Methods of making chimeric antigen receptor-expressing cells

Abstract

To provide methods of making immune effector cells expressing a chimeric antigen receptor (CAR) and compositions prepared by such methods.SOLUTION: A method is provided for preparing a population of cells expressing a CAR (e.g., T cells), the method comprising the steps of: (i) contacting (e.g., binding) a population of cells (e.g., T cells, e.g., T cells isolated from a frozen or fresh leucocyte apheresis product) to an agent that stimulates CD3 / TCR complex and / or an agent that stimulates a co-stimulatory molecule on the surface of the cells; (ii) contacting the population of cells (e.g., T cells) to a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding the CAR, thereby providing a population of cells (e.g., T cells) containing the nucleic acid molecules; and (iii) collecting the population of cells (e.g., T cells) for storage (e.g., reformulating the cell population in a cryopreservation medium) or for administration.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application is a joint venture of U.S. Provisional Patent Application No. 62 / 726,155, filed August 31, 2018. Specification, U.S. Provisional Patent Application No. 62 / 773,679, filed November 30, 2018 The specification and the description of U.S. Provisional Patent Application No. 62 / 858,482, filed June 7, 2019 The entire contents of each of which is incorporated herein by reference. Assert.

[0002] Sequence Listing This application has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on August 16, 2019. The file is named N2067-7153WO_SL.txt and is 260,532 in size. It's a part-time job.

[0003] The present invention generally relates to an immunoglobulin that is engineered to express a chimeric antigen receptor (CAR). The present invention relates to a method for producing vector cells (e.g., T cells or NK cells) and compositions comprising the same. do. [Background technology]

[0004] Adoptive cell transfer (ACT) of T cells, especially chimeric antigen receptor (CAR)-transduced T cells T cell therapy has shown promise in trials for several blood cancers. Manufacturing of CAR-expressing cell therapy products is currently a complex process. What is needed are methods and processes that improve the quality and maximize the therapeutic benefit of products. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure provides immune effector cells (e.g., T cells) engineered to express a CAR. The present invention relates to methods for generating human NK cells (human or NK cells) and compositions produced using such methods. Also disclosed are methods of using such compositions to treat a disease, such as cancer, in a subject. can be.

[0006] In some embodiments, the present invention provides a method for the production of a chimeric antigen receptor (CAR)-expressing cell (e.g., The present invention features a method for producing a population of cells (e.g., T cells), the method comprising: (i) selecting a cell (e.g., T cells, A population of T cells (e.g., isolated from frozen or fresh leukapheresis products) , an agent that stimulates the CD3 / TCR complex and / or a costimulatory molecule on the surface of the cell (ii) contacting a population of cells (e.g., T cells) with an agent; The subject is contacted with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding a CAR, thereby (iii) providing a population of cells (e.g., T cells) that contain the nucleic acid molecule; and Preservation (e.g., reformulating a population of cells in a cryopreservation medium) or administering the cells ( (a) step (ii) comprises the step of: Together with step (i) or within 20 hours after the initiation of step (i), e.g. Within 12, 13, 14, 15, 16, 17, or 18 hours after initiation, for example, step ( and step (iii) is carried out within 18 hours after the initiation of step (i). Within 26 hours after initiation, for example 22, 23, 24 or 25 hours after initiation of step (i). (b) step ( ii) may be performed together with step (i) or within 20 hours after the start of step (i), e.g. Within 12, 13, 14, 15, 16, 17 or 18 hours after the initiation of step (i); For example, step (i) is carried out within 18 hours after the initiation of step (i), and step (iii) is Within 30 hours after the initiation of step (ii), e.g., within 22, 2 (c) is performed within 3, 24, 25, 26, 27, 28, 29, or 30 hours; The population of cells from step (iii) may be, for example, the population of cells at the start of step (i). compared with 5, 6, 7, 8, 9, 1 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 Or by no more than 40%, for example no more than 10%. The nucleic acid molecule in (ii) is a DNA molecule. In some embodiments, the nucleic acid molecule in step (ii) is an RNA molecule. The nucleic acid molecