and 37C: Single-cell RNA-seq data for input cells (Figure 37A), day 1 cells (Figure 37B), and day 9 cells (Figure 37C). The "nGene" graph shows the number of expression cells per cell. The "nUMI" graph shows the number of unique molecular identifiers (UMIs) per cell. Figures 38A, 38B, 38C, and 38D are T-distribution stochastic neighbor embedding (TSNE) plots comparing the proliferation signatures of input cells (Figure 38A), day 1 cells (Figure 38B), and day 9 cells (Figure 38C), where the proliferation signature was determined based on the expression of genes CCNB1, CCND1, CCNE1, PLK1, and MKI67. Each point represents a cell in that sample. Cells shown in light gray do not express proliferation genes, while darkly shaded cells express one or more proliferation genes. Figure 38D is a violin plot showing the distribution of gene set scores for gene sets containing genes that characterize resting T cell states versus activated T cell states for day 1 cells, day 9 cells, and input cells. In Figure 38D, a higher gene set score (Up resting vs. Down activated) indicates an increase in the resting T cell phenotype, while a lower gene set score (Up resting vs. Down activated) indicates an increase in the activated T cell phenotype. Input cells were generally more resting than day 9 and day 1 cells. Day 1 cells showed the highest activation gene set score. Figures 39A, 39B, 39C, 39D, and 39E: Gene set analysis of input cells, day 1 cells, and day 9 cells. In Figure 39A, a higher gene set score "Up TEM vs. Down TSCM" indicates an increase in the effector memory T cell (TEM) phenotype of cells in that sample, while a lower gene set score indicates an increase in the stem cell memory T cell (TSCM) phenotype. In Figure 39B, a higher gene set score "Up Treg vs. Down Teff" indicates an increase in the regulatory T cell (Treg) phenotype, while a lower gene set score indicates an increase in the effector T cell (Teff) phenotype. In Figure 39C, a lower gene set score "Down stemness" indicates an increase in the stem cell phenotype. In Figure 39D, a higher gene set score "Up hypoxia" indicates an increase in the hypoxic phenotype. In Figure 39E, a higher gene set score "Up autophagy" indicates an increase in the autophagy phenotype. Day 1 cells appeared similar to input cells in terms of memory, stem-like characteristics, and differentiation signature. In contrast, day 9 cells showed high enrichment in response to metabolic stress. Figures 40A, 40B, and 40C: Gene cluster analysis of input cells. Figures 40A–40C are violin plots showing gene set scores from gene set analysis of four clusters of input cells. Each dot overlapping the violin plot in Figures 40A–40C represents the gene set score of the cell. In Figure 40A, a higher gene set score of "Up Treg vs. Down Teff" indicates an increase in the Treg cell phenotype, and a lower gene set score of "Up Treg vs. Down Teff" indicates an increase in the Teff cell phenotype. In Figure 40B, a higher gene set score of "Progressively up in memory differentiation" indicates an increase in the late memory T cell phenotype, and a lower gene set score of "Progressively up in memory differentiation" indicates an increase in the early memory T cell phenotype. In Figure 40C, a higher gene set score "Up TEM vs. Down TN" indicates an increase in the effector memory T cell phenotype, while a lower gene set score "Up TEM vs. Down TN" indicates an increase in the naive T cell phenotype. Compared to the cells in clusters 1 and 2, which are in an early memory, poorly differentiated T cell state, the cells in cluster 3 are in a late memory, more differentiated T cell state. Cluster 0 is thought to be in an intermediate T cell state. Overall, this data indicates a considerable level of heterogeneity in the input cells. Figures 41A, 41B, and 41C: TCR sequencing and measurement of chronotype diversity. Day 9 cells have a flatter distribution (higher diversity) of chronotype frequencies. Figure 42: A flowchart showing the plan for a Phase I clinical trial testing BCMA CART cells produced using the ARM process in adult patients with relapsed and / or refractory multiple myeloma. Figure 43: This graph shows FACS analysis of ARM-BCMA CAR expression at various collection points after virus addition, in the presence or absence of two different concentrations (30 μM and 100 μM) of AZT. The lentiviral vector was added 1 hour later than AZT treatment at activation and cell inoculation. Figures 44A and 44B: Graphs showing the evaluation of ARM-BCMA CAR expression at thawing (Figure 44A) and 48 hours post-thaw, as well as the CCR7 / CD45RO marker of the 48-hour post-thaw product, and the evaluation of TM-BCMA CAR at day 9 (Figure 44B). The data shown are representative from two experiments conducted using T cells from two different donors. Figures 45A and 45B: Graphs showing cytokine concentrations in cell culture supernatant. ARM-BCMA CAR, TM-BCMA CAR, and representative UTDs were co-cultured with KMS-11. Supernatants were collected after 24 hours. The data shown are representative from two experiments conducted using T cells from two different donors. Figure 46: This graph shows an overview of the xenotransplant efficacy trial used to test ARM-BCMA. Figure 47: A graph comparing the efficacy of ARM-BCMA CARs with that of TM-BCMA CARs in a xenograft model. NSG mice were injected with the MM cell line KMS11, which expresses the luciferase reporter gene. Tumor load is expressed as total bioluminescence (p / s) and shown as mean tumor load + SEM. On day 8 after tumor inoculation, mice were treated with either ARM-BCMA CAR or TM-BCMA CAR at their respective doses (number of viable CARs + T cells). Vehicle (PBS) and UTD T cells were used as negative controls. N=5 mice were present in all groups except for the ARM-BCMA CAR (1e4 cells), PBS, and UTD groups, which had N=4. Figures 48A, 48B, and 48C are graphs showing plasma IFN-γ dynamics in mice treated with ARM-BCMA CAR or TM-BCMA CAR. They show the UTD for each CAR-T dose and plasma IFN-γ levels in KMS11-luc tumor-bearing mice treated with ARM-BCMA CAR or TM-BCMA CAR. All IFN-γ levels are shown as mean ± SEM. Blood samples were collected from the mice, and plasma cytokines were measured using the Meso Scale Discovery (MSD) assay. Figure 49: Graphs showing the cellular dynamics of ARM-BCMA CAR and TM-BCMA CAR in vivo. Peripheral blood cellular dynamics of KMS11 tumor-bearing mice treated with TM UTD, ARM UTD, ARM-BCMA CAR, and TM-BCMA CAR at different doses. Cell count is expressed as mean cell count + SD. Mice were treated with either ARM-BCMA CAR or TM-BCMA CAR at each dose (number of surviving CARs + T cells) on day 8 after tumor inoculation. Vehicle (PBS) and UTD T cells were used as negative controls. Blood samples were collected 7, 14, and 21 days after CAR-T injection and analyzed by flow cytometry at planned time points. N=5 mice were present in all groups except for the ARM-BCMA CAR (1e4 cells), PBS, and UTD groups (N=4). Figures 50A and 50B are a pair of graphs showing the 24-hour survival rate (%) (Figure 50A) and the 24-hour recovery rate (%) (Figure 50B). The columns shown in Figures 50A and 50B, from left to right, represent CAR19 (MOI of 1), CAR19 (MOI of 2), CAR19.HilD (MOI of 1), CAR19.HilD (MOI of 2), UTD (MOI of 1), and UTD (MOI of 2). Figures 51A, 50B, 51C, and 51D: These graphs show the CAR expression rate (%) in CAR19 cells (Figures 50A and 50B) or CAR19.HilD cells (Figures 51C and 51D) in the presence of lenalidomide or DMSO, as shown in the figures. [Modes for carrying out the invention]

[0151] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0152] As used herein, "controllable chimeric antigen receptor (CCAR)" refers to a CAR whose level and / or activity can be regulated. In some embodiments, the expression or activity level of the CCAR can be adjusted to enhance CAR function and / or reduce toxicity. In some embodiments, the CCAR is regulated at the transcriptional, translational or post-translational level. In some embodiments, the CCAR is regulated by an On switch that results in stabilization of the CAR or turns on the expression and / or activity of the CAR. In some embodiments, the CCAR is regulated by an Off switch that results in ubiquitination and degradation of the CAR or turns off the expression and / or activity of the CAR. In some embodiments, the CCAR is regulated by both an On switch and an Off switch. In some embodiments, the CCAR comprises a degron tag as disclosed in International Publication No. WO 2019 / 079569, which is hereby incorporated by reference in its entirety. In some embodiments, the CCAR is a regulatable CAR (RCAR) as disclosed in International Publication No. WO 2015 / 090229, which is hereby incorporated by reference in its entirety. In some embodiments, the CCAR is a conditional active heterodimeric CAR as disclosed in International Publication No. WO 2014 / 127261, which is hereby incorporated by reference in its entirety. In some embodiments, the CCAR is a sortase synthetic CAR as disclosed in International Publication No. WO 2016 / 014553, which is hereby incorporated by reference in its entirety.

[0153] As used herein, “regulatory molecule” means a molecule having regulatory activity or a molecule that can be used to mediate regulatory activity. In some embodiments, the regulatory molecule can be co-expressed with CAR in cells to regulate the expression and / or activity of CAR directly (e.g., by directly acting on the expression level or functional activity) or indirectly (e.g., by regulating the survival or activity of cells expressing CAR). In some embodiments, the regulatory molecule can be used to induce cell death in cells, e.g., CAR-expressing cells, e.g., apoptosis. In some embodiments, the regulatory molecule can be used to activate cells, e.g., CAR-expressing cells. In some embodiments, the regulatory molecule is a marker used to label cells, e.g., CAR-expressing cells, for depletion, e.g., a cell surface marker. In some embodiments, the regulatory molecule is a caspase, e.g., inducible caspase 9, e.g., inducible caspase 9 disclosed in International Publication No. 2011146862, No. 2014164348, or No. 2016100236 (all of which are incorporated herein by reference). In some embodiments, the regulatory molecule is a cleavage-type EGFR, for example, a cleavage-type EGFR disclosed in International Publication No. 2011056894 or International Publication No. 2013123061 (both of which are incorporated herein by reference).

[0154] The terms "a" and "an" refer to one or more (i.e., at least one) of the grammatical referents of the articles. For example, "element" means one or more elements.

[0155] The term “approximately” means that, when referring to a measurable value such as quantity or duration of time, a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value is included as appropriate for carrying out the method of this disclosure.

[0156] The compositions and methods of this disclosure include polypeptides and nucleic acids having a specified sequence or substantially identical or similar sequences, for example, sequences that are at least 85%, 90%, or 95% identical to the specified sequence. In relation to amino acid sequences, the term “substantially identical” is used herein to mean an amino acid sequence having a common structural domain that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence, for example, a sequence described herein, such that the first and second amino acid sequences may have a common structural domain and / or common functional activity.

[0157] In relation to nucleotide sequences, the term “substantially identical” is used herein to mean a nucleotide sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a first nucleic acid sequence, e.g., a reference sequence, e.g., a sequence described herein, such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or a common structural polypeptide domain or common functional polypeptide activity, and containing a sufficient or minimum number of nucleotides identical to the aligned nucleotides in the second nucleic acid sequence.

[0158] The term "mutant" refers to a polypeptide having an amino acid sequence substantially identical to a reference amino acid sequence, or encoded by a substantially identical nucleotide sequence. In some embodiments, the mutant is a functional mutant.

[0159] The term "functional variant" refers to a polypeptide that has an amino acid sequence substantially identical to a reference amino acid sequence, or is encoded by a substantially identical nucleotide sequence, and that may possess one or more of the activities of the reference amino acid sequence.

[0160] The term cytokine (e.g., IL-2, IL-7, IL-15, IL-21, or IL-6) encompasses full-length naturally occurring cytokines, fragments, or variants, such as functional variants (including fragments and functional variants of naturally occurring cytokines having at least 10%, 30%, 50%, or 80% of the activity, e.g., immunomodulatory activity). In some embodiments, the cytokine has an amino acid sequence encoded by a nucleotide sequence that is substantially identical to a naturally occurring cytokine (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) or substantially identical to a naturally occurring nucleotide sequence encoding the cytokine (e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity). In some embodiments, as understood from the context, the cytokine further comprises a receptor domain, such as a cytokine receptor domain (e.g., IL-15 / IL-15R).

[0161] The term “chimeric antigen receptor” or alternatively “CAR” refers to a recombinant polypeptide construct comprising a cytoplasmic signaling domain (also referred to herein as the “intracellular signaling domain”) which includes at least an extracellular antigen-binding domain, a transmembrane domain, and a functional signaling domain derived from an irritant molecule, as defined below. In some embodiments, the domains in the CAR polypeptide construct include, for example, a chimeric fusion protein within the same polypeptide chain. In some embodiments, the domains in the CAR polypeptide construct are not adjacent to each other and are provided, for example, within different polypeptide chains, for example, within an RCAR as described herein. In some embodiments, the CAR is a CCAR, for example, a CCAR disclosed herein.

[0162] In some embodiments, the cytoplasmic signaling domain includes a primary signaling domain (e.g., the primary signaling domain of CD3ζ). In some embodiments, the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In some embodiments, the co-stimulatory molecule is selected from 41BB (i.e., CD137), CD27, ICOS, and / or CD28. In some embodiments, the CAR includes a chimeric fusion protein containing an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain containing functional signaling domains derived from a stimulatory molecule. In some embodiments, the CAR includes a chimeric fusion protein containing an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain containing functional signaling domains derived from a co-stimulatory molecule and a stimulatory molecule. In some embodiments, the CAR includes a chimeric fusion protein containing an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain containing two functional signaling domains derived from one or more co-stimulatory molecules and a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein including an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain including at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR includes an optional leader sequence at the amino terminus (N terminus) of the CAR fusion protein. In some embodiments, the CAR further includes a leader sequence at the N terminus of the extracellular antigen recognition domain, which is optionally cleaved from the antigen recognition domain (e.g., scFv) during cell processing and localization of the CAR to the cell membrane.

[0163] A CAR containing an antigen-binding domain (e.g., scFv, single-domain antibody, or TCR (e.g., TCRα-binding domain or TCRβ-binding domain)) that targets a specific tumor antigen X (where X may be a tumor marker as described herein), such as those described herein, is also referred to as an XCAR. For example, a CAR containing an antigen-binding domain that targets BCMA is referred to as a BCMA CAR. A CAR can be expressed on any cell, such as immune effector cells as described herein (e.g., T cells or NK cells).

[0164] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within a cell to regulate cellular activity via a specific signaling pathway, either by generating second messengers or by acting as an effector in response to such messengers.

[0165] The term “antibody,” as used herein, refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins, and may be derived from natural or recombinant sources. Antibodies may be tetramers of immunoglobulin molecules.

[0166] The term "antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, and also to an antigen-binding domain sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen, such as the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, scFv antibody fragments, linear antibodies, single-domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and polyspecific antibodies formed from antibody fragments such as two or more fragments linked by disulfide crosslinks at the hinge region, such as two Fab fragments or two or more linked antibodies, such as a bivalent fragment containing two isolated CDRs or other epitope fragments. Antibody fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antibody fragments can also be grafted onto polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies). The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are closely linked by a short, mobile polypeptide linker, and the scFv can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, when used herein, scFv may have VL and VH variable regions in either order relative to, for example, the N-terminal and C-terminal ends of a polypeptide, and scFv may include VL-linker-VH or VH-linker-VL.

[0167] In some embodiments, scFv is NH2-V L-Linker-V H -COOH or NH2-V H -Linker-V L - May include the COOH structure.

[0168] As used herein, the terms “complementarity-determining region” or “CDR” refer to the amino acid sequence within the antibody variable region that gives antigen specificity and binding affinity. For example, generally, each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) has three CDRs, and each light chain variable region (LCDR1, LCDR2, and LCDR3) has three CDRs. The exact amino acid sequence boundary of a given CDR can be determined using one of many well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest”, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), or a combination thereof. In the numbering scheme combining Kabat and Chothia, in some embodiments, a CDR corresponds to an amino acid residue that is part of a Kabat CDR, part of a Chothia CDR, or both.

[0169] The portion of the CAR composition of this disclosure, comprising an antibody or an antibody fragment thereof, may exist in various forms, for example, wherein the antigen-binding domain is expressed as part of a polypeptide chain, including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), or, for example, a humanized or humanized antibody (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the antigen-binding domain of the CAR of this disclosure comprises an antibody fragment. In some embodiments, the CAR comprises an antibody fragment comprising an scFv.

[0170] As used herein, the terms “binding domain” or “antibody molecule” (also referred to herein as “antitarget binding domain”) refer to a protein, such as an immunoglobulin chain or a fragment thereof, that contains at least one immunoglobulin variable domain sequence. The terms “binding domain” or “antibody molecule” encompass antibodies and antibody fragments. In some embodiments, the antibody molecule is a multispecific antibody molecule, for example, which contains multiple immunoglobulin variable domain sequences, where a first of the multiple immunoglobulin variable domain sequences has binding specificity to a first epitope, and a second of the multiple immunoglobulin variable domain sequences has binding specificity to a second epitope. In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity to two or fewer antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope.

[0171] The terms "bispecific antibody" and "multiple bispecific antibodies" refer to a molecule that conjugates the antigen-binding sites of two antibodies within a single molecule. Therefore, a bispecific antibody can conjugate to two different antibodies simultaneously or sequentially. Methods for producing bispecific antibodies are known in the art. Various formats for conjugating two antibodies are also known in the art. Forms of bispecific antibodies in this disclosure include, but are not limited to, diabodies, single-chain antibodies, Fab dimers (Fab-Fab), Fab-scFv, and tandem antibodies, as are well known to those skilled in the art.

[0172] The term "antibody heavy chain" refers to the larger of the two polypeptide chains present in an antibody molecule in its naturally occurring conformation, and this usually determines the class to which the antibody belongs.

[0173] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformation. Kappa (κ) and lambda (λ) light chains are the two main antibody light chain isotypes.

[0174] The term "recombinant antibody" refers to antibodies created using recombinant DNA technology, such as antibodies expressed by bacteriophages or yeast expression systems. This term should also be interpreted as meaning an antibody created by the synthesis of a DNA molecule that encodes an antibody and expresses an antibody protein, or an amino acid sequence that specifies the antibody, and the DNA or amino acid sequence is obtained using well-known recombinant DNA or amino acid sequence technologies available in the art.

[0175] The terms “antigen” or “Ag” refer to a molecule that elicits an immune response. This immune response may include either antibody production or activation of specific immune-qualified cells, or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can be an antigen. Furthermore, antigens may originate from recombinant DNA or genomic DNA. Those skilled in the art will therefore understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response will encode an “antigen” as the term is used herein. Furthermore, those skilled in the art will understand that an antigen does not have to be encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent that this disclosure includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences may be sequenced in various combinations to encode a polypeptide that elicits a desired immune response. Furthermore, those skilled in the art will understand that an antigen does not have to be encoded by a “gene” at all. It will be readily apparent that antigens may be synthesized, obtained from biological samples, or may be macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or bodily fluids along with other biological components.

[0176] The terms “antitumor effect” and “antiancé effect” are used interchangeably and are not limited to, but refer to biological effects that may be manifested by a variety of means, including, for example, a reduction in tumor volume or cancer volume, a reduction in the number of tumor cells or cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in tumor cell proliferation or cancer cell proliferation, a reduction in tumor cell survival or cancer cell survival, or an improvement in a variety of physiological symptoms associated with cancerous conditions. “Antitumor effect” or “antiancé effect” may also first manifest by the ability of the peptides, polynucleotides, cells and antibodies of this disclosure to prevent the development of tumors or cancers.

[0177] The term "self" refers to any material originating from the same individual that will later be reintroduced into that individual.

[0178] The term "allogeneic" refers to any material originating from different animals of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic if they do not have identical genes at one or more loci. In some embodiments, allogeneic material from individuals of the same species may be genetically distinct enough to interact antigenically.

[0179] The term "heterogeneous" refers to grafts derived from animals of different species.

[0180] As used herein, the term "apheresis" refers to an in vitro procedure approved in the art, which involves collecting blood from a donor or patient, passing it through a device that separates specific components of a chosen type, and then returning the remainder to the donor or patient, for example, by retransfusion. Accordingly, in relation to "apheresis sample," it refers to a sample obtained using apheresis.

[0181] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Various examples of cancers described herein, but not limited to, include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer. In some embodiments, the cancers treated by the methods described herein include multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.

[0182] The terms “tumor” and “cancer” are used synonymously herein, and both terms, for example, encompass solid and liquid tumors, such as diffuse or circulating tumors. When used herein, the terms “cancer” or “tumor” include precancerous and malignant cancers and tumors.

[0183] "Derived from," as used herein, refers to the relationship between a first molecule and a second molecule. This generally refers to the structural similarity between the first and second molecules and does not imply or include limiting methods or sources relating to the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from the CD3ζ molecule, the intracellular signaling domain retains a sufficient CD3ζ structure to have the desired function, i.e., the ability to generate a signal under appropriate conditions. This does not imply or include limiting to a specific method of constructing the intracellular signaling domain, and does not mean, for example, that it is necessary to start with the CD3ζ sequence and delete or confer mutations to arrive at the intracellular signaling domain.

[0184] The term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding properties of the antibody or antibody fragment containing that amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody or antibody fragment of this disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative substitution is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Accordingly, one or more amino acid residues in a CAR of this disclosure can be replaced with other amino acid residues from the same side chain family, and the modified CAR can be tested using the functional assays described herein.

[0185] In relation to stimulation by stimuli and / or co-stimulatory molecules, the term “stimulus” refers to a response, such as a primary or secondary response, induced by the binding of a stimulant molecule (e.g., the TCR / CD3 complex) and / or a co-stimulatory molecule (e.g., C28 or 4-1BB) to its homologous ligand, thereby mediating signaling events, including, but not limited to, signaling via the TCR / CD3 complex. Stimuli can mediate changes in the expression of specific molecules and / or reorganization of the cytoskeleton structure, among other things.

[0186] The term “stimulating molecule” refers to a molecule expressed by T cells that provides a primary cytoplasmic signaling sequence that modulates primary activation of the TCR complex in at least some aspects of the T cell signaling pathway. In some embodiments, the ITAM-containing domain within the CAR replicates primary TCR signaling independently of the endogenous TCR complex. In some embodiments, the primary signal is initiated, for example, by the binding of the TCR / CD3 complex to a peptide-laden MHC molecule, which results in mediating T cell responses, including, but not limited to, proliferation, activation, and differentiation. Primary cytoplasmic signaling sequences that act in a stimulating manner (also referred to as “primary signaling domains”) may include an immunoreceptor tyrosine-based activation motif or a signaling motif known as an ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs that are particularly useful in this disclosure include, but are not limited to, those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI and CD66d, DAP10 and DAP12. In a specific CAR of this disclosure, the intracellular signaling domain of any one or more CARS of this disclosure includes an intracellular signaling sequence, such as the primary signaling sequence of CD3-ζ. The term "antigen-presenting cell" or "APC" refers to immune system cells (e.g., B cells, dendritic cells, etc.) such as accessory cells that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells can recognize these complexes using their T cell receptor (TCR). APCs process the antigen and present it to T cells.

[0187] The term "intracellular signaling domain" refers to the intracellular portion of a molecule when used herein. In embodiments, the intracellular signaling domain transduces effector functional signals, directing cells to perform specific functions. While the entire intracellular signaling domain can be used, it is often not necessary to use the entire chain. Within the scope of using cleaved portions of the intracellular signaling domain, such cleaved portions may be used in place of the complete chain, as long as they transmit effector functional signals. The term "intracellular signaling domain" is therefore intended to include any cleaved portion of the intracellular signaling domain sufficient to transmit effector functional signals.

[0188] The intracellular signaling domain generates signals that promote the immune effector function of CAR-containing cells, such as CART cells. Examples of immune effector function in CART cells include helper activity, including cytolytic activity and cytokine secretion.

[0189] In some embodiments, the intracellular signaling domain may include a primary intracellular signaling domain. Exemplary primary intracellular signaling domains may include those derived from molecules involved in the primary stimulus or antigen-dependent stimulus. In some embodiments, the intracellular signaling domain may include a co-stimulatory intracellular domain. Exemplary co-stimulatory intracellular signaling domains may include those derived from molecules involved in the co-stimulatory signal or antigen-independent stimulus. For example, in the case of CART, the primary intracellular signaling domain may include the cytoplasmic sequence of the T cell receptor, and the co-stimulatory intracellular signaling domain may include the cytoplasmic sequence from the co-receptor or co-stimulatory molecule.

[0190] The primary intracellular signaling domain may contain signaling motifs known as immunoreceptor-activated tyrosine motifs or ITAMs. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, CD66d, DAP10, and DAP12.

[0191] The terms "ζ" or alternatively "ζ chain," "CD3-ζ," or "TCR-ζ" refer to CD247. Swiss-Prot accession number P20963 provides an exemplary human CD3ζ amino acid sequence. "ζ stimulating domain" or alternatively "CD3-ζ stimulating domain" or "TCR-ζ stimulating domain" refer to the stimulating domain of CD3ζ or its variants (e.g., molecules having mutations, e.g., point mutations, fragments, insertions, or deletions). In some embodiments, the cytoplasmic domain of ζ includes residues 52-164 of GenBank accession number BAG36664.1 or its variants (e.g., molecules having mutations, e.g., point mutations, fragments, insertions, or deletions). In some embodiments, "ζ stimulating domain" or "CD3-ζ stimulating domain" is the sequence provided as Sequence ID No. 9 or 10 or its variants (e.g., molecules having mutations, e.g., point mutations, fragments, insertions, or deletions).

[0192] The term "costimulatory molecule" refers to a congenital binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response, including but not limited to T cell proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for an efficient immune response. Examples of costimulatory molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphoid activators (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, and CD S, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD1 9, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, IT GAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, T NFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160( This includes, but is not limited to, ligands that specifically bind to BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB, and CD83.

[0193] The co-stimulatory intracellular signaling domain refers to the intracellular portion of a co-stimulatory molecule.

[0194] An intracellular signaling domain may include the entire intracellular portion of the molecule from which it originates, the entire intrinsic intracellular signaling domain, or a functional fragment thereof.

[0195] The term "4-1BB" refers to CDR137 or tumor necrosis factor receptor superfamily member 9. Swiss-Prot accession number P20963 provides an exemplary human 4-1BB amino acid sequence. "4-1BB co-stimulatory domain" refers to the 4-1BB co-stimulatory domain or its variants (e.g., molecules having mutations, e.g., point mutations, fragments, insertions, or deletions). In some embodiments, the "4-1BB co-stimulatory domain" is the sequence provided as Sequence ID No. 7 or its variants (e.g., molecules having mutations, e.g., point mutations, fragments, insertions, or deletions).

[0196] When the term is used herein, “immune effector cells” refers to cells involved in promoting an immune response, such as an immune effector response. Examples of immune effector cells include T cells, such as α / βT cells and γ / δT cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytic cells.

[0197] When used herein, “immune effector function or immune effector response” refers to the function or response of, for example, immune effector cells that enhance or promote the immune attack of target cells. For example, immune effector function or response refers to the properties of T cells or NK cells that promote the death or inhibition of growth or proliferation of target cells. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector function or response.

[0198] The term "effector function" refers to a specialized function of a cell. The effector function of a T cell may include, for example, cytolytic activity or helper activity, including cytokine secretion.

[0199] The term “coding” refers to the inherent properties of a specific nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, and the biological properties it provides, which serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence. Thus, a gene, cDNA, or RNA codes for a protein when the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a transcriptional template for a gene or cDNA, can be said to code for a protein or other product of the cDNA of that gene.

[0200] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" includes all nucleotide sequences that encode the same amino acid sequence, including degenerate versions of each other. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns, insofar as the nucleotide sequence encoding that protein may contain one or more introns in any version.

[0201] The terms “effective dose” or “therapeutic dose” are used interchangeably herein and refer to the amount of a compound, formulation, material, or composition described herein that is effective in achieving a particular biological outcome.

[0202] The term "endogenous" refers to any substance produced from or within a living organism, cell, tissue, or system.

[0203] The term "exogenous" refers to any material introduced from or produced outside of an organism, cell, tissue, or system.

[0204] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence. In some embodiments, expression includes the translation of mRNA introduced into a cell.

[0205] The term "transfer vector" refers to a composition containing isolated nucleic acid that can be used for the delivery of isolated nucleic acid into a cell. In the art, many vectors are known, including, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "transfer vector" includes self-replicating plasmids or viruses. This term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral transfer vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and lentiviral vectors.

[0206] The term "expression vector" refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence operably ligated to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, with other expression elements being supplied by a host cell or an in vitro expression system. Expression vectors include all known in the art, including cosmids, plasmids (e.g., naked or liposome-containing) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0207] The term "lentivirus" refers to a genus of the family Retroviridae. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and they can deliver large amounts of genetic information to the host cell's DNA, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0208] The term "lentiviral vector" refers specifically to vectors derived from at least a portion of a lentiviral genome, including self-inactivating lentiviral vectors as provided in Milone et al., Mol.Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors available for clinical use include, but are not limited to, the LENTIVECTOR® gene delivery technology from Oxford BioMedica and the LENTIMAX® vector system from Lentigen. Non-clinical lentiviral vectors are also available and will be known to those skilled in the art.

[0209] The terms "homologous" or "identical" refer to the subunit sequence identity between two polymer molecules, between two nucleic acid molecules, or between two polypeptide molecules, such as two DNA molecules or two RNA molecules. When the subunit positions in both molecules are occupied by the same monomeric subunit, for example, when each position in two DNA molecules is occupied by adenine, they are homologous or identical at that position. Homologousity between two sequences is a direct function of the number of matching or homologous positions. For example, if half of the positions in two sequences (e.g., five positions in a polymer of 10 subunits) are homologous, then those two sequences are 50% homologous. If 90% of the positions (e.g., nine out of ten) are matching or homologous, then those two sequences are 90% homologous.

[0210] Humanized non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as the antibody's Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences) containing minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies and their antibody fragments are those in which residues from the recipient's complementarity-determining region (CDR) in a human immunoglobulin (recipient antibody or antibody fragment) are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, that possess the desired specificity, affinity, and capability. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments may contain residues not found in the recipient antibody or the transferred CDR or framework sequence. These modifications can further refine and optimize the performance of the antibody or antibody fragment. Generally, humanized antibodies or antibody fragments will contain substantially all of at least one and typically two variable domains, with all or substantially all of the CDR region corresponding to that of a non-human immunoglobulin, and all or most of the FR region being a human immunoglobulin sequence. Humanized antibodies or antibody fragments may also contain the immunoglobulin constant region (Fc), typically at least a portion of the Fc of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992.

[0211] "Completely human" refers to immunoglobulins such as antibodies or antibody fragments whose entire molecule is of human origin, or whose amino acid sequence is identical to that of human antibodies or immunoglobulins.

[0212] The term "isolated" means that something has been modified or removed from its natural state. For example, nucleic acids or peptides that are naturally present in living animals are not "isolated," but the same nucleic acids or peptides that have been partially or completely separated from the coexisting material in their natural state are "isolated." Isolated nucleic acids or proteins can exist in a substantially purified form or in a non-natural environment, such as in a host cell.

[0213] In connection with this disclosure, the following abbreviations are used for commonly existing nucleic acid bases: "A" refers to adenosine, "C" to cytosine, "G" to guanosine, "T" to thymidine, and "U" to uridine.

[0214] The terms "operably linked" or "transcriptional regulation" refer to a functional link between a regulatory sequence and a heterogeneous nucleic acid sequence that results in the expression of the latter. For example, when a first nucleic acid sequence is functionally related to a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, when a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Operafully linked DNA sequences can be contiguous and, for example, within the same reading frame when it is necessary to link two protein-coding regions together.

[0215] The term "parenteral" administration of immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, intratumoral, or infusion techniques.

[0216] The terms “nucleic acid,” “nucleic acid molecule,” “polypeptide,” or “polynucleotide molecule” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single-stranded or double-stranded forms, and polymers thereof. Unless specifically limited, the terms include nucleic acids, including known analogues of natural nucleotides, that have similar binding properties to the reference nucleic acid and are metabolized in the same way as naturally occurring nucleotides. In some embodiments, “nucleic acid,” “nucleic acid molecule,” “polypeptide,” or “polynucleotide molecule” include nucleotide / nucleoside derivatives or analogues. Unless otherwise indicated, a particular nucleic acid sequence implicitly includes its conservedly modified variants (e.g., degenerate codon substitutions, e.g., conserved substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions, such as conservative substitutions, can be achieved by constructing sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0217] The terms “peptide,” “polypeptide,” and “protein” are used synonymously and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can be included in a protein sequence or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, this term refers to both short chains, commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and longer chains of many types, commonly referred to in the art as proteins. “Polypeptides” particularly include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, or combinations thereof.

[0218] The term "promoter" refers to a DNA sequence recognized by a cellular synthetic mechanism or introduced synthetic mechanism, which is necessary to initiate the specific transcription of a polynucleotide sequence.

[0219] The term "promoter / regulatory sequence" refers to a nucleic acid sequence necessary for the expression of a gene product that is operably ligated to that promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, while in others, it may also include enhancer sequences and other regulatory elements necessary for the expression of the gene product. A promoter / regulatory sequence may, for example, be responsible for tissue-specific expression of a gene product.

[0220] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or designating a gene product, causes the cell to produce the gene product under most or all physiological conditions.

[0221] The term "inducible" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or designating a gene product, causes the production of a gene product in a cell only if a substantially equivalent inducer is present in the cell.

[0222] The term "tissue-specific" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or designated by a gene, causes the production of a gene product in a cell only if the cell is substantially the tissue type corresponding to the promoter.

[0223] The terms “cancer-associated antigen,” “tumor antigen,” “hyperproliferative disorder antigen,” and “antigen associated with hyperproliferative disorder” are interchangeable and refer to antigens common to a particular hyperproliferative disorder. In some embodiments, these terms refer to molecules (typically proteins, carbohydrates, or lipids) that are expressed on the surface of cancer cells, either completely or as fragments (e.g., MHC / peptides), and that are useful for preferential targeting of pharmacological agents against cancer cells. In some embodiments, a tumor antigen is a marker expressed by both normal and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, a tumor antigen is a cell surface molecule that is overexpressed in cancer cells compared to normal cells (e.g., 1x overexpression, 2x overexpression, 3x or more overexpression compared to normal cells). In some embodiments, a tumor antigen is a cell surface molecule that is improperly synthesized in cancer cells, e.g., a molecule with deletions, additions, or mutations compared to a molecule expressed on normal cells. In some embodiments, tumor antigens are expressed only on the surface of cancer cells, either completely or as fragments (e.g., MHC / peptides), and are not synthesized or expressed on the surface of normal cells. In some embodiments, the hyperproliferative disorder antigens of this disclosure are derived from primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and breast cancer, prostate cancer (e.g., castration-resistant or treatment-resistant prostate cancer or metastatic prostate cancer), ovarian cancer, pancreatic cancer, etc., or plasmacytoproliferative disorders, such as asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma), monoclonal gammaglobulinemia of unknown significance (MGUS), Waldenström macroglobulinemia, plasmacytoma (e.g., plasmacytoproliferative disorder, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma and multiple plasmacytoma), systemic light chain amyloidosis and POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome). In some embodiments, the CARs of this disclosure include a CAR comprising an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presenting peptide.Typically, peptides derived from endogenous proteins fit into the pocket of a major histocompatibility complex (MHC) class I molecule and are recognized by the T cell receptor (TCR) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes constitute a unique class of cell surface targets for immunotherapy. TCR-like antibodies targeting viral or tumor antigen-derived peptides in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, for example, Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibodies can be identified by library screening, such as a human scFv phage display library.

[0224] The term "tumor-supporting antigen" or "cancer-supporting antigen" interchangeably refers to molecules (typically proteins, carbohydrates, or lipids) that are expressed on the surface of non-cancerous cells but support cancer cells by, for example, promoting their proliferation or survival, or their resistance to immune cells. Exemplary cells of this type include stromal cells and myeloid-derived immunosuppressive cells (MDSCs). Tumor-supporting antigens themselves do not need to play a supporting role for cancer cells as long as the antigen is present on the cells supporting the cancer cells.

[0225] The term “mobile polypeptide linker” or “linker,” when used in relation to scFv, refers to a peptide linker consisting of amino acids, such as glycine and / or serine residues, used alone or in combination to link a variable heavy chain region and a variable light chain region together. In some embodiments, the mobile polypeptide linker is a Gly / Ser linker and includes the amino acid sequence (Gly-Gly-Gly-Ser)n (wherein n is a positive integer of 1 or more, e.g., n=1, n=2, n=3, n=4, n=5 and n=6, n=7, n=8, n=9 and n=10) (SEQ ID NO: 41). In some embodiments, the mobile polypeptide linker includes, but is not limited to, (Gly4 Ser)4 (SEQ ID NO: 27) or (Gly4 Ser)3 (SEQ ID NO: 28). In some embodiments, the linker includes multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO: 29). Furthermore, the linker described in International Publication No. 2012 / 138475 (incorporated herein by reference) is also included within the scope of this disclosure.

[0226] When used herein, the 5' cap (RNA cap, RNA7-methylguanosine cap or RNA m) 7 A 5' cap (also known as a G-cap) is a modified guanine nucleotide added to the "pre" or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of a terminal group that is linked to the first transcription nucleotide. Its presence is important for ribosome recognition and protection from RNases. Capping is linked to transcription and occurs synchronously, with each influencing the other. Immediately after transcription initiation, a cap synthesis complex associated with RNA polymerase binds to the 5' end of the synthesized mRNA. This enzyme complex catalyzes the chemical reactions necessary for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping region can be modified to adjust the function of the mRNA, such as its stability or translation efficiency.

[0227] As used herein, “in vitro transcription RNA” refers to RNA synthesized in vitro. In some embodiments, RNA is mRNA. Generally, in vitro transcription RNA is prepared from an in vitro transcription vector. An in vitro transcription vector contains a template used to prepare in vitro transcription RNA.

[0228] As used herein, "poly(A)" refers to a series of adenosines bound to mRNA by polyadenylation. In some embodiments of the construct for transient expression, poly(A) is 50 to 5000 (SEQ ID NO: 30). In some embodiments, poly(A) is greater than 64. In some embodiments, poly(A) is greater than 100. In some embodiments, poly(A) is greater than 300. In some embodiments, poly(A) is greater than 400. The poly(A) sequence can be chemically or enzymatically modified to modulate mRNA function such as localization, stability, or translation efficiency.

[0229] As used herein, “polyadenylation” refers to the covalent bonding of a polyadenylyl portion or a modified variant thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at their 3' end. The 3' poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the mRNA precursor by the action of the enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to the transcript containing a specific sequence, the polyadenylation signal. The poly(A) tail and the protein bound to it help protect mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, mRNA export from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription from DNA to RNA, but can also occur later in the cytoplasm. After transcription termination, the mRNA strand is cleaved by the action of endonuclease complexes associated with RNA polymerase. The cleavage site is usually characterized by the presence of the nucleotide sequence AAUAAA near the cleavage site. After the mRNA is cleaved, an adenosine residue is added to the free 3' end of the cleavage site.

[0230] As used herein, “transient” refers to the expression of a non-integrated transgene over a period of several hours, several days, or several weeks, the duration of which is shorter than the duration of gene expression when integrated into the genome in a host cell or contained in a stable plasmid replicon.

[0231] As used herein, the terms “to treat,” “treatment,” and “treating” refer to a reduction or improvement in the progression, severity, and / or duration of a proliferative disorder or improvement in one or more symptoms (preferably one or more recognizable symptoms) of a proliferative disorder, brought about by the administration of one or more therapies (e.g., one or more therapeutic agents such as CARs of this disclosure). In specific embodiments, the terms “to treat,” “treatment,” and “treating” refer to an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which is not necessarily recognizable to the patient. In other embodiments, the terms “to treat,” “treatment,” and “treating” refer to either or both physical inhibition of the progression of a proliferative disorder, such as stabilization of recognizable symptoms, or physiological inhibition, such as stabilization of a physical parameter. In other embodiments, the terms “to treat,” “treatment,” and “treating” refer to a reduction or stabilization of tumor size or the number of cancer cells.

[0232] The term "signaling pathway" refers to the biochemical relationships between various signaling molecules that play a role in the transmission of signals from one part of a cell to another. The term "cell surface receptor" includes molecules and molecular complexes that receive signals and have the ability to transmit those signals across the cell membrane.

[0233] The term "subject" is intended to include living organisms capable of eliciting an immune response (e.g., mammals, e.g., humans).

[0234] The term "substantially purified" refers to cells that essentially contain no other cell types. Substantially purified cells also refer to cells that are separated from the other cell types to which they normally bind in their natural state. In some examples, a substantially purified cell population refers to a homogeneous cell population. In other examples, the term simply refers to cells that are separated from the cells to which they naturally bind in their natural state. In some embodiments, these cells are cultured in vitro. In some embodiments, these cells are not cultured in vitro.

[0235] As used herein, the term "therapeutic" means a treatment. Therapeutic effects are achieved by reducing, suppressing, relieving, or eradicating a disease.

[0236] As used herein, the term "prevention" means the prevention of a disease or condition, or preventive measures.

[0237] The terms "transfected," "transformed," or "transduced" refer to the process of introducing or transferring exogenous nucleic acids into host cells. A "transfected," "transformed," or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acids. Cells include primary target cells and their offspring.

[0238] The term "specifically binding" refers to an antibody or ligand that recognizes and binds to cognitive-binding partner proteins (e.g., stimuli and / or co-stimuli molecules present in T cells) in the sample, but substantially does not recognize or bind to other molecules in the sample.

[0239] As used herein, the term “modulated chimeric antigen receptor (RCAR)” refers to a set of polypeptides, typically two polypeptides in the simplest embodiment, that, when present in immune effector cells, confer specificity to target cells, typically cancer cells, and intracellular signaling to the cells. In some embodiments, the RCAR includes at least one extracellular antigen-binding domain, a transmembrane domain, and a cell tumor signaling domain (also referred to herein as the “intracellular signaling domain”) comprising a functional signaling domain derived from stimulating and / or co-stimulating molecules as defined herein in relation to the CAR molecule. In some embodiments, these set of polypeptides in the RCAR are not adjacent to each other and are, for example, located in different polypeptide chains. In some embodiments, the RCAR includes a dimerization switch that, in the presence of a dimerizing molecule, can cause the polypeptides to bind to each other, for example, by binding the antigen-binding domain to the intracellular signaling domain. In some embodiments, the RCAR is expressed in cells described herein (e.g., immune effector cells), for example, RCAR-expressing cells (also referred to as “RCARX cells”). In some embodiments, the RCARX cells are T cells and are referred to as RCART cells. In some embodiments, RCARX is an NK cell and is referred to as an RCARN cell. RCAR can confer specificity and regulated intracellular signaling or proliferation to RCAR-expressing cells, which can optimize the immunoeffector properties of RCAR-expressing cells. In several embodiments, RCAR cells rely at least partially on the antigen-binding domain to obtain specificity to target cells containing an antigen bound to the antigen-binding domain.

[0240] When used herein, “membrane anchor” or “membrane tethering domain” refers to a polypeptide or moiety sufficient to bind an extracellular or intracellular domain to the plasma membrane, such as a myristoyl group.

[0241] When the term “switch domain” is used herein, for example, to refer to RCAR, it refers to an entity, typically a polypeptide-based entity, that binds to another switch domain in the presence of a dimerizing molecule. This binding results in a functional coupling between a first entity linked to, e.g., fused to, the first switch domain, and a second entity linked to, e.g., fused to, the second switch domain. The first and second switch domains are collectively called a dimerizing switch. In several embodiments, the first and second switch domains are identical to each other, e.g., both are polypeptides having the same primary amino acid sequence, and are collectively called a homodimerizing switch. In several embodiments, the first and second switch domains are different to each other, e.g., they are polypeptides having different primary amino acid sequences, and are collectively called a heterodimerizing switch. In several embodiments, the switch is intracellular. In several embodiments, the switch is extracellular. In several embodiments, the switch domain is a polypeptide-based entity, e.g., FKBP or FRB-based, and the dimerizing molecule is a small molecule, e.g., a rapalog. In several embodiments, the switch domain is a polypeptide-based entity, such as an scFv that binds to a myc peptide, and the dimerized molecule is a polypeptide, a fragment thereof, or a polypeptide polymer, such as a myc ligand or a polymer of myc ligands that bind to one or more myc scFvs. In several embodiments, the switch domain is a polypeptide-based entity, such as a myc receptor, and the dimerized molecule is an antibody or a fragment thereof, such as a myc antibody.

[0242] The term “dimerizing molecule” refers to a molecule that facilitates the binding of the first switch domain to the second switch domain, when used herein, for example, to refer to RCAR. In some embodiments, the dimerizing molecule is either not naturally present in the subject or is not present in concentrations that cause significant dimerization. In some embodiments, the dimerizing molecule is a small molecule, such as rapamycin or rapalog, such as RAD001.

[0243] The “low immunoenhancing dose” refers to a dose of an mTOR inhibitor that partially, but not completely, inhibits mTOR activity, as measured, for example, by inhibition of P70 S6 kinase activity, when used in conjunction with an mTOR inhibitor, such as an allosteric mTOR inhibitor, such as RAD001 or rapamycin, or a catalytic mTOR inhibitor. For example, a method for assessing mTOR activity by inhibition of P70 S6 kinase activity is detailed herein. This dose is insufficient to produce complete immunosuppression but sufficient to enhance the immune response. In some embodiments, a low immunoenhancing dose of an mTOR inhibitor results in a decrease in the number of PD-1 positive T cells and / or an increase in the number of PD-1 negative T cells or an increase in the PD-1 negative T cell / PD-1 positive T cell ratio. In some embodiments, a low immunoenhancing dose of an mTOR inhibitor results in an increase in the number of naive T cells. In some embodiments, a low immunoenhancing dose of an mTOR inhibitor is For example, the following marker in memory T cells, or memory T cell precursors: CD62L high CD127 high CD27 + and increased expression of one or more BCL2; For example, decreased expression of KLRG1 in memory T cells, such as memory T cell precursors; and For example, the following features: CD62L high Increase, CD127 high Increase, CD27 + An increase in the number of memory T cell precursors, characterized by an increase in one or a combination of the following: an increase in KLRG1, a decrease in KLRG1, and an increase in BCL2. This results in one or more of the aforementioned changes, where any of the aforementioned changes occur, for example, at least temporarily, compared to the untreated subject.

[0244] As used herein, "refractory" refers to a disease that does not respond to treatment, such as cancer. In some embodiments, refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, refractory cancer may become resistant during treatment. Refractory cancer is also referred to as resistant cancer.

[0245] As used herein, “relapsed” or “recurrence” refers to the recurrence or reappearance of signs and symptoms of a disease (e.g., cancer) or a disease such as cancer, after a period of improvement or response, following a previous treatment, for example, with a certain therapy (e.g., cancer treatment). The initial response period may include a decrease in the level of cancer cells to below a certain threshold, for example, less than 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Reappearance may include an increase in the level of cancer cells above a certain threshold, for example, more than 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, reappearance may include, for example, in relation to B-ALL, the reappearance of blasts after a complete response, for example, in the blood, bone marrow (>5%), or any extramedullary location. In this context, a complete response may include <5% BM blasts. More generally, in some embodiments, a response (e.g., a complete response or a partial response) may include the absence of detectable MRD (minimal residual disease). In one embodiment, the initial response period lasts for at least 1, 2, 3, 4, 5, or 6 days; at least 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10, or 12 months; or at least 1, 2, 3, 4, or 5 years.

[0246] Scope: Throughout this disclosure, various embodiments of the disclosure may be presented in the form of scope. It should be understood that descriptions in the form of scope are for convenience and conciseness only and should not be interpreted as a definitive limitation on the scope of the disclosure. Accordingly, a scope description should be understood to have all possible sub-scopes specifically disclosed and the individual numbers within those scopes. For example, a scope description such as 1-6 should be understood to have specifically disclosed sub-scopes such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6 and the individual numbers within those scopes, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a scope such as 95-99% identity includes those having 95%, 96%, 97%, 98%, or 99% identity, and includes sub-scopes such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the range width.

[0247] When used herein, the term "gene editing system" refers to a system, such as one or more molecules, that instructs and carries out the modification, such as deletion, of one or more nucleic acids at or near a site of genomic DNA targeted by the system. Gene editing systems are well known in the art and are described in more detail below.

[0248] "Administered in combination," as used herein, means that two or more different treatments are delivered to a subject during the course of the subject's suffering due to a disease, for example, after the subject has been diagnosed with a disease and before the disease is cured or eliminated, or before the treatment is discontinued for any other reason. In some embodiments, the delivery of one treatment is still ongoing when the delivery of a second treatment begins, thereby creating an overlap in administration. This may be referred to herein as "simultaneous" or "simultaneous delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatments are more effective when administered in combination. For example, the second treatment is more effective, for example, an equivalent effect can be achieved with fewer second treatments, or the second treatment alleviates symptoms to a greater extent than what would be seen if the second treatment were administered without the first treatment, or a similar situation to that seen with the first treatment. In some embodiments, the delivery is such that other parameters relating to symptom relief or impairment are higher than those observed when one is delivered without the other. The effects of the two treatments may be partially additive, fully additive, or more than additive. The delivery may be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered.

[0249] The terms “depletion” or “to deplete” as used interchangeably herein refer to a decrease or reduction in the level or amount of cells, proteins, or macromolecules in a sample after a process, such as a selection step, such as negative selection, has been carried out. Depletion may be complete or partial depletion of cells, proteins, or macromolecules. In some embodiments, depletion is a decrease or reduction of at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the level or amount of cells, proteins, or macromolecules in the sample before the process was carried out.

[0250] As used herein, “naive T cell” refers to an antigen-inexperienced T cell. In some embodiments, the antigen-inexperienced T cell has encountered its cognitive antigen in the thymus but not in the periphery. In some embodiments, the naive T cell is a precursor of memory T cells. In some embodiments, the naive T cell expresses both CD45RA and CCR7 but not CD45RO. In some embodiments, the naive T cell may be characterized by the expression of CD62L, CD27, CCR7, CD45RA, CD28, and CD127, and the absence of CD95 or CD45RO isoforms. In some embodiments, the naive T cell expresses CD62L, IL-7 receptor α, IL-6 receptor, and CD132, but not CD25, CD44, CD69, or CD45RO. In some embodiments, naive T cells express CD45RA, CCR7, and CD62L, but do not express CD95 or IL-2 receptor β. In some embodiments, the surface expression levels of the markers are evaluated using flow cytometry.

[0251] The term "central memory T cells" refers to a subset of T cells in humans that are CD45RO-positive and express CCR7. In some embodiments, central memory T cells express CD95. In some embodiments, central memory T cells express IL-2R, IL-7R, and / or IL-15R. In some embodiments, central memory T cells express CD45RO, CD95, IL-2 receptor β, CCR7, and CD62L. In some embodiments, the surface expression levels of markers are evaluated using flow cytometry.

[0252] The terms “stem memory T cells,” “stem cell memory T cells,” “stem cell-like memory T cells,” “memory stem T cells,” “T memory stem cells,” “T stem cell memory cells,” or “TSCM cells” refer to a subset of memory T cells that possess stem cell-like capabilities, such as the ability to regenerate and / or the pluripotency to reconstitute memory and / or effector T cell subsets. In some embodiments, stem memory T cells express CD45RA, CD95, IL-2 receptor β, CCR7, and CD62L. In some embodiments, the surface expression levels of markers are evaluated using flow cytometry. In some embodiments, exemplary stem memory T cells are disclosed in Gattinoni et al., Nat Med. 2017 January 06;23(1):18-27, the entirety of which is incorporated herein by reference.

[0253] For clarification purposes, unless otherwise noted, classifying cells or groups of cells as "not expressing," "not having," or "negative for" a particular marker does not necessarily mean the absence of the marker. Those skilled in the art can easily classify cells or groups of cells as not expressing or negative for a marker when cells have an expression level below a predetermined threshold, or when a group of cells has an overexpression level below a predetermined threshold, using conventional detection methods, such as flow cytometry, as described in the examples herein. For example, a typical gating strategy is shown in Figure 1. For example, CCR7-positive, CD45RO-negative cells are shown in the upper left quadrant of Figure 1G.

[0254] As used herein, the term "GeneSetScore (Up TEM vs. Down TSCM)" for a cell refers to a score that represents the degree to which the cell exhibits the effector memory T cell (TEM) phenotype compared to the stem cell memory T cell (TSCM) phenotype. A higher GeneSetScore (Up TEM vs. Down TSCM) indicates an increase in the TEM phenotype, while a lower GeneSetScore (Up TEM vs. Down TSCM) indicates an increase in the TSCM phenotype. In some embodiments, GeneSetScore (Up TEM vs. Down TSCM refers to one or more genes that are upregulated in TEM cells and / or downregulated in TSCM, such as MXRA7, CLIC1, NAT13, TBC1D2B, GLCCI1, DUSP10, APOBEC3D, CACNB3, ANXA2P2, TPRG1, EOMES, MATK, ARHGAP10, ADAM8, MAN1A1, SLFN12L, SH2D2A, EIF2C4, CD58, MYO1F, RA The GeneSetScore (Up TEM vs. Down TSCM) is determined by measuring the expression of one or more genes selected from the group consisting of B27B, ERN1, NPC1, NBEAL2, APOBEC3G, SYTL2, SLC4A4, PIK3AP1, PTGDR, MAF, PLEKHA5, ADRB2, PLXND1, GNAO1, THBS1, PPP2R2B, CYTH3, KLRF1, FLJ16686, AUTS2, PTPRM, GNLY, and GFPT2. In some embodiments, the GeneSetScore (Up TEM vs. Down TSCM) is determined for each cell using RNA-seq, such as single-cell RNA-seq (scRNA-seq), as illustrated in Example 10 with reference to Figure 39A, for example. In some embodiments, the GeneSetScore (Up TEM vs. Down TSCM) is calculated by obtaining the mean log-normalized gene expression values ​​for all genes in the gene set.

[0255] As used herein, the term "GeneSetScore (Up Treg vs. Down Teff)" of a cell refers to a score that represents the degree to which the cell exhibits regulatory T cell (Treg) characteristics compared to effector T cell (Teff) characteristics. A higher GeneSetScore (Up Treg vs. Down Teff) indicates an increase in the Treg phenotype, while a lower GeneSetScore (Up Treg vs. Down Teff) indicates an increase in the Teff phenotype. In some embodiments, the term GeneSetScore (Up Treg vs. Down Teff) Teff) refers to one or more genes that are upregulated in Treg cells and / or downregulated in Teff cells, e.g., C12orf75, SELPLG, SWAP70, RGS1, PRR11, SPATS2L, SPATS2L, TSHR, C14orf145, CASP8, SYT11, ACTN4, ANXA5, GLRX, HLA-DMB, PMCH, RAB11FIP1, IL32, FAM160B1, SHMT2, FRMD4B, CCR3, TNFRSF13B, NTNG2, CLDND1, BARD1, FCER1G, TYMS, ATP1B1, GJB6, FGL2, TK1, SLC2A8, CDKN2 A, SKAP2, GPR55, CDCA7, S100A4, GDPD5, PMAIP1, ACOT9, CEP55, SGMS1, ADPRH, AKAP2, HDAC9, IKZF4, CARD17, VAV 3, OBFC2A, ITGB1, CIITA, SETD7, HLA-DMA, CCR10, KIAA0101, SLC14A1, PTTG3P, DUSP10, FAM164A, PYHIN1, MYO1 F, SLC1A4, MYBL2, PTTG1, RRM2, TP53INP1, CCR5, ST8SIA6, TOX, BFSP2, ITPRIPL1, NCAPH, HLA-DPB2, SYT4, NINJ2 , FAM46C, CCR4, GBP5, C15orf53, LMCD1, MKI67, NUSAP1, PDE4A, E2F2, CD58, ARHGEF12, LOC100188949, FAS, HLA- DPB1, SELP, WEE1, HLA-DPA1, FCRL1, ICA1, CNTNAP1, OAS1, METTL7A, CCR6, HLA-DRB4, ANXA2P3, STAM, HLA-DQB2,The GeneSetScore (Up Treg vs. Down Teff) is determined by measuring the expression of one or more genes selected from the group consisting of LGALS1, ANXA2, PI16, DUSP4, LAYN, ANXA2P2, PTPLA, ANXA2P1, ZNF365, LAIR2, LOC541471, RASGRP4, BCAS1, UTS2, MIAT, PRDM1, SEMA3G, FAM129A, HPGD, NCF4, LGALS3, CEACAM4, JAKMIP1, TIGIT, HLA-DRA, IKZF2, HLA-DRB1, FANK1, RTKN2, TRIB1, FCRL3, and FOXP3. In some embodiments, the GeneSetScore (Up Treg vs. Down Teff) is determined using RNA-seq, for example, single-cell RNA-seq (scRNA-seq), as illustrated in Example 10 with reference to Figure 39B. In some embodiments, the GeneSetScore (Up Treg vs. Down Teff) is calculated by obtaining the average log-normalized gene expression values ​​for all genes in the gene set.

[0256] As used herein, the term "GeneSetScore(Down stemness)" of a cell refers to a score that represents the degree to which the cell exhibits a stem cell phenotype. A lower GeneSetScore(Down stemness) indicates an increase in the stem cell phenotype. In some embodiments, GeneSetScore(Down stemness) is measured by measuring the expression of one or more genes selected from the group consisting of, for example, ACE, BATF, CDK6, CHD2, ERCC2, HOXB4, MEOX1, SFRP1, SP7, SRF, TAL1, and XRCC5, which are downregulated in hematopoietic stem cells but upregulated in differentiated stem cells. In some embodiments, GeneSetScore(Down stemness) is determined using RNA-seq, for example, single-cell RNA-seq (scRNA-seq), as illustrated in Example 10 with reference to Figure 39C. In some embodiments, GeneSetScore(Down stemness) is calculated by obtaining the mean log-normalized gene expression values ​​of all genes in the gene set.

[0257] As used herein, the term "GeneSetScore(UP hypoxia)" of a cell refers to a score representing the degree to which the cell exhibits a hypoxic phenotype. A higher GeneSetScore(UP hypoxia) indicates an increase in the hypoxic phenotype. In some embodiments, GeneSetScore(UP hypoxia) is upregulated for one or more genes in a hypoxic cell, e.g., ABCB1, ACAT1, ADM, ADORA2B, AK2, AK3, ALDH1A1, ALDH1A3, ALDOA, ALDOC, ANGPT2, ANGPTL4, ANXA1, ANXA2, ANXA5, ARHGAP5, ARSE, ART1, BACE2, BATF3, BCL2L1, BCL2L2, BHLHE40, BHLHE41, BIK, BIRC2, BNIP3, B NIP3L, BPI, BTG1, C11orf2, C7orf68, CA12, CA9, CALD1, CCNG2, CCT6A, CD99, CDK1, CDKN1A, CDKN1B, CITED2, CLK1, CNOT7, COL4A5, COL5A1, COL5A2, COL5A3, CP, CTSD, CXCR4, D4S234E, DDIT3, DDIT4, 1-Dec, DKC1, DR1, EDN1, EDN2, EFNA1, EGF, EGR1, EIF4A3, ELF3, ELL2, ENG, ENO1, ENO3, ENPEP, EPO, ERRFI1, ETS1, F3, FABP5, FGF3, FKBP4, FLT1, FN1, FOS, FTL, GAPDH, GBE1, GLRX, GPI, GPRC5A, HA P1, HBP1, HDAC1, HDAC9, HERC3, HERPUD1, HGF, HIF1A, HK1, HK2, HLA-DQB1, HMOX1, HMOX2, HSPA5, HSPD1, HSPH1, HYOU1, ICAM1, ID2, IFI27, IGF2, IGFBP1, IGFBP2, IGFBP3, IGFBP5, IL6, IL8, INSIG1, IRF6, ITGA5, JUN, KDR, KRT14, KRT18, KRT19, LDHA, LDHB, LEP, LG ALS1, LONP1, LOX, LRP1, MAP4, MET, MIF, MMP13, MMP2, MMP7, MPI, MT1L, MTL3P, ​​MUC1, MXI1, NDRG1, NFIL3, NFKB1, NFKB2, NOS1, NOS2,NOS2P1, NOS2P2, NOS3, NR3C1, NR4A1, NT5E, ODC1, P4HA1, P4HA2, PAICS, PDGFB, PDK3, PFKFB1, PFKFB3, PFKFB4, PFKL, PGAM1, PGF, PGK1, PGK2, PGM1, PIM1, PIM2, PKM2, PLAU, PLAUR, PLIN2, P LOD2, PNN, PNP, POLM, PPARA, PPAT, PROK1, PSMA3, PSMD9, PTGS1, PTGS2, QSOX1, RBPJ, RELA, RIOK3, RNASEL, RPL36A, RRP9, SAT1, SERPINB2, SERPINE1, SGSM2, SIAH2, SIN3A, SIRPA, SLC16A1 This is determined by measuring the expression of one or more genes selected from the group consisting of SLC16A2, SLC20A1, SLC2A1, SLC2A3, SLC3A2, SLC6A10P, SLC6A16, SLC6A6, SLC6A8, SORL1, SPP1, SRSF6, SSSCA1, STC2, STRA13, SYT7, TBPL1, TCEAL1, TEK, TF, TFF3, TFRC, TGFA, TGFB1, TGFB3, TGFBI, TGM2, TH, THBS1, THBS2, TIMM17A, TNFAIP3, TP53, TPBG, TPD52, TPI1, TXN, TXNIP, UMPS, VEGFA, VEGFB, VEGFC, VIM, VPS11, and XRCC6. In some embodiments, the GeneSetScore(UP hypoxia) is determined using RNA-seq, such as single-cell RNA-seq (scRNA-seq), as illustrated in Example 10 with reference to Figure 39D. In some embodiments, the GeneSetScore(UP hypoxia) is calculated by obtaining the mean log-normalized gene expression values ​​for all genes in the gene set.

[0258] As used herein, the term “GeneSetScore(UP autophagy)” of a cell refers to a score representing the degree to which the cell exhibits an autophagy phenotype. A higher GeneSetScore(UP autophagy) indicates an increase in the autophagy phenotype. In some embodiments, GeneSetScore(UP autophagy) refers to one or more genes that are upregulated in a cell undergoing autophagy, such as ABL1, ACBD5, ACIN1, ACTRT1, ADAMTS7, AKR1E2, ALKBH5, ALPK1, AMBRA1, ANXA5, ANXA7, ARSB, ASB2, ATG10, ATG12, ATG13, ATG14, ATG16L1, ATG16L2, ATG2A, ATG2B, ATG3, ATG4A, ATG4B, ATG4C, ATG4D , ATG5, ATG7, ATG9A, ATG9B, ATP13A2, ATP1B1, ATPAF1-AS1, ATPIF1, BECN1, BECN1P1, BLOC1S1, BMP2KL, BNIP1, BNIP3, BOC, C11o rf2, C11orf41, C12orf44, C12orf5, C14orf133, C1orf210, C5, C6orf106, C7orf59, C7orf68, C8orf59, C9orf72, CA7, CALCB, CAL COCO2, CAPS, CCDC36, CD163L1, CD93, CDC37, CDKN2A, CHAF1B, CHMP2A, CHMP2B, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP6, CHST3, C ISD2, CLDN7, CLEC16A, CLN3, CLVS1, COX8A, CPA3, CRNKL1, CSPG5, CTSA, CTSB, CTSD, CXCR7, DAP, DKKL1, DNAAF2, DPF3, DRAM1, DRA M2, DYNLL1, DYNLL2, DZANK1, EI24, EIF2S1, EPG5, EPM2A, FABP1, FAM125A, FAM131B, FAM134B, FAM13B, FAM176A, FAM176B, FAM48 A, FANCC, FANCF, FANCL, FBXO7, FCGR3B, FGF14, FGF7, FGFBP1, FIS1, FNBP1L, FOXO1, FUNDC1, FUNDC2, FXR2, GABARAP, GABARAPL1,GABARAPL2、GABARAPL3、GABRA5、GDF5、GMIP、HAP1、HAPLN1、HBXIP、HCAR1、HDAC6、HGS、HIST1H3A、HIST1H3B、HIST1H3C、HIST1H3D、HIST1H3E、HIST1H3F 、HIST1H3G、HIST1H3H、HIST1H3I、HIST1H3J、HK2、HMGB1、HPR、HSF2BP、HSP90AA1、HSPA8、IFI16、IPPK、IRGM、IST1、ITGB4、ITPKC、KCNK3、KCNQ1、KIAA022 6、KIAA1324、KRCC1、KRT15、KRT73、LAMP1、LAMP2、LAMTOR1、LAMTOR2、LAMTOR3、LARP1B、LENG9、LGALS8、LIX1、LIX1L、LMCD1、LRRK2、LRSAM1、LSM4、MAP1A 、MAP1LC3A、MAP1LC3B、MAP1LC3B2、MAP1LC3C、MAP1S、MAP2K1、MAP3K12、MARK2、MBD5、MDH1、MEX3C、MFN1、MFN2、MLST8、MRPS10、MRPS2、MSTN、MTERFD1、MT MR14、MTMR3、MTOR、MTSS1、MYH11、MYLK、MYOM1、NBR1、NDUFB9、NEFM、NHLRC1、NME2、NPC1、NR2C2、NRBF2、NTHL1、NUP93、OBSCN、OPTN、P2RX5、PACS2、PARK2 、PARK7、PDK1、PDK4、PEX13、PEX3、PFKP、PGK2、PHF23、PHYHIP、PI4K2A、PIK3C3、PIK3CA、PIK3CB、PIK3R4、PINK1、PLEKHM1、PLOD2、PNPO、PPARGC1A、PPY、P RKAA1、PRKAA2、PRKAB1、PRKAB2、PRKAG1、PRKAG2、PRKAG3、PRKD2、PRKG1、PSEN1、PTPN22、RAB12、RAB1A、RAB1B、RAB23、RAB24、RAB33B、RAB39、RAB7A、RB1 CC1、RBM18、REEP2、REP15、RFWD3、RGS19、RHEB、RIMS3、RNF185、RNF41、RPS27A、RPTOR、RRAGA、RRAGB、RRAGC、RRAGD、S100A8、S100A9、SCN1A、SERPIN10、SESN2, SFRP4, SH3GLB1, SIRT2, SLC1A3, SLC1A4, SLC22A3, SLC25A19, SLC35B3, SLC35C1, SLC37A4, SLC6A1, SLCO1A2, SMURF1 , SNAP29, SNAPIN, SNF8, SNRPB, SNRPB2, SNRPD1, SNRPF, SNTG1, SNX14, SPATA18, SQSTM1, SRPX, STAM, STAM2, STAT2, STBD1, ST K11, STK32A, STOM, STX12, STX17, SUPT3H, TBC1D17, TBC1D25, TBC1D5, TCIRG1, TEAD4, TECPR1, TECPR2, TFEB, TM9SF1, TMBIM 6, TMEM203, TMEM208, TMEM39A, TMEM39B, TMEM59, TMEM74, TMEM93, TNIK, TOLLIP, TOMM20, TOMM22, TOMM40, TOMM5, TOMM6, TOM M7, TOMM70A, TP53INP1, TP53INP2, TRAPPC8, TREM1, TRIM17, TRIM5, TSG101, TXLNA, UBA52, UBB, UBC, UBQLN1, UBQLN2, UBQLN 4, ULK1, ULK2, ULK3, USP10, USP13, USP30, UVRAG, VAMP7, VAMP8, VDAC1, VMP1, VPS11, VPS16, VPS18, VPS25, VPS28, VPS33A, VP The GeneSetScore(UP autophagy) is determined by measuring the expression of one or more genes selected from the group consisting of S33B, VPS36, VPS37A, VPS37B, VPS37C, VPS37D, VPS39, VPS41, VPS4A, VPS4B, VTA1, VTI1A, VTI1B, WDFY3, WDR45, WDR45L, WIPI1, WIPI2, XBP1, YIPF1, ZCCHC17, ZFYVE1, ZKSCAN3, ZNF189, ZNF593, and ZNF681. In some embodiments, the GeneSetScore(UP autophagy) is determined using RNA-seq, for example, single-cell RNA-seq (scRNA-seq), as illustrated in Example 10 with reference to Figure 39E. In some embodiments, the GeneSetScore(UP autophagy) is determined by measuring the expression of one or more genes selected from the group consisting of S33B, VPS36, VPS37A, VPS37B, VPS37C, VPS37D, VPS39, VPS41, VPS4A, VPS4B, VTA1, VTI1A, VTI1B, WDFY3, WDR45, WDR45L, WIPI1, WIPI2, XBP1, YIPF1, ZCCHC17, ZFYVE1, ZKSCAN3, ZNF189, ZNF593, and ZNF681.It is calculated by obtaining the average log-normalized gene expression values ​​for all genes in the gene set.

[0259] As used herein, the term "GeneSetScore (Up resting vs. Down activated)" of a cell refers to a score representing the degree to which the cell exhibits a resting T cell phenotype versus an activated T cell phenotype. A higher GeneSetScore (Up resting vs. Down activated) indicates an increase in the resting T cell phenotype, while a lower GeneSetScore (Up resting vs. Down activated) indicates an increase in the activated T cell phenotype. In some embodiments, GeneSetScore (Up resting vs. Down activated) refers to one or more genes that are upregulated in resting T cells and / or downregulated in activated T cells, e.g., ABCA7, ABCF3, ACAP2, AMT, ANKH, ATF7IP2, ATG14, ATP1A1, ATXN7, ATXN7L3B, BCL7A, BEX4, BSDC1, BTG1, BTG2, BTN3A1, C11orf21, C19orf22, C21orf2, CAMK2G, CARS2, CCNL2, CD248, CD5, CD55, CEP164, CHKB, CLK1, CLK4, CTSL1, DBP, DCUN1D2, DENND1C, DGKD, DLG 1, DUSP1, EAPP, ECE1, ECHDC2, ERBB2IP, FAM117A, FAM134B, FAM134C, FAM169A, FAM190B, FAU, FLJ10038, FOXJ 2, FOXJ3, FOXL1, FOXO1, FXYD5, FYB, HLA-E, HSPA1L, HYAL2, ICAM2, IFIT5, IFITM1, IKBKB, IQSEC1, IRS4, KIAA 0664L3, KIAA0748, KLF3, KLF9, KRT18, LEF1, LINC00342, LIPA, LIPT1, LLGL2, LMBR1L, LPAR2, LTBP3, LYPD3, L ZTFL1, MAMBA, MAP2K6, MAP3K1, MARCH8, MAU2, MGEA5, MMP8, MPO, MSL1, MSL3, MYH3, MYLIP, NAGPA, NDST2, NISC H, NKTR, NLRP1, NOSIP, NPIP, NUMA1, PAIP2B, PAPD7, PBXIP1, PCIF1, PI4KA, PLCL2, PLEKHA1, PLEKHF2, PNISR,PPFIBP2, PRKCA, PRKCZ, PRKD3, PRMT2, PTP4A3, PXN, RASA2, RASA3, RASGRP2, RBM38, REPIN1, RNF3 8, RNF44, ROR1, RPL30, RPL32, RPLP1, RPS20, RPS24, RPS27, RPS6, RPS9, RXRA, RYK, SCAND2, SEMA4C , SETD1B, SETD6, SETX, SF3B1, SH2B1, SLC2A4RG, SLC35E2B, SLC46A3, SMAGP, SMARCE1, SMPD1, SNP H, SP140L, SPATA6, SPG7, SREK1IP1, SRSF5, STAT5B, SVIL, SYF2, SYNJ2BP, TAF1C, TBC1D4, TCF20, T This is determined by measuring the expression of one or more genes selected from the group consisting of ECTA, TES, TMEM127, TMEM159, TMEM30B, TMEM66, TMEM8B, TP53TG1, TPCN1, TRIM22, TRIM44, TSC1, TSC22D1, TSC22D3, TSPYL2, TTC9, TTN, UBE2G2, USP33, USP34, VAMP1, VILL, VIPR1, VPS13C, ZBED5, ZBTB25, ZBTB40, ZC3H3, ZFP161, ZFP36L1, ZFP36L2, ZHX2, ZMYM5, ZNF136, ZNF148, ZNF318, ZNF350, ZNF512B, ZNF609, ZNF652, ZNF83, ZNF862, and ZNF91. In some embodiments, the GeneSetScore (Up resting vs. Down activated) is determined using RNA-seq, such as single-cell RNA-seq (scRNA-seq), as illustrated in Example 10 with reference to Figure 38D. In some embodiments, the GeneSetScore (Up resting vs. Down activated) is calculated by obtaining the average log-normalized gene expression values ​​for all genes in the gene set.

[0260] As used herein, the "GeneSetScore (Progressively up in memory differentiation)" of a cell refers to a score representing the cell's stage in memory differentiation. A higher GeneSetScore (Progressively up in memory differentiation) indicates an increase in late memory T cell phenotypes, while a lower GeneSetScore (Progressively up in memory differentiation) indicates an increase in early memory T cell phenotypes. In some embodiments, GeneSetScore (UP autophagy) refers to one or more genes upregulated during memory differentiation, such as MTCH2, RAB6C, KIAA0195, SETD2, C2orf24, NRD1, GNA13, COPA, SELT, TNIP1, CBFA2T2, LRP10, PRKCI, BRE, ANKS1A, PNPLA6, ARL6IP1, WDFY1, MAPK1, GPR153, SHKBP1, MAP 1LC3B2, PIP4K2A, HCN3, GTPBP1, TLN1, C4orf34, KIF3B, TCIRG1, PPP3CA, ATG4D, TYMP, TRAF6, C17orf76, WI PF1, FAM108A1, MYL6, NRM, SPCS2, GGT3P, GALK1, CLIP4, ARL4C, YWHAQ, LPCAT4, ATG2A, IDS, TBC1D5, DMPK, S T6GALNAC6, REEP5, ABHD6, KIAA0247, EMB, TSEN54, SPIRE2, PIWIL4, ZSCAN22, ICAM1, CHD9, LPIN2, SETD8, Z C3H12A, ULBP3, IL15RA, HLA-DQB2, LCP1, CHP, RUNX3, TMEM43, REEP4, MEF2D, ABL1, TMEM39A, PCBP4, PLCD1, CHST12, RASGRP1, C1orf58, C11orf63, C6orf129, FHOD1, DKFZp434F142, PIK3CG, ITPR3, BTG3, C4orf50, CN NM3, IFI16, AK1, CDK2AP1, REL, BCL2L1, MVD, TTC39C, PLEKHA2, FKBP11, EML4, FANCA, CDCA4, FUCA2, MFSD10,TBCD、CAPN2、IQGAP1、CHST11、PIK3R1、MYO5A、KIR2DL3、DLG3、MXD4、RALGDS 、S1PR5、WSB2、CCR3、TIPARP、SP140、CD151、SOX13、KRTAP5-2、NF1、PEA15、P ARP8、RNF166、UEVLD、LIMK1、CACNB1、TMX4、SLC6A6、LBA1、SV2A、LLGL2、IRF 1、PPP2R5C、CD99、RAPGEF1、PPP4R1、OSBPL7、FOXP4、SLA2、TBC1D2B、ST7、JAZ F1、GGA2、PI4K2A、CD68、LPGAT1、STX11、ZAK、FAM160B1、RORA、C8orf80、APO BEC3F、TGFBI、DNAJC1、GPR114、LRP8、CD69、CMIP、NAT13、TGFB1、FLJ00049、A NTXR2、NR4A3、IL12RB1、NTNG2、RDX、MLLT4、GPRIN3、ADCY9、CD300A、SCD5、A BI3、PTPN22、LGALS1、SYTL3、BMPR1A、TBK1、PMAIP1、RASGEF1A、GCNT1、GABAR APL1, STOM, CALHM2, ABCA2, PPP1R16B, SYNE2, PAM, C12orf75, CLCF1, MXRA7, APOBEC3C, CLSTN3, ACOT9, HIP1, LAG3, TNFAIP3, DCBLD1, KLF6, CACNB3, RNF 19A、RAB27A、FADS3、DLG5、APOBEC3D、TNFRSF1B、ACTN4、TBKBP1、ATXN1、ARA P2、ARHGEF12、FAM53B、MAN1A1、FAM38A、PLXNC1、GRLF1、SRGN、HLA-DRB5、B4G ALT5、WIPI1、PTPRJ、SLFN11、DUSP2、ANXA5、AHNAK、NEO1、CLIC1、EIF2C4、MA P3K5、IL2RB、PLEKHG1、MYO6、GTDC1、EDARADD、GALM、TARP、ADAM8、MSC、HNRPL L、SYT11、ATP2B4、NHSL2、MATK、ARHGAP18、SLFN12L、SPATS2L、RAB27B、PIK3 R3、TP53INP1、MBOAT1、GYG1、KATNAL1、FAM46C、ZC3HAV1L、ANXA2P2、CTNNA1、NPC1, C3AR1, CRIM1, SH2D2A, ERN1, YPEL1, TBX21, SLC1A4, FASLG, PHACTR2, GALNT3, ADRB2, PIK3AP1, TLR3, PLEKHA5, DUSP10, GNAO1, PTGDR, FRMD4B, A NXA2, EOMES, CADM1, MAF, TPRG1, NBEAL2, PPP2R2B, PELO, SLC4A4, KLRF1, FOSL2, RGS2, TGFBR3, PRF1, MYO1F, GAB3, C17orf66, MICAL2, CYTH3, TOX, HLA -The GeneSetScore (Progressively up in memory differentiation) is determined by measuring the expression of one or more genes selected from the group consisting of DRA, SYNE1, WEE1, PYHIN1, F2R, PLD1, THBS1, CD58, FAS, NETO2, CXCR6, ST6GALNAC2, DUSP4, AUTS2, C1orf21, KLRG1, TNIP3, GZMA, PRR5L, PRDM1, ST8SIA6, PLXND1, PTPRM, GFPT2, MYBL1, SLAMF7, FLJ16686, GNLY, ZEB2, CST7, IL18RAP, CCL5, KLRD1, and KLRB1. In some embodiments, the GeneSetScore (Progressively up in memory differentiation) is determined using RNA-seq, for example, single-cell RNA-seq (scRNA-seq), as illustrated in Example 10 with reference to Figure 40B. In some embodiments, the GeneSetScore (Progressively up in memory differentiation) is calculated by obtaining the average log-normalized gene expression values ​​for all genes in the gene set.

[0261] As used herein, the term "GeneSetScore (Up TEM vs. Down TN)" for a cell refers to a score that represents the degree to which the cell exhibits the effector memory T cell (TEM) phenotype compared to the naive T cell (TN) phenotype. A higher GeneSetScore (Up TEM vs. Down TN) indicates an increase in the TEM phenotype, while a lower GeneSetScore (Up TEM vs. Down TN) indicates an increase in the TN phenotype.In some embodiments, GeneSetScore(Up TEM vs. Down TN) is one or more genes that are upregulated in TEM cells and / or downregulated in TN cells, e.g., MYO5A, MXD4, STK3, S1PR5, GLCCI1, CCR3, SOX13, KRTAP5-2, PEA15, PARP8, RNF166, UEVLD, LIMK1, SLC6A6, SV2A, KPNA2, OSBPL7, ST7, GGA2, PI4K2A, CD68, ZAK, RORA, TGFBI, DNAJC1, JOSD1, ZFYVE28, LRP8, OS BPL3, CMIP, NAT13, TGFB1, ANTXR2, NR4A3, RDX, ADCY9, CHN1, CD300A, SCD5, PTPN22, LGALS1, RASGEF1A, GCNT1, GLUL, ABCA2, CLDND1 , PAM, CLCF1, MXRA7, CLSTN3, ACOT9, METRNL, BMPR1A, LRIG1, APOBEC3G, CACNB3, RNF19A, RAB27A, FADS3, ACTN4, TBKBP1, FAM53B, MAN 1A1, FAM38A, GRLF1, B4GALT5, WIPI1, DUSP2, ANXA5, AHNAK, CLIC1, MAP3K5, ST8SIA1, TARP, ADAM8, MATK, SLFN12L, PIK3R3, FAM46C, ANXA2P2, CTNNA1, NPC1, SH2D2A, ERN1, YPEL1, TBX21, STOM, PHACTR2, GBP5, ADRB2, PIK3AP1, DUSP10, PTGDR, EOMES, MAF, TPRG1, NBE It is determined by measuring the expression of one or more genes selected from the group consisting of AL2, NCAPH, SLC4A4, FOSL2, RGS2, TGFBR3, MYO1F, C17orf66, CYTH3, WEE1, PYHIN1, F2R, THBS1, CD58, AUTS2, FAM129A, TNIP3, GZMA, PRR5L, PRDM1, PLXND1, PTPRM, GFPT2, MYBL1, SLAMF7, ZEB2, CST7, CCL5, GZMK, and KLRB1.In some embodiments, the GeneSetScore (Up TEM vs. Down TN) is determined using RNA-seq, such as single-cell RNA-seq (scRNA-seq), as illustrated in Example 10 with reference to Figure 40C. In some embodiments, the GeneSetScore (Up TEM vs. Down TN) is calculated by obtaining the mean log-normalized gene expression values ​​for all genes in the gene set.

[0262] In relation to the GeneSetScore value (e.g., the GeneSetScore median), when a positive GeneSetScore decreases by 100%, this value becomes 0. When a negative GeneSetScore increases by 100%, this value also becomes 0. For example, in Figure 39A, the GeneSetScore median for the day 1 sample is -0.084; the GeneSetScore median for the day 9 sample is 0.035; and the GeneSetScore median for the input sample is -0.1. In Figure 39A, a 100% increase in the input sample's GeneSetScore median results in a GeneSetScore value of 0; and a 200% increase in the input sample's GeneSetScore median results in a GeneSetScore value of 0.1. In Figure 39A, a 100% decrease in the median GeneSetScore of the Day 9 sample results in a GeneSetScore value of 0; a 200% decrease in the median GeneSetScore of the Day 9 sample results in a GeneSetScore value of -0.035.

[0263] As used herein, the term “beads” refers to individual particles having a solid surface with a size ranging from approximately 0.1 μm to several millimeters in diameter. Beads may be spherical (e.g., microspheres) or have an irregular shape. Beads may include a variety of materials, but are not limited to, paramagnetic materials, ceramics, plastics, glass, polystyrene, methylstyrene, acrylic polymers, titanium, latex, Sepharose®, cellulose, nylon, and the like. In some embodiments, the beads are relatively uniform, spherical, superparamagnetic polystyrene beads with a diameter of approximately 4.5 μm, coated, or bound, with a mixture of antibodies against, for example, CD3 (e.g., CD3ε) and CD28. In some embodiments, the beads are Dynabeads®. In some embodiments, both anti-CD3 and anti-CD28 antibodies are bound to the same bead to mimic the stimulation of T cells by antigen-presenting cells. The properties of Dynabeads® and their use for cell isolation and expansion are known in the art; see, for example, Neurauter et al., Cell isolation and expansion using Dynabeads, Adv Biochem Eng Biotechnol 2007;106:41-73, the entire contents of which are incorporated herein by reference.

[0264] As used herein, the term “nanomatrix” refers to a nanostructure comprising a matrix of mobile polymer chains. Nanomatrixes are 1 to 500 nm in size, for example, 10 to 200 nm. In some embodiments, the matrix of mobile polymer chains is bound to one or more agonists, e.g., agonist anti-CD3 and / or anti-CD28 antibodies, which provide an activation signal to T cells. In some embodiments, the nanomatrix comprises a covalently bonded colloidal polymer nanomatrix bound to agonists of one or more stimulating molecules and / or agonists of one or more co-stimulating molecules. In some embodiments, the agonists of one or more stimulating molecules are CD3 agonists (e.g., anti-CD3 agonist antibodies). In some embodiments, the agonists of one or more stimulating molecules are CD28 agonists (e.g., anti-CD28 agonist antibodies). In some embodiments, the nanomatrix is ​​characterized by the absence of solid surfaces as binding sites for agonists, such as anti-CD3 and / or anti-CD28 antibodies. In some embodiments, the nanomatrix is ​​a nanomatrix disclosed in International Publication No. 2014 / 048920A1 or a nanomatrix provided in the MACS® GMP T Cell TransAct® kit manufactured by Miltenyi Biotcc GmbH, the entire contents of which are incorporated herein by reference. MACS® GMP T Cell TransAct® consists of a colloidal polymer nanomatrix covalently bound to humanized recombinant agonist antibodies against human CD3 and CD28.

[0265] As used herein, "ubiquitination" refers to the addition of a ubiquitin molecule, for example, the addition of a single ubiquitin molecule (monoubiquitination) or the addition of two or more ubiquitin molecules (for example, a chain of ubiquitin molecules or polyubiquitination). Ubiquitination can be carried out by an enzymatic mechanism comprising one or more ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3).

[0266] As used herein, the term "CRBN" refers to a protein encoded in humans by the CRBN gene or a fragment or variant thereof (for example, an amino acid sequence substantially identical thereto, e.g., having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto). Swiss-Prot accession number Q96SW2 provides an exemplary human CRBN amino acid sequence.

[0267] As used herein, "IKZF polypeptide" refers to IKZF or its fragments or variants (for example, an amino acid sequence substantially identical thereto, for example, having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto).

[0268] As used herein, “IKZF3” refers to the protein encoded by the IKZF3 gene in humans. Swiss-Prot accession number Q9UKT9 provides an exemplary human IKZF3 amino acid sequence. An exemplary human IKZF3 amino acid sequence is provided in SEQ ID NO: 328. The term “IKZF3 polypeptide” refers to IKZF3 or its fragments or variants (e.g., amino acid sequences substantially identical thereto, e.g., having at least 85, 87, 90, 95, 97, 98, 99 or 100% identity thereto).

[0269] As used herein, “IKZF1” refers to the protein encoded by the IKZF1 gene in humans. Swiss-Prot accession number Q13422 provides an exemplary human IKZF1 amino acid sequence. An exemplary human IKZF1 amino acid sequence is provided in SEQ ID NO: 329. The term “IKZF1 polypeptide” refers to IKZF1 or its fragments or variants (for example, an amino acid sequence substantially identical thereto, e.g., having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto).

[0270] As used herein, “IKZF2” refers to the protein encoded by the IKZF2 gene in humans. Swiss-Prot accession number Q9UKS7 provides an exemplary human IKZF2 amino acid sequence. An exemplary human IKZF2 amino acid sequence is provided in SEQ ID NO: 330. The term “IKZF2 polypeptide” refers to IKZF2 or its fragments or variants (e.g., amino acid sequences substantially identical thereto, e.g., having at least 85, 87, 90, 95, 97, 98, 99 or 100% identity thereto).

[0271] As used herein, “IKZF4” refers to the protein encoded by the IKZF4 gene in humans. Swiss-Prot accession number Q9H2S9 provides an exemplary human IKZF4 amino acid sequence. An exemplary human IKZF4 amino acid sequence is provided in SEQ ID NO: 331. The term “IKZF4 polypeptide” refers to IKZF4 or its fragments or variants (for example, an amino acid sequence substantially identical thereto, e.g., having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto).

[0272] As used herein, “IKZF5” refers to the protein encoded by the IKZF5 gene in humans. Swiss-Prot accession number Q9H5V7 provides an exemplary human IKZF5 amino acid sequence. An exemplary human IKZF5 amino acid sequence is provided in SEQ ID NO: 332. The term “IKZF5 polypeptide” refers to IKZF5 or its fragments or variants (e.g., amino acid sequences substantially identical thereto, e.g., having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto).

[0273] As used herein, “fusion polypeptide” or “chimeric polypeptide” refers to a polypeptide comprising two or more heterogeneous amino acid sequences and / or protein domains in a single continuous polypeptide. In some embodiments, the two or more heterogeneous protein domains are linked directly or indirectly via linkers by covalent bonds.

[0274] As used herein, the term “estrogen receptor (ER)” refers to the protein encoded by the ESR1 gene in humans. Swiss-Prot accession number P03372 provides an exemplary human estrogen receptor (ER) amino acid sequence. “Estrogen receptor (ER) domain” refers to the estrogen receptor or a fragment or variant thereof (e.g., an amino acid sequence substantially identical thereto, e.g., having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto). Exemplary estrogen receptor (ER) domain amino acid sequences are provided in SEQ ID NOs: 340, 342, and 344. Exemplary estrogen receptor (ER) domain nucleotide sequences are provided in SEQ ID NOs: 341, 343, and 345.

[0275] As used herein, “FKB protein (FKBP) domain” refers to FKBP or its fragments or variants. An exemplary FKB protein (FKBP) domain amino acid sequence is provided in SEQ ID NO: 346.

[0276] As used herein, the term “dihydrofolate reductase (DHFR)” refers to the protein encoded by the DHFR gene in humans. Swiss-Prot accession number P00374 provides an exemplary human dihydrofolate reductase (DHFR) amino acid sequence. “Dihydrofolate reductase (DHFR) domain” refers to DHFR or its fragments or variants. An exemplary dihydrofolate reductase (DHFR) domain amino acid sequence is provided in SEQ ID NO: 347.

[0277] As used herein, the term “degradation domain” refers to a domain of a fusion polypeptide that adopts a stable conformation when expressed in the presence of a stabilizing compound. When expressed in a cell of interest, without a stable conformation, most degradation domains (and typically any protein to which they are fused) would be degraded by endogenous cellular mechanisms. In particular, degradation domains are not naturally occurring domains of a protein, but rather are engineered to be unstable without contact with a stabilizing compound. Thus, degradation domains can be identified by the following characteristics: (1) they are not naturally occurring; (2) their expression is regulated cotranslatically or posttranslatically by an increase or decrease in degradation rate; and (3) their degradation rate is substantially reduced in the presence of a stabilizing compound. In some embodiments, in the absence of a stabilizing compound, the degradation domain or other domains of a fusion polypeptide are substantially undetectable intracellularly or on the cell. In some embodiments, the degradation domain is destabilized in the absence of a stabilizing compound. In some embodiments, the degradation domain does not self-associate in the absence of a stabilizing compound, for example, it does not homodimerize. In some embodiments, the degradation domain is fused to a heteroprotease cleavage site, in which case cleavage of the heteroprotease cleavage site is more efficient in the presence of the stabilizing compound than in the absence of the stabilizing compound.

[0278] Decomposed domains are not aggregated domains as defined in PCT application number PCT / U.S. Patent Application Publication 2017 / 027778.

[0279] A "stabilization compound" or "stabilizing compound" refers to a compound that, when added to cells expressing a degradation domain, stabilizes the degradation domain and any protein fused to it, thereby reducing the rate of subsequent degradation. Stabilization compounds or stabilizing compounds may be naturally occurring or can be synthesized.

[0280] Furthermore, a "heterogeneous protease cleavage site" refers to a protease cleavage site that has a different origin from one or more protein domains to which it is fused (for example, one that is not naturally fused to at least one of other reference domains).

[0281] A "protease" is a protein that cuts another protein based on the presence of a cleavage site in the protein it intends to cleave.

[0282] "Intracellular protease" refers to a protease that is naturally expressed within the target cell.

[0283] "Extracellular protease" refers to a protease that is naturally expressed in an organism (e.g., a mammal) and is secreted or exposed outside the cell (e.g., in the blood or on the surface of the skin).

[0284] As used herein, the term “cleavage” refers to the cleavage of covalent bonds, such as within the backbone of a nucleic acid molecule or through hydrolysis of peptide bonds. Cleavage can be initiated in a variety of ways, including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand breaks are possible. Double-strand breaks can occur as a result of two different single-strand break events.

[0285] Definitions of specific functional groups and chemical terms are explained in more detail below. Chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Edition, and inside cover, and specific functional groups are generally defined as described therein. Furthermore, general principles of organic chemistry, as well as specific functional parts and reactivity, are described below: Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

[0286] As used herein, the term "alkyl" means a monovalent saturated, linear or branched hydrocarbon, for example, a linear or branched group having 1 to 12, 1 to 10, or 1 to 6 carbon atoms, respectively, as used herein. 12 Alkyl, C1-C 10 These are called alkyl and C1-C6 alkyl groups. Examples of alkyl groups, though not limited to them, include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, sec-pentyl, isopentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, and sec-hexyl.

[0287] As used herein, the terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groups that are similar in length and possible substitutions to the alkyl groups described above, but each contains at least one double or triple bond. Exemplary alkenyl groups include, but are not limited to, -CH=CH2 and -CH2CH=CH2.

[0288] As used herein, the term "alkoxy" refers to a linear or branched saturated hydrocarbon having 1 to 12 carbon atoms and containing a terminal "O" in the chain, such as -O(alkyl). Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, t-butoxy, or pentoxy groups.

[0289] As used herein, the term "aryl" refers to a monocyclic, bicyclic, or polycyclic hydrocarbon ring system in which at least one ring is aromatic. Typical aryl groups include complete aromatic ring systems, such as phenyl (e.g., (C6)aryl) and naphthyl (e.g., (C6)aryl). 10 )aryl) and anthracenyl (e.g., (C 14 Examples include ring systems in which an aryl group and an aromatic carbon ring are fused to one or more non-aromatic carbon rings, such as indanyl, phthalimidyl, naphthymidyl, or tetrahydronaphthyl.

[0290] As used herein, the term "carbocyclyl" refers to monocyclic, condensed, spirocondensed, and / or bridging bicyclic or polycyclic hydrocarbon ring systems containing 3 to 18 carbon atoms, where each ring is either fully saturated or contains one or more unsaturated units, but none of the rings are aromatic. Typical carbocyclyl groups include cycloalkyl groups (e.g., cyclopentyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.) and cycloalkenyl groups (e.g., cyclopentenyl, cyclohexenyl, cyclopentadienyl, etc.).

[0291] As used herein, the term "carbonyl" refers to -C=O.

[0292] As used herein, the term "cyano" refers to -CN.

[0293] As used herein, the terms "halo" or "halogen" refer to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iod, -I).

[0294] As used herein, the term "haloalkyl" refers to a monovalent saturated linear or branched alkyl chain in which at least one carbon atom in the chain is substituted with one or more halogen atoms. In some embodiments, a haloalkyl may contain, for example, 1 to 12, 1 to 10, or 1 to 6 carbon atoms, and as used herein, C1 to C 12 Haloalkyl, C1~C 10 These are called haloalkyls and C1-C6 haloalkyls. Examples of haloalkyls, though not limited to them, include trifluoromethyl, difluoromethyl, pentafluoroethyl, and trichloromethyl.

[0295] The term "haloalkoxy" refers to a linear or branched saturated hydrocarbon having 1 to 12 carbon atoms and containing a terminal "O" in the chain, where at least one carbon atom in the chain is substituted with one or more halogens. Examples of haloalkoxy groups, but not limited to, include trifluoromethoxy, difluoromethoxy, pentafluoroethoxy, and trichloromethoxy.

[0296] As used herein, the term "heteroalkyl" refers to a monovalent saturated linear or branched alkyl chain in which at least one carbon atom in the chain is substituted with a heteroatom such as O, S, or N, provided that in the case of substitution, the chain contains at least one carbon atom. In some embodiments, the heteroalkyl may contain, for example, 1 to 12, 1 to 10, or 1 to 6 carbon atoms, and as used herein, C1 to C 12 Heteroalkyl, C1~C 10These are called heteroalkyls and C1-C6 heteroalkyls. In certain cases, a heteroalkyl group contains one, two, three, or four independently selected heteroatoms instead of one, two, three, or four individual carbon atoms in the alkyl chain. Typical heteroalkyl groups include -CH2NHC(O)CH3, -CH2CH2OCH3, -CH2CH2NHCH3, and -CH2CH2N(CH3)CH3.

[0297] As used herein, the terms "alkylene," "alkenylene," "alkynylene," and "heteroalkylene" refer to the divalent groups of alkyl, alkenyl, alkynyl, or heteroalkyl groups, respectively. Any monovalent alkyl, alkenyl, alkynyl, or heteroalkyl group may be an alkylene, alkenylene, alkynylene, or heteroalkyl group obtained by removing the second hydrogen atom from the above alkyl, alkenyl, alkynyl, or heteroalkyl group.

[0298] As used herein, the term "heteroaryl" refers to a monocyclic, bicyclic, or polyc...

Claims

1. A first nucleic acid molecule encoding a controllable chimeric antigen receptor (CCAR), or A second nucleic acid molecule encoding a chimeric antigen receptor (CAR) and regulatory molecules. A method for producing a population of cells (e.g., T cells) that include, (i) The step of bringing a population of cells (e.g., T cells, e.g., T cells isolated from frozen or fresh leukocyte apheresis products) into contact with (e.g., bind to) a drug that stimulates the CD3 / TCR complex and / or a drug that stimulates costimulatory molecules on the surface of the cells; (ii) The steps of bringing a population of cells (e.g., T cells) into contact with a first nucleic acid molecule (e.g., a DNA or RNA molecule) encoding a CCAR or a second nucleic acid molecule (e.g., a DNA or RNA molecule) encoding a CAR and regulatory molecule, thereby providing a population of cells (e.g., T cells) containing the first or second nucleic acid molecule, and (iii) The step of collecting the population of cells (e.g., T cells) for preservation (e.g., by reformulating the population of cells in a cryopreservation medium) or administration. Includes, (a) Step (ii) is carried out together with Step (i), or within 20 hours after the start of Step (i), for example within 12, 13, 14, 15, 16, 17 or 18 hours after the start of Step (i), for example within 18 hours after the start of Step (i), and Step (iii) is to be carried out within 30 (e.g., 26) hours after the start of Step (i), for example, within 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours after the start of Step (i), for example, within 24 hours after the start of Step (i), (b) Step (ii) is carried out together with Step (i), or within 20 hours after the start of Step (i), for example within 12, 13, 14, 15, 16, 17 or 18 hours after the start of Step (i), for example within 18 hours after the start of Step (i), and Step (iii) is to be carried out within 30 hours after the start of Step (ii), for example, within 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours after the start of Step (ii), or (c) The population of cells from step (iii) is evaluated by the number of viable cells, for example, as not to have proliferated compared to the population of cells at the start of step (i), or has proliferated by 5, 10, 15, 20, 25, 30, 35, or 40%, for example, 10% or less. A method comprising, optionally, the first or second nucleic acid molecule in step (ii) being on a viral vector, optionally, the first or second nucleic acid molecule in step (ii) being an RNA molecule on a viral vector, and optionally, step (ii) transducing a population of cells (e.g., T cells) with a viral vector containing the first or second nucleic acid molecule.

2. The agent that stimulates the CD3 / TCR complex is a CD3-stimulating agent (e.g., an anti-CD3 antibody), and the agent that stimulates the co-stimulatory molecule is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof, and optionally, the agent that stimulates the CD3 / TCR complex or the agent that stimulates the co-stimulatory molecule may be an antibody (e.g., a single-domain antibody (e.g., a heavy-chain variable-domain antibody), a peptide body, a Fab fragment or scFv), a small molecule or a ligand (e.g., a naturally occurring ligand, a recombinant ligand, etc.). The method according to claim 1, wherein the agent is selected from (or a chimeric ligand), and optionally, the agent that stimulates the CD3 / TCR complex or the agent that stimulates the co-stimulatory molecule does not contain beads, optionally, the agent that stimulates the CD3 / TCR complex contains an anti-CD3 antibody, and the agent that stimulates the co-stimulatory molecule contains an anti-CD28 antibody, optionally, the agent that stimulates the CD3 / TCR complex contains an anti-CD3 antibody covalently bound to a colloidal polymer nanomatrix, and the agent that stimulates the co-stimulatory molecule contains an anti-CD28 antibody covalently bound to a colloidal polymer nanomatrix, and optionally, the agent that stimulates the CD3 / TCR complex and the agent that stimulates the co-stimulatory molecule contain T Cell TransAct®.

3. The method according to claim 1 or 2, wherein step (i) increases the percentage of cells containing the first or second nucleic acid molecule in the population of cells from step (iii), for example, the population of cells from step (iii) exhibits a higher percentage (e.g., at least 10, 20, 30, 40, 50, or 60%) of cells containing the first or second nucleic acid molecule compared to cells produced by the same method except without step (i).

4. (a) The percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells in the cell population from step (iii) is the same as the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells in the cell population at the start of step (i), or differs by only 5 or 10% or less; (b) The percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells in the cell population from step (iii) is increased by, for example, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3 times compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells in the cell population at the start of step (i); (c) The percentage of naive T cells containing the first or second nucleic acid molecule in the population of cells, for example, CD45RA+CD45RO-CCR7+ T cells containing the first or second nucleic acid molecule, increases during the period of step (ii), for example, by at least 30, 35, 40, 45, 50, 55, or 60% within 18 to 24 hours after the start of step (ii); or (d) The method according to any one of claims 1 to 3, wherein the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells in the cell population from step (iii) is not reduced, or is reduced by 5 or 10% or less, compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells in the cell population at the start of step (i).

5. (a) The population of cells from step (iii) shows a higher percentage (e.g., at least 10, 20, 30, or 40%) of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, compared to cells produced by the same method except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (b) The percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells in the population of cells from step (iii) is higher (e.g., at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3 times higher) than the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells in cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (c) The percentage of naive T cells containing the first or second nucleic acid molecule, e.g., CD45RA+CD45RO-CCR7+ T cells containing the first or second nucleic acid molecule, in the population of cells from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 times higher) than the percentage of naive T cells containing the first or second nucleic acid molecule, e.g., CD45RA+CD45RO-CCR7+ T cells containing the first or second nucleic acid molecule, in cells produced by the same method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i) (e.g., at least 4, 6, 8, 10, or 12 times higher); (d) The population of cells from step (iii) exhibits a higher percentage (e.g., at least 10, 20, 30, or 40%) of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, compared to cells produced by a similar method, except that the method further includes a step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, e.g., 5, 6, 7, 8, or 9 days, e.g., naive T cells; (e) The percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells in the population of cells from step (iii) is higher (e.g., at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3 times higher) in cells produced by the same method, except that the method further includes a step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, e.g., 5, 6, 7, 8, or 9 days, e.g., 3 times higher); or (f) The method according to any one of claims 1 to 4, wherein the percentage of naive T cells containing the first or second nucleic acid molecule, e.g., CD45RA+CD45RO-CCR7+ T cells containing the first or second nucleic acid molecule, in the population of cells from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 times higher) than the percentage of naive T cells containing the first or second nucleic acid molecule, e.g., CD45RA+CD45RO-CCR7+ T cells containing the first or second nucleic acid molecule, in cells produced by the same method except further comprising the step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii)

6. (a) The percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population from step (iii) is the same as the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population at the start of step (i), or differs by only 5 or 10% or less; (b) The percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the cell population from step (iii) is reduced by at least 20, 25, 30, 35, 40, 45, or 50% compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the cell population at the beginning of step (i); (c) The percentage of central memory T cells containing the first or second nucleic acid molecule, for example, CCR7+CD45RO+ cells containing the first or second nucleic acid molecule, decreases during the period of step (ii), for example, by at least 8, 10, 12, 14, 16, or 20% within 18 to 24 hours after the start of step (ii); or (d) The method according to any one of claims 1 to 5, wherein the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells in the cell population from step (iii) is not increased compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells in the cell population at the start of step (i), or increases by 5 or 10% or less.

7. (a) The population of cells from step (iii) shows a lower percentage (e.g., at least 10, 20, 30, or 40%) of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, compared to cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (b) The percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 20, 30, 40, or 50%) than the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (c) The percentage of central memory T cells containing the first or second nucleic acid molecule, e.g., CCR7+CD45RO+ T cells containing the first or second nucleic acid molecule, in the population of cells from step (iii) is lower (e.g., at least 10, 20, 30, or 40%) than the percentage of central memory T cells containing the first or second nucleic acid molecule, e.g., CCR7+CD45RO+ T cells containing the first or second nucleic acid molecule, in cells produced by the same method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i) (e.g., at least 10, 20, 30, or 40%); (d) The population of cells from step (iii) exhibits a lower percentage (e.g., at least 10, 20, 30, or 40%) of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, compared to cells produced by a similar method, except that the method further includes a step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after and before step (iii); (e) The percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells in the population of cells from step (iii) is lower (e.g., at least 20, 30, 40, or 50%) than the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells in cells produced by the same method, except that the method further includes a step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, e.g., 5, 6, 7, 8, or 9 days, e.g., 5, 6, 7, 8, or 9 days, e.g., 20, 30, 40, or 50%) lower than the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells in cells produced by the same method, except that the method further includes a step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, e.g., 5, 6, 7, 8, or 9 days, e.g., 20, 30, 40, or 50%) lower than the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells in the population of cells from step (iii) is lower than the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells in cells produced by the same method, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells in the population of cells from step (iii) is lower than the percentage of central memory cells, (f) The method according to any one of claims 1 to 6, wherein the percentage of central memory T cells containing the first or second nucleic acid molecule, e.g., CCR7+CD45RO+ T cells containing the first or second nucleic acid molecule, in the population of cells from step (iii) is lower (e.g., at least 10, 20, 30, or 40%) than the percentage of central memory T cells containing the first or second nucleic acid molecule, e.g., CCR7+CD45RO+ T cells containing the first or second nucleic acid molecule, in cells produced by the same method except further comprising the step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii) (e.g., at least 10, 20, 30, or 40%).

8. (a) Is the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the cell population from step (iii) increased compared to the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the cell population at the start of step (i)? (b) The percentage of stem memory T cells containing the first or second nucleic acid molecule in the population of cells from step (iii) is increased compared to the percentage of stem memory T cells containing the first or second nucleic acid molecule in the population of cells at the start of step (i), for example, CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells containing the first or second nucleic acid molecule; (c) The percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the population of cells from step (iii) is higher than the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), for example more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or (d) The percentage of stem memory T cells containing the first or second nucleic acid molecule, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells containing the first or second nucleic acid molecule, in the population of cells from step (iii) is higher than the percentage of stem memory T cells containing the first or second nucleic acid molecule, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells containing the first or second nucleic acid molecule, in cells produced by the same method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); (e) The percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the population of cells from step (iii) is higher than the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in cells produced by the same method, except that the step further includes growing the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii); or (f) The method according to any one of claims 1 to 7, wherein the percentage of stem memory T cells containing the first or second nucleic acid molecule, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells containing the first or second nucleic acid molecule, in the population of cells from step (iii) is higher than the percentage of stem memory T cells containing the first or second nucleic acid molecule, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells containing the first or second nucleic acid molecule, in cells produced by the same method except further comprising the step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

9. (a) The GeneSetScore median (Up TEM vs. Down TSCM) of the cell population from step (iii) is approximately the same as the GeneSetScore median (Up TEM vs. Down TSCM) of the cell population at the start of step (i), or differs by only about 25, 50, 75, 100, or 125% or less (for example, increased by only less than or equal to that); (b) The GeneSetScore median (Up TEM vs. Down TSCM) of the cell population from step (iii) is: Cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i), or Cells produced by the same method, except that the step after step (ii) and before step (iii) further includes growing the population of cells (e.g., T cells) in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days. Lower than the GeneSetScore median (Up TEM vs. Down TSCM) (for example, at least 100, 150, 200, 250, or 300% lower); (c) The GeneSetScore median (Up Treg vs. Down Teff) of the cell population from step (iii) is approximately the same as the GeneSetScore median (Up Treg vs. Down Teff) of the cell population at the start of step (i), or differs by only about 25, 50, 100, 150, or 200% or less (for example, increased by only less than or equal to that); (d) The GeneSetScore median (Up Treg vs. Down Teff) of the cell population from step (iii) is: Cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i), or Cells produced by the same method, except that the step after step (ii) and before step (iii) further includes growing the population of cells (e.g., T cells) in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days. It is lower than the GeneSetScore median (Up Treg vs. Down Teff) (for example, at least 50, 100, 125, 150, or 175% lower); (e) The GeneSetScore median (Down stemness) of the cell population from step (iii) is approximately the same as the GeneSetScore median (Down stemness) of the cell population at the start of step (i), or differs by only about 25, 50, 100, 150, 200, or 250% or less (for example, increased by only less than or equal to that); (f) The GeneSetScore median (Down stemness) of the cell population from step (iii) is: Cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i), or Cells produced by the same method, except that the step after step (ii) and before step (iii) further includes growing the population of cells (e.g., T cells) in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days. It is lower than the GeneSetScore median (Down Stress) (for example, at least about 50, 100, or 125% lower); (g) The GeneSetScore median (Up hypoxia) of the cell population from step (iii) is approximately the same as the GeneSetScore median (Up hypoxia) of the cell population at the start of step (i), or differs by only about 125, 150, 175, or 200% or less (for example, increased by only that amount); (h) The GeneSetScore median (Up hypoxia) of the cell population from step (iii) is: Cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i), or Cells produced by the same method, except that the step after step (ii) and before step (iii) further includes growing the population of cells (e.g., T cells) in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days. It is lower than the GeneSetScore median (Up hypoxia) (for example, at least 40, 50, 60, 70, or 80% lower); (j) The GeneSetScore median (Up autophgy) of the cell population from step (iii) is approximately the same as the GeneSetScore median (Up autophgy) of the cell population at the start of step (i), or differs by only about 180, 190, 200, or 210% or less (for example, increased by only that much); or (k) The GeneSetScore median (Up autophgy) of the cell population from step (iii) is: Cells produced by the same method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i), or Cells produced by the same method, except that the step after step (ii) and before step (iii) further includes growing the population of cells (e.g., T cells) in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days. The method according to any one of claims 1 to 8, wherein the GeneSetScore median (Up autophgy) is lower (for example, at least about 20, 30, or 40%).

10. The method according to any one of claims 1 to 9, wherein the population of cells from step (iii) is incubated with cells expressing the antigen recognized by the CCAR or CAR, and then secretes IL-2 at a higher level (e.g., at least 2, 4, 6, 8, 10, 12, or 14 times higher) than cells produced by the same method except that step (iii) is performed more than 26 hours after the start of step (i), for example more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), or cells produced by the same method except that the step further includes a step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, for example 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

11. The method according to any one of claims 1 to 10, wherein the population of cells from step (iii), after being administered in vivo, lasts longer or proliferates at a higher level than cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), or compared to cells produced by a similar method, except that it further includes a step of proliferating the population of cells (e.g., T cells) in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days after step (ii) and before step (iii) (for example, as evaluated using the method described in Example 1 with respect to Figure 4C).

12. The population of cells from step (iii) is administered in vivo, and step (iii) is performed more than 26 hours after the start of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), except that the cells are produced by the same method or more potent antitumor activity than cells produced by the same method, except that step (iii) is further performed after and before step (ii) to proliferate the population of cells (e.g., T cells) in vitro for more than 3 days, for example, 5, 6, 7, 8, or 9 days. ６ , 0.2 × 10 ６ , 0.25 × 10 ６ or 0.3 × 10 ６ The method according to any one of claims 1 to 11, which exhibits more potent antitumor activity in viable cells containing the first or second nucleic acid molecule at doses of one or fewer units.

13. The method according to any one of claims 1 to 12, wherein the population of cells from step (iii) is not proliferated compared to the population of cells at the start of step (i), for example, by the number of viable cells, or is proliferated by 5, 10, 15, 20, 25, 30, 35, or 40% or less, and optionally, the number of viable cells in the population of cells from step (iii) is reduced from the number of viable cells in the population of cells at the start of step (i).

14. The method according to any one of claims 1 to 13, wherein the population of cells from step (iii) is not grown compared to the population of cells at the start of step (i), or is grown for less than 2 hours, for example, less than 1 or 1.5 hours.

15. The method according to any one of claims 1 to 14, wherein step (i) and / or (ii) is carried out in a cell medium (e.g., serum-free medium) containing IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-7, IL-21, IL-6 (e.g., IL-6 / sIL-6Ra), an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof.

16. The method according to any one of claims 1 to 15, wherein step (i) and / or (ii) is carried out in a serum-free cell medium containing a serum substitute.

17. The method according to claim 16, wherein the serum substitute is CTS® Immune Cell Serum Replacement (ICSR).

18. Before step (i), (iv) (Optional) A step of receiving fresh leukocyte apheresis products (or alternative sources of hematopoietic tissue such as fresh whole blood products, fresh bone marrow products, or fresh tumor or organ biopsy or excision (e.g., fresh products from thymectomy)) from an entity, e.g., a laboratory, a hospital or a healthcare provider, and (v) The step of isolating the population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) that were in contact in step (i) from a fresh leukocyte apheresis product (or an alternative source of hematopoietic tissue such as fresh whole blood product, fresh bone marrow product, or fresh tumor or organ biopsy or excision (e.g., fresh product from thymectomy)). It further includes, optionally, Step (iii) is to be carried out within 35 hours after the start of Step (v), for example, within 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours after the start of Step (v), for example, within 30 hours after the start of Step (v), or The method according to any one of claims 1 to 17, wherein the population of cells from step (iii) is not proliferated, or is proliferated by 5, 10, 15, 20, 25, 30, 35, or 40%, for example, 10%, compared to the population of cells at the end of step (v).

19. The method according to any one of claims 1 to 17, further comprising the step of receiving, prior to step (i), cryopreserved T cells isolated from leukocyte apheresis products (or alternative sources of hematopoietic tissue such as cryopreserved T cells isolated from whole blood, bone marrow, or tumor or organ biopsy or excision (e.g., thymectomy)) from an entity, for example, a laboratory, a hospital or a healthcare provider.

20. Before step (i), (iv) (Optional) A step of receiving cryopreserved leukocyte apheresis products (or alternative sources of hematopoietic tissue such as cryopreserved whole blood products, cryopreserved bone marrow products, or cryopreserved tumor or organ biopsy or excision (e.g., cryopreserved products from thymectomy)) from an entity, e.g., a laboratory, a hospital or a healthcare provider, and (v) The step of isolating a population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) that were in contact in step (i) from cryopreserved leukocyte apheresis products (or alternative sources of hematopoietic tissue such as cryopreserved whole blood products, cryopreserved bone marrow products, or cryopreserved tumor or organ biopsy or excision (e.g., cryopreserved products from thymectomy)). It further includes, optionally, Step (iii) is to be carried out within 35 hours after the start of Step (v), for example, within 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours after the start of Step (v), for example, within 30 hours after the start of Step (v), or The method according to any one of claims 1 to 17, wherein the population of cells from step (iii) is not proliferated, or is proliferated by 5, 10, 15, 20, 25, 30, 35, or 40%, for example, 10%, compared to the population of cells at the end of step (v).

21. Step (vi): Culturing a portion of the cell population from step (iii) for at least 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 6, 6.5, or 7 days, for example, at least 2 days and no more than 7 days, and measuring the CAR (e.g., CCAR) expression level in the portion (for example, measuring the percentage of viable CAR-expressing cells (e.g., CCAR-expressing cells) in the portion). It further includes, optionally, The method according to any one of claims 1 to 20, wherein step (iii) comprises collecting and freezing a population of cells (e.g., T cells), and step (vi) comprises thawing a portion of the population of cells from step (iii), culturing the portion for at least 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 6, 6.5, or 7 days, for example, at least 2 days and no more than 7 days, and measuring the CAR (e.g., CCAR) expression level in the portion (e.g., measuring the percentage of viable CAR-expressing cells (e.g., CCAR-expressing cells) in the portion).

22. A first nucleic acid molecule encoding a controllable chimeric antigen receptor (CCAR), or A second nucleic acid molecule encoding a chimeric antigen receptor (CAR) and regulatory molecules. A method for producing a population of cells (e.g., T cells) that include, (1) A step of contacting a population of cells (e.g., T cells, e.g., T cells isolated from frozen leukocyte apheresis products) with a cytokine selected from IL-2, IL-7, IL-15, IL-21, IL-6, or a combination thereof, (2) The steps of bringing a population of cells (e.g., T cells) into contact with a first nucleic acid molecule encoding CCAR (e.g., a DNA or RNA molecule) or a second nucleic acid molecule encoding CAR and regulatory molecules (e.g., a DNA or RNA molecule), thereby providing a population of cells (e.g., T cells) containing the first or second nucleic acid molecule, and (3) Step of collecting the cell population (e.g., T cells) for preservation (e.g., by reformulating the cell population in a cryopreservation medium) or administration. Includes, (a) Step (2) is carried out together with Step (1), or within 5 hours after the start of Step (1), for example, within 1, 2, 3, 4, or 5 hours after the start of Step (1), and Step (3) is carried out within 26 hours after the start of Step (1), for example within 22, 23, or 24 hours after the start of Step (1), for example within 24 hours after the start of Step (1), or (b) The population of cells from step (3) is evaluated by the number of viable cells as being not proliferated compared to the population of cells at the start of step (1), for example, by 5, 10, 15, 20, 25, 30, 35 or 40%, for example 10% or less. A method comprising, optionally, the first or second nucleic acid molecule in step (2) being on a viral vector, optionally, the first or second nucleic acid molecule in step (ii) being an RNA molecule on a viral vector, and optionally, step (ii) transducing a population of cells (e.g., T cells) with a viral vector containing the first or second nucleic acid molecule.

23. The method according to claim 22, wherein step (1) includes bringing the population of cells (e.g., T cells) into contact with IL-2.

24. The method according to claim 22, wherein step (1) includes bringing the population of cells (e.g., T cells) into contact with IL-7.

25. The method according to claim 22, wherein step (1) comprises bringing the population of cells (e.g., T cells) into contact with IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)).

26. The method according to claim 22, wherein step (1) includes bringing the population of cells (e.g., T cells) into contact with IL-21.

27. The method according to claim 22, wherein step (1) comprises bringing the population of cells (e.g., T cells) into contact with IL-6 (e.g., IL-6 / sIL-6Ra).

28. The method according to claim 22, wherein step (1) is to bring the population of cells (e.g., T cells) into contact with IL-7 and IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)).

29. The method according to claim 22, wherein step (1) includes bringing the population of cells (e.g., T cells) into contact with IL-7 and IL-21.

30. The method according to claim 22, wherein step (1) comprises bringing the population of cells (e.g., T cells) into contact with IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)) and IL-21.

31. The method according to claim 22, wherein step (1) comprises bringing the population of cells (e.g., T cells) into contact with IL-7, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)) and IL-21.

32. The method according to claim 22, wherein step (1) comprises bringing a population of cells (e.g., T cells) into contact with IL-6 (e.g., IL-6 / sIL-6Ra) and IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)).

33. The method according to claim 22, wherein step (1) comprises bringing the population of cells (e.g., T cells) into contact with IL-2 and IL-6 (e.g., IL-6 / sIL-6Ra).

34. The method according to any one of claims 22 to 33, wherein the population of cells from step (3) exhibits a higher percentage (e.g., at least 10, 15, 20, 25, 30, 35, or 40%) of naive cells among the cells containing the first or second nucleic acid molecule, compared to cells produced by a similar method, except further comprising the step of contacting the population of cells with, for example, an anti-CD3 antibody.

35. The percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the population of cells from step (3) is: (a) The percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells in the cell population at the start of step (1) is the same as, or differs by only 5 or 10% or less, or (b) The method according to any one of claims 22 to 34, wherein the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells in the cell population at the start of step (1) is increased, e.g., by at least 10 or 20%.

36. The method according to any one of claims 22 to 35, wherein the population of cells from step (3) exhibits a higher percentage (e.g., at least 10, 20, 30, or 40%) of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, compared to cells produced by a similar method, except that step (3) is performed more than 26 hours after the start of step (1), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (1).

37. The method according to any one of claims 22 to 36, wherein the population of cells from step (3) exhibits a higher percentage (e.g., at least 10, 20, 30, or 40%) of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, compared to cells produced by a similar method, except that the method further includes a step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, for example 5, 6, 7, 8, or 9 days, after step (2) and before step (3).

38. The method according to any one of claims 22 to 37, wherein the population of cells from step (3), after being administered in vivo, persists longer or proliferates at a higher level than cells produced by a similar method, except that step (3) is performed more than 26 hours after the start of step (1), for example, more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (1) (as evaluated, for example, using the method described in Example 1 with respect to Figure 4C).

39. The method according to any one of claims 22 to 38, wherein the population of cells from step (3) persists longer or proliferates at a higher level than cells produced by a similar method, except that the method further includes a step of growing the population of cells (e.g., T cells) in vitro for more than 3 days, for example 5, 6, 7, 8, or 9 days, after administration in vivo, after step (2) and before step (3).

40. The method according to any one of claims 22 to 39, wherein the population of cells from step (3) is not proliferated compared to the population of cells at the start of step (1), for example, by the number of surviving cells, or is proliferated by 5, 10, 15, 20, 25, 30, 35, or 40%, for example, 10%, or less, and optionally, the number of surviving cells in the population of cells from step (3) is reduced from the number of surviving cells in the population of cells at the start of step (1).

41. The method according to any one of claims 22 to 40, wherein the population of cells from step (3) is not proliferated compared to the population of cells at the start of step (1), or is proliferated for less than 2 hours, for example, less than 1 or 1.5 hours.

42. The method according to any one of claims 22 to 41, wherein the population of cells is not in vitro contacted with a drug that stimulates the CD3 / TCR complex and / or a drug that stimulates a co-stimulatory molecule on the surface of the cells, or if it is contacted, the contact step is less than 2 hours, for example, 1 or 1.5 hours or less.

43. The method according to claim 42, wherein the agent stimulating the CD3 / TCR complex is an agent stimulating CD3 (e.g., an anti-CD3 antibody), and the agent stimulating the co-stimulatory molecule is an agent stimulating CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof, and optionally, the agent stimulating the CD3 / TCR complex or the agent stimulating the co-stimulatory molecule is selected from antibodies (e.g., single-domain antibodies (e.g., heavy-chain variable-domain antibodies), peptide bodies, Fab fragments, or scFv), small molecules, or ligands (e.g., naturally occurring ligands, recombinant ligands, or chimeric ligands).

44. Step (1) and / or (2) are: Serum with a concentration of 5, 4, 3, 2, 1 or 0% or less, and optionally, steps (1) and / or (2) are carried out in a cell medium containing approximately 2% serum, or LSD1 inhibitor or MALT1 inhibitor The method according to any one of claims 22 to 43, carried out in a cell culture medium containing the following.

45. The method according to any one of claims 22 to 44, further comprising the step of receiving a cryopreserved leukocyte apheresis product (or an alternative source of hematopoietic tissue such as cryopreserved whole blood product, cryopreserved bone marrow product, or cryopreserved tumor or organ biopsy or excision (e.g., cryopreserved product from thymectomy)) from an entity, for example, a laboratory, hospital, or healthcare provider.

46. The method according to any one of claims 1 to 45, wherein the population of cells at the start of step (i) or step (1) is enriched with IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ).

47. The method according to any one of claims 1 to 46, wherein the population of cells at the start of step (i) or step (1) comprises 50, 60, or 70% or more IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ).

48. The method according to any one of claims 1 to 47, wherein steps (i) and (ii) or steps (1) and (2) are carried out in a cell culture medium containing IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)).

49. The method according to claim 48, wherein IL-15 increases the ability of the cell population to proliferate, for example, after 10, 15, 20, or 25 days.

50. The method according to claim 48, wherein IL-15 increases the percentage of IL6Rβ-expressing cells in the cell population.

51. The method according to any one of claims 1 to 50, wherein the CCAR or CAR comprises an antigen-binding domain, a transmembrane domain and / or an intracellular signaling domain.

52. The antigen-binding domains include CD19, CD20, CD22, BCMA, mesothelin, EGFRvIII, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptide, sTn-O-glycopeptide, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, Regman, GD3, CD171, IL-11Ra, PSCA, and MAD-C. T-1, MAD-CT-2, VEGFR2, Lewis Y, CD24, PDGFR-β, SSEA-4, folate receptor α, ERBB (e.g., ERBB2), Her2 / neu, MUC1, EGFR, NCAM, Ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, FAP, Regmine, HPV The method according to claim 51, wherein the antigen is bound to an antigen selected from any peptide of the antigens presented to E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, β-human chorionic gonadotropin, AFP, thyroglobulin, PLAC1, globo H, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, mut hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4, or MHC.

53. The antigen-binding domain comprises a CDR, VH, VL, or scFv sequence as disclosed herein, optionally, (a) The antigen-binding domain binds to BCMA and includes a CDR, VH, VL, scFv, or CAR sequence disclosed in Tables 3 to 15, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto; (b) The antigen-binding domain binds to CD19 and includes a CDR, VH, VL, scFv, or CAR sequence disclosed in Table 2, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity therewith; (c) The antigen-binding domain binds to CD20 and includes a CDR, VH, VL, scFv, or CAR sequence disclosed herein or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity therewith; or (d) The method according to claim 51 or 52, wherein the antigen-binding domain binds to CD22 and comprises a CDR, VH, VL, scFv, or CAR sequence disclosed herein or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity therewith.

54. The method according to any one of claims 51 to 53, wherein the antigen-binding domain comprises VH and VL, the VH and VL are linked by a linker, and optionally the linker comprises the amino acid sequence of SEQ ID NO: 63 or 104.

55. (a) The transmembrane domain includes the transmembrane domain of a protein selected from the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, (b) The transmembrane domain includes the transmembrane domain of CD8, (c) The transmembrane domain contains the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or (d) The method according to any one of claims 51 to 54, wherein the first or second nucleic acid molecule comprises a nucleic acid sequence encoding the transmembrane domain, the nucleic acid sequence comprising the nucleic acid sequence of Sequence ID No. 17 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

56. The antigen-binding domain is linked to the transmembrane domain by a hinge region, and optionally, (a) The hinge region contains the amino acid sequence of SEQ ID NO: 2, 3, or 4 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or (b) The method according to any one of claims 51 to 55, wherein the first or second nucleic acid molecule comprises a nucleic acid sequence encoding the hinge region, the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 13, 14, or 15 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

57. The intracellular signaling domain includes a primary signaling domain, and optionally, the primary signaling domain includes a functional signaling domain derived from CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, or CD66d, and optionally, (a) The primary signaling domain includes a functional signaling domain derived from CD3ζ, (b) The primary signaling domain comprises the amino acid sequence of SEQ ID NO: 9 or 10 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or (c) The method according to any one of claims 51 to 56, wherein the first or second nucleic acid molecule comprises a nucleic acid sequence encoding the primary signaling domain, the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 20 or 21 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

58. The intracellular signaling domain includes a co-stimulatory signaling domain, which may optionally include an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activator molecule (SLAM protein), an activated NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, C DS, ICAM-1, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), N Kp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, C D49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (C The ligands include D229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB, or CD83, and optionally include, (a) The co-stimulus signaling domain includes a functional signaling domain derived from 4-1BB, (b) The co-stimulus signaling domain includes the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or (c) The method according to any one of claims 51 to 57, wherein the first or second nucleic acid molecule comprises a nucleic acid sequence encoding the costimulatory signaling domain, the nucleic acid sequence comprising the nucleic acid sequence of Sequence ID No. 18 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

59. The method according to any one of claims 51 to 58, wherein the intracellular signaling domain comprises a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3ζ, and optionally the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and the amino acid sequence of SEQ ID NO: 9 or 10 (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto), and optionally the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or 10.

60. The method according to any one of claims 51 to 59, wherein the CCAR or CAR further comprises a leader sequence including the amino acid sequence of SEQ ID NO:

1.

61. A population of cells containing the first or second nucleic acid molecule (for example, autologous or allogeneic T cells or NK cells containing the first or second nucleic acid molecule) prepared by the method according to any one of claims 1 to 60.

62. A first nucleic acid molecule encoding CCAR, or A second nucleic acid molecule encoding CAR and regulatory molecules A population of cells manipulated to include, (a) Approximately the same percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells, as the percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells, in the same population of cells before they are manipulated to contain the first or second nucleic acid molecule; (b) A change of approximately 5% to approximately 10% of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ cells, compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ cells, in the same population of cells before they are manipulated to contain the first or second nucleic acid molecule; (c) A percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ cells, that is increased by, for example, at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3 times compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ cells, in the same population of cells before being manipulated to include the first or second nucleic acid molecule; (d) Approximately the same percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, as the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the same population of cells before they are manipulated to include the first or second nucleic acid molecule; (e) A change of approximately 5% to approximately 10% of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the same population of cells before being manipulated to include the first or second nucleic acid molecule; (f) A percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, reduced by, for example, at least 20, 25, 30, 35, 40, 45, or 50%, compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the same population of cells before being manipulated to include the first or second nucleic acid molecule; (g) Approximately the same percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, as the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the same population of cells before being manipulated to contain the first or second nucleic acid molecule; (h) A change of within approximately 5% to approximately 10% of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, compared to the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the same population of cells before they are manipulated to contain the first or second nucleic acid molecule; or (i) An increased percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, compared to the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells in the same cell population before being manipulated to include the first or second nucleic acid molecule. A group of cells that include this.

63. A first nucleic acid molecule encoding CCAR, or A second nucleic acid molecule encoding CAR and regulatory molecules A population of cells manipulated to include, (a) The GeneSetScore median (Up TEM vs. Down TSCM) of the cell population is approximately the same as the GeneSetScore median (Up TEM vs. Down TSCM) of the same cell population before it was manipulated to contain the first or second nucleic acid molecule, or differs by only about 25, 50, 75, 100, or 125% or less (e.g., increased by only that amount); (b) The GeneSetScore median (Up Treg vs. Down Teff) of the cell population is approximately the same as the GeneSetScore median (Up Treg vs. Down Teff) of the cell population before it was manipulated to contain the first or second nucleic acid molecule, or differs by only about 25, 50, 100, 150, or 200% or less (e.g., increased by only that amount); (c) The GeneSetScore median (Down stemness) of the cell population is approximately the same as the GeneSetScore median (Down stemness) of the cell population before it was manipulated to contain the first or second nucleic acid molecule, or differs by only about 25, 50, 100, 150, 200, or 250% or less (for example, increased by only that amount); (d) The GeneSetScore median (Up hypoxia) of the cell population is approximately the same as the GeneSetScore median (Up hypoxia) of the cell population before it was manipulated to contain the first or second nucleic acid molecule, or differs by only about 125, 150, 175, or 200% or less (for example, increased by only that amount or less); or (e) A population of cells whose GeneSetScore median (Upautophgy) is approximately the same as the GeneSetScore median (Upautophgy) of the population of cells before it is manipulated to contain the first or second nucleic acid molecule, or differs by only about 180, 190, 200, or 210% or less (e.g., increased by only that amount or less).

64. The population of cells according to any one of claims 1 to 60 or any one of claims 61 to 63, wherein the population of cells comprises the first nucleic acid molecule encoding CCAR.

65. The method according to claim 64 or the population of cells according to claim 64, wherein the CCAR is a fusion polypeptide comprising a degraded polypeptide (e.g., a degraded polypeptide disclosed herein) and a CAR polypeptide (e.g., a CAR polypeptide disclosed herein).

66. (i) The degraded polypeptide contains or comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 310-315, 320-324, 337-339, 360-361, 367-369 and 374 (or sequences having at least 85, 87, 90, 95, 97, 98, 99 or 100% identity therewith), and optionally, the degraded polypeptide contains or comprises the amino acid sequence of SEQ ID NO: 312; (ii) The degraded polypeptide comprises a β-turn of IKZF1 or IKZF3 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity therewith), and optionally comprises a β-hairpin or β-chain of IKZF1 or IKZF3 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity therewith); (iii) The degraded polypeptide comprises an α-helix of IKZF1 or IKZF3 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity therewith); (iv) The degraded polypeptide comprises a first β-chain, a β-hairpin, a second β-chain, and a first α-helix from the N-terminus to the C-terminus of IKZF1 or IKZF3 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto); (v) The degraded polypeptide comprises, from the N-terminus to the C-terminus, a first β-chain, a β-hairpin, a second β-chain, a first α-helix, and a second α-helix of IKZF1 or IKZF3 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto), wherein optionally, the β-hairpin and the second α-helix are separated by 60, 50, 40, or 30 or fewer amino acid residues; (vi) The degraded polypeptide comprises about 10 to about 95 amino acid residues, about 15 to about 90 amino acid residues, about 20 to about 85 amino acid residues, about 25 to about 80 amino acid residues, about 30 to about 75 amino acid residues, about 35 to about 70 amino acid residues, about 40 to about 65 amino acid residues, about 45 to about 65 amino acid residues, about 50 to about 65 amino acid residues, or about 55 to about 65 amino acid residues of IKZF1 or IKZF3 (or sequences having at least 85, 87, 90, 95, 97, 98, 99 or 100% identity therewith); (vii) The degraded polypeptide comprises at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, at least 65 amino acids, at least 70 amino acids, at least 75 amino acids, at least 80 amino acids, at least 85 amino acids, at least 90 amino acids, at least 90 amino acids, or at least 95 amino acids of IKZF1 or IKZF3 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99 or 100% identity therewith); (viiii) The association of the fusion polypeptide with cereblon (CRBN) in the absence of COF1 or COF2, for example, an immunomodulatory imid (IMiD), for example, lenalidomide, pomalidomide, or thalidomide, is less than or equal to, for example, 0.01%, 0.1%, 1%, 5%, 10%, 15%, or 20%, of the association of the fusion polypeptide with CRBN in the presence of COF1 or COF2, for example, an IMiD, for example, lenalidomide, pomalidomide, or thalidomide; (ix) The ubiquitination of the fusion polypeptide in the absence of COF1 or COF2, e.g., IMiD, e.g., lenalidomide, pomalidomide, or thalidomide, is less than or equal to, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the ubiquitination of the fusion polypeptide in the presence of COF1 or COF2, e.g., IMiD, e.g., lenalidomide, pomalidomide, or thalidomide; (x) The degradation of the fusion polypeptide in the absence of COF1 or COF2, e.g., IMiD, e.g., lenalidomide, pomalidomide or thalidomide, is less than or equal to, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the degradation of the fusion polypeptide in the presence of COF1 or COF2, e.g., IMiD, e.g., lenalidomide, pomalidomide or thalidomide; and / or (xi) The method according to claim 65 or the population of cells according to claim 65, wherein the expression level of the fusion polypeptide in the presence of COF1 or COF2, e.g., IMiD, e.g., lenalidomide, pomalidomide or thalidomide, is reduced by, for example, at least 40, 50, 60, 70, 80, 90 or 99% compared to the expression level of the fusion polypeptide in the absence of COF1 or COF2, e.g., IMiD, e.g., lenalidomide, pomalidomide or thalidomide.

67. (i) The degraded polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 375 to 377 (or sequences having at least 85, 87, 90, 95, 97, 98, 99 or 100% identity therewith), and optionally, the degraded polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 375; (ii) The degraded polypeptide comprises a β-turn of IKZF2 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity therewith), and optionally comprises a β-hairpin or β-chain of IKZF2 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity therewith); (iii) The degraded polypeptide comprises an α-helix of IKZF2 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity therewith); (iv) The degraded polypeptide comprises a first β-chain, a β-hairpin, a second β-chain, and a first α-helix from the N-terminus to the C-terminus of IKZF2 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity therewith); (v) The degraded polypeptide comprises, from the N-terminus to the C-terminus, a first β-chain, a β-hairpin, a second β-chain, a first α-helix, and a second α-helix of IKZF2 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity therewith), wherein optionally, the β-hairpin and the second α-helix are separated by 60, 50, 40, or 30 or fewer amino acid residues; (vi) The degraded polypeptide comprises about 10 to about 95 amino acid residues, about 15 to about 90 amino acid residues, about 20 to about 85 amino acid residues, about 25 to about 80 amino acid residues, about 30 to about 75 amino acid residues, about 35 to about 70 amino acid residues, about 40 to about 65 amino acid residues, about 45 to about 65 amino acid residues, about 50 to about 65 amino acid residues, or about 55 to about 65 amino acid residues of IKZF2 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99 or 100% identity therewith); (vii) The degraded polypeptide comprises at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, at least 65 amino acids, at least 70 amino acids, at least 75 amino acids, at least 80 amino acids, at least 85 amino acids, at least 90 amino acids, at least 90 amino acids, or at least 95 amino acids of IKZF2 (or a sequence having at least 85, 87, 90, 95, 97, 98, 99 or 100% identity therewith); (viiii) The association of the fusion polypeptide with cereblon (CRBN) in the absence of COF3, for example compound I-112 disclosed in Table 29, is less than or equal to, for example 0.01%, 0.1%, 1%, 5%, 10%, 15%, or 20%, of the association of the fusion polypeptide with CRBN in the presence of COF3, for example compound I-112 disclosed in Table 29; (ix) The ubiquitination of the fusion polypeptide in the absence of COF3, for example compound I-112 disclosed in Table 29, is less than or equal to, for example, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the ubiquitination of the fusion polypeptide in the presence of COF3, for example compound I-112 disclosed in Table 29; (x) The degradation of the fusion polypeptide in the absence of COF3, for example compound I-112 disclosed in Table 29, is less than or equal to, for example 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the degradation of the fusion polypeptide in the presence of COF3, for example compound I-112 disclosed in Table 29; and / or (viiii) The method according to claim 65 or the population of cells according to claim 65, wherein the expression level of the fusion polypeptide in the presence of COF3, for example compound I-112 disclosed in Table 29, is reduced by, for example, at least 40, 50, 60, 70, 80, 90, or 99% compared to the expression level of the fusion polypeptide in the absence of COF3, for example compound I-112 disclosed in Table 29.

68. (i) The degraded polypeptide is fused to the CAR polypeptide; (ii) The degraded polypeptide and the CAR polypeptide are linked by a peptide bond; (iii) The degraded polypeptide and the CAR polypeptide are linked by a bond other than a peptide bond; (iv) The degraded polypeptide is directly linked to the CAR polypeptide; (v) The degraded polypeptide is indirectly linked to the CAR polypeptide; (vi) The degraded polypeptide and the CAR polypeptide are operably linked via a linker, for example, a glycine-serine linker, for example, a linker having the amino acid sequence GGGGGGGGTGGGGGSG (SEQ ID NO: 335); (vii) The degraded polypeptide is linked to the C-terminus or N-terminus of the CAR polypeptide; or (viiii) The degraded polypeptide is located in the center of the CAR polypeptide, the method according to any one of claims 65 to 67 or the population of cells according to any one of claims 65 to 67.

69. The method according to claim 64 or the population of cells according to claim 64, wherein the CCAR is a fusion polypeptide comprising a degradation domain (e.g., a degradation domain disclosed herein) and a CAR polypeptide (e.g., a CAR polypeptide disclosed herein), wherein optionally the degradation domain is separated from the CAR polypeptide by a heteroprotease cleavage site, and optionally the CCAR comprises the degradation domain, the heteroprotease cleavage site and the CAR polypeptide from the N-terminus to the C-terminus.

70. (i) The degradation domain has a first state associated with the expression of a first level of the fusion polypeptide and a second state associated with the expression of a second level of the fusion polypeptide, wherein the second level is increased by, for example, at least 2, 3, 4, 5, 10, 20 or 30 times relative to the first level in the presence of the stabilizing compound, and optionally, (a) In the absence of the stabilizing compound, the fusion polypeptide is degraded by a cytodegradation pathway, for example, by at least 50%, 60%, 70%, 80%, 90%, or more of the fusion polypeptide being degraded; (b) In the presence of the stabilizing compound, the degradation domain adopts a conformation that is more resistant to cytodegradation than the conformation in the absence of the stabilizing compound; and / or (c) In the presence of the stabilizing compound, the conformation of the fusion polypeptide is more tolerant of cleavage at the heteroprotease cleavage site than the conformation in the absence of the stabilizing compound; (ii) The degradation domain is selected from the estrogen receptor (ER) domain, the FKB protein (FKBP) domain, or the dihydrofolate reductase (DHFR) domain, and is optionally selected. (a) The degradation domain is an estrogen receptor (ER) domain, for example, the degradation domain comprises the amino acid sequence of SEQ ID NO: 342 or 344 or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto, and optionally the stabilizing compound is bazedoxifene or 4-hydroxytamoxifene (4-OHT) or a pharmaceutically acceptable salt thereof; (b) The degradation domain is an FKB protein (FKBP) domain, for example, the degradation domain comprises the amino acid sequence of SEQ ID NO: 346 or a sequence having at least 85, 87, 90, 95, 97, 98, 99 or 100% identity thereto, and optionally the stabilizing compound is Shield-1 or a pharmaceutically acceptable salt thereof; or (c) The method according to claim 69 or the population of cells according to claim 69, wherein the degradation domain is a dihydrofolate reductase (DHFR) domain, for example, the degradation domain comprises the amino acid sequence of Sequence ID No. 347 or a sequence having at least 85, 87, 90, 95, 97, 98, 99 or 100% identity therewith, and optionally the stabilizing compound is trimethoprim or a pharmaceutically acceptable salt thereof.

71. (i) The heterologous protease cleavage site is cleaved by mammalian intracellular proteases, and optionally, (a) The heterologous protease cleavage site is cleaved by a protease selected from the group consisting of furin, PCSK1, PCSK5, PCSK6, PCSK7, cathepsin B, granzyme B, factor XA, enterokinase, genenase, saltase, precision protease, thrombin, TEV protease, and elastase 1; (b) The heterologous protease cleavage sites are the RX(K / R)R consensus motif (where X can be any amino acid; SEQ ID NO: 348), the RXXX[KR]R consensus motif (where X can be any amino acid; SEQ ID NO: 349), the RRX consensus motif (SEQ ID NO: 350), the I-E-P-D-X consensus motif (SEQ ID NO: 351), the Ile-Glu / Asp-Gly-Arg (SEQ ID NO: 352), the Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 353), The sequence contains a cleavage motif selected from the group consisting of Pro-Gly-Ala-Ala-His-Tyr (SEQ ID NO: 354), LPXTG / A consensus motif (SEQ ID NO: 355), Leu-Glu-Val-Phe-Gln-Gly-Pro (SEQ ID NO: 356), Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO: 357), E-N-L-Y-F-Q-G (SEQ ID NO: 358), and [AGSV]-X (where X can be any amino acid; SEQ ID NO: 359); or (c) The heteroprotease cleavage site includes a furin cleavage site selected from the group consisting of RTKR (SEQ ID NO: 378); GTGAEEDPRPSRKRRSLGDVG (SEQ ID NO: 379); GTGAEEDPRPSRKRR (SEQ ID NO: 381); LQWLEQQVAKRRTKR (SEQ ID NO: 383); GTGAEEDPRPSRKRRSLGG (SEQ ID NO: 385); GTGAEEDPRPSRKRRSLG (SEQ ID NO: 387); SLNLTESHNSRKKR (SEQ ID NO: 389); CKINGYPKRGRKRR (SEQ ID NO: 391); and SARNRQKR (SEQ ID NO: 336); or (iii) The heterologous protease cleavage site is cleaved by a mammalian extracellular protease, and optionally, (a) The heterologous protease cleavage site is cleaved by a protease selected from the group consisting of factor XA, enterokinase, genenase, saltase, precision protease, thrombin, TEV protease, and elastase 1; or (b) The method according to claim 69 or 70 or the population of cells according to claim 69 or 70, wherein the heterologous protease cleavage site comprises an amino acid sequence selected from the group consisting of Ile-Glu / Asp-Gly-Arg (SEQ ID NO: 352), Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 353), Pro-Gly-Ala-Ala-His-Tyr (SEQ ID NO: 354), LPXTG / A consensus motif (SEQ ID NO: 355), Leu-Glu-Val-Phe-Gln-Gly-Pro (SEQ ID NO: 356), Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO: 357), E-N-L-Y-F-Q-G (SEQ ID NO: 358), and [AGSV]-X (where X may be any amino acid; SEQ ID NO: 359).

72. The method according to claim 64 or the population of cells according to claim 64, wherein the CCAR is a controllable CAR (RCAR) (for example, an RCAR disclosed herein).

73. The aforementioned RCAR is, (i) intracellular signaling domains, such as intracellular signaling members including a primary intracellular signaling domain and a first switch domain; (ii) an antigen-binding member comprising an antigen-binding domain and a second switch domain; and (iii) A transmembrane domain which may optionally be located on the intracellular signaling member and / or the antigen-binding member. A method according to claim 72, or a population of cells according to claim 72, comprising:

74. The aforementioned RCAR is, (i) intracellular signaling domains, such as intracellular signaling members including a primary intracellular signaling domain and a first switch domain; (ii) an inhibitory extracellular domain member comprising an inhibitory extracellular domain (e.g., an inhibitory extracellular domain comprising an extracellular domain of B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM, LAG3, TIGIT, CTLA-4, BTLA, LAIR1, or the extracellular domain of the TGF-β receptor or a sequence having at least 85, 87, 90, 95, 97, 98, 99, or 100% identity thereto) and a second switch domain; and (iii) Transmembrane domains which may optionally be located on the intracellular signaling member and / or the inhibitory extracellular domain member. A method according to claim 72, or a population of cells according to claim 72, comprising:

75. The aforementioned RCAR is, (i) intracellular signaling domains, such as intracellular signaling members including a primary intracellular signaling domain and a first switch domain; (ii) a costimulatory extracellular domain member comprising a costimulatory extracellular domain (e.g., an extracellular domain of ICOS, CD28, VEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2 or CD226, or a sequence having at least 85, 87, 90, 95, 97, 98, 99 or 100% identity thereto) and a second switch domain; and (iii) Transmembrane domains which may optionally be located on the intracellular signaling member and / or the costimulatory extracellular domain member. A method according to claim 72, or a population of cells according to claim 72, comprising:

76. The first and second switch domains can, for example, form a dimerizing switch in the presence of a dimerizing molecule, and optionally, (i) The dimerization switch is an intracellular dimerization switch or an extracellular dimerization switch; (ii) The dimerization switch is a homodimerization switch or a heterodimerization switch; (iii) The dimerizing switch comprises an FKBP-FRB based switch, for example, the dimerizing switch comprises a switch domain comprising an FRB-binding fragment or an analog of FKBP, and a switch domain comprising an FKBP-binding fragment or an analog of FRB, wherein optionally, the FKBP-binding fragment or FRB analog comprises one or more mutations disclosed herein (e.g., one or more mutations selected from the F2032 mutation, the T2098 mutation, or the E2032 and T2098 mutations), wherein optionally, the dimerizing molecule is an mTOR inhibitor, e.g., a rapamycin analog, e.g., RAD001; and / or (iv) The method according to any one of claims 73 to 75, wherein the antigen-binding domain binds to a target antigen but does not promote the immune effector response of T cells until the dimerization molecule is present, or the population of cells according to any one of claims 73 to 75.

77. (i) The intracellular signaling member comprises a primary intracellular signaling domain, for example, a primary intracellular signaling domain disclosed herein, for example, a CD3ζ domain; (ii) The intracellular signaling member comprises a co-stimulus signaling domain, for example, a co-stimulus signaling domain disclosed herein, for example, a 4-1BB domain or a CD28 domain; (iii) The antigen-binding member does not include a primary intracellular signaling domain; for example, the antigen-binding member includes a co-stimulatory signaling domain and does not include a primary intracellular signaling domain; (iv) The inhibitory extracellular domain member does not include a primary intracellular signaling domain, for example, the inhibitory extracellular domain member includes a co-stimulatory signaling domain and does not include a primary intracellular signaling domain; and / or (v) The method according to any one of claims 73 to 76 or the population of cells according to any one of claims 73 to 76, wherein the costimulatory extracellular domain member does not include a primary intracellular signaling domain, for example, the costimulatory extracellular domain member includes a costimulatory signaling domain and does not include a primary intracellular signaling domain.

78. The population of cells according to any one of claims 1 to 60 or any one of claims 61 to 63, wherein the population of cells comprises the second nucleic acid molecule encoding CAR and regulatory molecules.

79. The second nucleic acid molecule includes a nucleic acid sequence encoding the CAR and a nucleic acid sequence encoding the regulatory molecule, and optionally, the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the regulatory molecule are (i) Arranged on a single nucleic acid molecule, for example, the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the regulatory molecule are separated by a nucleic acid sequence encoding a self-cleavage site; or (ii) The method according to claim 78 or the population of cells according to claim 78, wherein the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the regulatory molecule are arranged on individual nucleic acid molecules.

80. The method according to claim 78 or 79, or the population of cells according to claim 78 or 79, wherein the regulatory molecule comprises a chimeric protein comprising (i) a multimeric ligand-binding domain and (ii) nine caspase molecules.

81. The method according to claim 80 or the population of cells according to claim 80, wherein the caspase 9 molecule is a cleavage-type caspase 9, and optionally, the caspase 9 molecule lacks a caspase recruitment domain.

82. The method according to claim 80 or 81, or the population of cells according to claim 80 or 81, wherein the multimeric ligand-binding region is selected from the group consisting of FKBP, cyclophyllin receptor, steroid receptor, tetracycline receptor, heavy-chain antibody subunit, light-chain antibody subunit, single-chain antibodies composed of tandem heavy-chain and light-chain variable regions separated by a flexible linker domain, and variant sequences thereof, and optionally the multimeric ligand-binding region is an FKBP12 region.

83. The method according to claim 78 or 79, or the population of cells according to claim 78 or 79, wherein the regulatory molecule comprises a cleaved epidermal growth factor receptor (EGFRt).

84. The aforementioned EGFRt has the following characteristics: (i) The EGFRt includes either or both of EGFR domain III and EGFR domain IV; (ii) The EGFRt does not contain one, two, three or all of the EGFR domain I, EGFR domain II, EGFR membrane proximity domain, and EGFR tyrosine kinase domain; (iii) The EGFRt does not mediate signal transduction or trafficking; (iv) The EGFRt does not bind to an endogenous EGFR ligand, such as epidermal growth factor (EGF); and (v) The EGFRt binds to an anti-EGFR antibody molecule (e.g., cetuximab, matsuzumab, necitumumab, and panitumumab), an EGFR-specific siRNA, or a small molecule that targets EGFR. The method according to claim 83 or a population of cells according to claim 83, having one, two, three, four or all of the above.

85. A pharmaceutical composition comprising a population of cells according to any one of claims 61 to 84 and a pharmaceutically acceptable carrier.

86. A method for increasing the target immune response, comprising a population of cells or according to any one of claims 61 to 84. A method comprising the step of administering the pharmaceutical composition according to claim 85 to the subject, thereby increasing the subject's immune response.

87. A method for treating a target cancer, wherein the population of cells described in any one of claims 61 to 84 or A method comprising the step of administering the pharmaceutical composition according to claim 85 to the subject, thereby treating the cancer in the subject.

88. The method according to claim 87, wherein the cancer is, for example, a solid tumor or metastasis thereof selected from one or more of the following: mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, skin cancer, melanoma, kidney cancer, liver cancer, brain tumor, thymoma, sarcoma, carcinoma, uterine cancer, kidney cancer, gastrointestinal cancer, urothelial carcinoma, pharyngeal cancer, head and neck cancer, rectal cancer, esophageal cancer, or bladder cancer.

89. The aforementioned cancers include, for example, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoblastic leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prelymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, and diffuse large B cell lymphoma. Cellular lymphoma (DLBCL), DLBCL with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasm, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (mucosa-associated lymphoid tissue type extranodal follicular marginal zone lymphoma), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, morphology Sodiumblastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, diffuse red medullary small cell B-cell lymphoma of the spleen, hairy cell leukemia - variant, lymphoplasmacytic lymphoma, H chain disease, plasma cell myeloma, solitary osteoplasmacytoma, extraskeletal plasmacytoma, nodular marginal zone lymphoma, pediatric nodular marginal zone lymphoma, primary cutaneous follicle The method according to claim 87, wherein the lymphoma is a humoral cancer selected from central lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma occurring in HHV8-associated multicentric Castleman disease, primary exudative lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or lymphoma that cannot be classified.

90. The method according to any one of claims 86 to 89, further comprising the step of administering a second therapeutic agent to the subject.

91. The method according to any one of claims 86 to 90, wherein the population of cells is administered in a dose determined based on the percentage of CAR-expressing cells (e.g., CCAR-expressing cells) measured in claim 21.

92. After administration of the aforementioned cell population or the aforementioned pharmaceutical composition, The step of administering an effective amount of IMiD (e.g., thalidomide and its derivatives, e.g., lenalidomide, pomalidomide, and thalidomide) or compound I-112 to the subject. It further includes, optionally, a) The subject has developed, is developing, or is expected to develop an adverse reaction after the administration of the cell population or the pharmaceutical composition. b) The administration of IMiD or compound I-112 is in response to the occurrence of an adverse reaction in the subject or the expectation of an adverse reaction in the subject, and / or c) The administration of IMiD or compound I-112 reduces or prevents adverse effects, The method according to any one of claims 86 to 91, wherein the population of cells is optionally the population of cells described in any one of claims 65 to 68.

93. A method for treating the target cancer, i) Ex vivo contact of a population of cells according to any one of claims 65 to 68 with IMiD (e.g., thalidomide and its derivatives, e.g., lenalidomide, pomalidomide and thalidomide) or compound I-112, optionally, In the presence of IMiD or compound I-112, the expression level of the CCAR is reduced by, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent compared to the expression level of the CCAR before the cell population was ex vivo contacted with IMiD or compound I-112, step and ii) The step of administering an effective amount of the population of cells to the subject. It includes, optionally, after step i) and before step ii), For example, a step of reducing the amount of IMiD or compound I-112 that comes into contact with the cell population, within and / or around the cell population. A method further comprising, for treating the cancer.

94. After step iii, iii) The step of administering an effective amount of IMiD or compound I-112 to the subject. The further includes, optionally, the administration of IMiD or compound I-112 reduces the expression level of the CCAR by, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent compared to the expression level of the CCAR after step ii) and before step iii), optionally, a) The subject has developed, is currently developing, or is expected to develop an adverse reaction. b) The administration of IMiD or compound I-112 is in response to the occurrence of an adverse reaction in the subject or the expectation of an adverse reaction in the subject, and / or c) The method according to claim 93, wherein the administration of IMiD or compound I-112 reduces or prevents adverse effects.

95. After step iii, iv) The step of discontinuing the administration of IMiD or compound I-112. The further includes, optionally, discontinuing the administration of IMiD or compound I-112 increases the expression level of the CCAR by, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times compared to the expression level of the CCAR after step iii) and before step iv) (for example, discontinuing the administration of IMiD or compound I-112 restores the expression level of the CCAR to the expression level after step ii) and before step iii)), optionally, a) The subject has either relapsed, is currently relapsed, or is expected to relapse, b) The discontinuation of the administration of IMiD or compound I-112 is in response to tumor recurrence in the subject or the expectation of recurrence in the subject, and / or c) The method according to claim 94, wherein the discontinuation of the administration of IMiD or compound I-112 treats or prevents tumor recurrence.

96. After step iv), v) A step that repeats step iii) and / or iv). The method according to claim 95, further comprising, and thereby treating the cancer.

97. A method for treating the target cancer, i) Administering an effective amount of a population of cells according to any one of claims 65 to 68 to the subject, wherein optionally, the population of cells is ex vivo contacted with IMiD (e.g., thalidomide and its derivatives, e.g., lenalidomide, pomalidomide and thalidomide) or compound I-112 prior to administration, optionally, In the presence of IMiD or compound I-112, the expression level of the CCAR is reduced by, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent compared to the expression level of the CCAR before the cell population is ex vivo contacted with IMiD or compound I-112, and optionally, the amount of IMiD or compound I-112 in contact with the cell population, for example, inside and / or around the cell population, after the cell population is ex vivo contacted with IMiD or compound I-112 and before the cell population is administered to the subject, step A method comprising, and thereby treating the cancer.

98. The method according to claim 97, wherein the population of cells is not ex vivo contacted with IMiD or compound I-112 before administration.

99. After step i), ii) The step of administering an effective amount of IMiD or compound I-112 to the subject. The further includes, optionally, the administration of IMiD or compound I-112 reduces the expression level of the CCAR by, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent compared to the expression level of the CCAR after step i) and before step ii), optionally, a) The subject has developed, is currently developing, or is expected to develop an adverse reaction. b) The administration of IMiD or compound I-112 is in response to the occurrence of an adverse reaction in the subject or the expectation of an adverse reaction in the subject, and / or c) The method according to claim 97 or 98, wherein the administration of IMiD or compound I-112 reduces or prevents adverse effects.

100. After step iii, iii) Step of discontinuing the administration of IMiD or compound I-112. The further includes, optionally, discontinuing the administration of IMiD or compound I-112 increases the expression level of the CCAR by, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times compared to the expression level of the CCAR after step ii) and before step iii) (for example, discontinuing the administration of IMiD or compound I-112 restores the expression level of the CCAR to the expression level after step i) and before step ii), optionally, a) The subject has either relapsed, is currently relapsed, or is expected to relapse, b) The discontinuation of the administration of IMiD or compound I-112 is in response to tumor recurrence in the subject or the expectation of recurrence in the subject, and / or c) The method according to claim 99, wherein the discontinuation of the administration of IMiD or compound I-112 treats or prevents tumor recurrence.

101. After step iii, iv) A step that repeats step ii) and / or iii). The method according to claim 100, further comprising, and thereby treating the cancer.

102. A method for treating the target cancer, i) Administering an effective amount of IMiD (e.g., thalidomide and its derivatives, e.g., lenalidomide, pomalidomide, and thalidomide) or compound I-112 to the subject. The subject includes a population of cells as described in any one of claims 65 to 68, and optionally the administration of IMiD or compound I-112 reduces the expression level of the CCAR by, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent compared to the expression level of the CCAR before the administration of IMiD or compound I-112, and optionally, a) The subject has developed, is currently developing, or is expected to develop an adverse reaction. b) The administration of IMiD or compound I-112 is in response to the occurrence of an adverse reaction in the subject or the expectation of an adverse reaction in the subject, and / or c) A method for reducing or preventing adverse effects by administering IMiD or compound I-112.

103. After step i), ii) The step of discontinuing the administration of IMiD or compound I-112. The further includes, optionally, discontinuing the administration of IMiD or compound I-112 increases the expression level of the CCAR by, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times compared to the expression level of the CCAR after step i) and before step ii) (for example, discontinuing the administration of IMiD or compound I-112 restores the expression level of the CCAR to the expression level before the administration of IMiD or compound I-112), optionally, a) The subject has either relapsed, is currently relapsed, or is expected to relapse, b) The discontinuation of the administration of IMiD or compound I-112 is in response to tumor recurrence in the subject or the expectation of recurrence in the subject, and / or c) The method according to claim 102, wherein the discontinuation of the administration of IMiD or compound I-112 treats or prevents tumor recurrence.

104. After step iii, iii) A step that repeats step i) and / or step ii). The method according to claim 103, further comprising, and thereby treating the cancer.

105. A method for treating the target cancer, i) The above target, (1) Stabilizing compounds, and (2) An effective amount of the population of cells according to any one of claims 69 to 71 A step of administering, optionally, The expression level of the CCAR in the presence of the stabilizing compound is, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times higher than the expression level of the CCAR in the absence of the stabilizing compound. A method comprising, and thereby treating the cancer.

106. After step i), ii) Step of discontinuing the administration of the stabilizing compound. The further includes, optionally, discontinuing the administration of the stabilizing compound reduces the expression level of the CCAR by, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times compared to the expression of the CCAR after step i) and before step ii), optionally, a) The subject has responded to the treatment in step i) (for example, the subject has a complete response to the treatment in step i), the subject shows a reduction in tumor mass, the subject shows a decrease in tumor cells, or the treatment in step i) is effective in the subject), and / or b) The method according to claim 105, wherein the discontinuation of the administration of the stabilizing compound is in accordance with the subject's response to the treatment in step i) (for example, the subject has a complete response to the treatment in step i), the subject shows a reduction in tumor mass, the subject shows a decrease in tumor cells, or the treatment in step i) is effective in the subject).

107. After step i), iii) Step of discontinuing the administration of the stabilizing compound. The further includes, optionally, discontinuing the administration of the stabilizing compound reduces the expression level of the CCAR by, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times compared to the expression of the CCAR after step i) and before step ii), optionally, a) The subject has developed, is currently developing, or is expected to develop an adverse reaction. b) The discontinuation of the administration of the stabilizing compound is in response to the occurrence of an adverse reaction in the subject or the expectation of an adverse reaction in the subject, and / or c) The method according to claim 105, wherein discontinuation of the administration of the stabilizing compound reduces or prevents adverse effects.

108. After step ii) or iii), iv) Step of administering an effective amount of the stabilizing compound. The further comprising, optionally, the administration of the stabilizing compound increases the expression level of the CCAR by, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times compared to the expression level of the CCAR after step ii) or iii) and before step iv), optionally, a) The subject has either relapsed, is currently relapsed, or is expected to relapse, b) The administration of the stabilizing compound is in accordance with the tumor recurrence in the subject or the expectation of recurrence in the subject, and / or c) The method according to claim 106 or 107, wherein the administration of the stabilizing compound treats or prevents tumor recurrence.

109. After step iv), v) A step that repeats step ii), iii), or iv). The method according to claim 108, further comprising, and thereby treating the cancer.

110. Step i) Before, vi) The step of ex vivo contacting the population of cells with a stabilizing compound. The method according to any one of claims 105 to 109, further comprising, optionally, the expression level of the CCAR in the presence of the stabilizing compound being, for example, at least about 1.5, 2, 3, 4, 5, 10, 20, 30, 40, or 50 times higher than the expression level of the CCAR in the absence of the stabilizing compound.

111. The method according to any one of claims 105 to 109, wherein the population of cells is not in ex vivo contact with the stabilizing compound before administration.

112. For use in a method to increase the immune response of a target, the method comprising the step of administering an effective amount of the population of cells or an effective amount of the pharmaceutical composition to the target, the population of cells according to any one of claims 61 to 84 or the pharmaceutical composition according to claim 85.

113. A method for use in treating a target cancer, wherein the method comprises the step of administering an effective amount of the cell population or an effective amount of the pharmaceutical composition to the target, according to any one of claims 61 to 84 or the pharmaceutical composition according to claim 85.

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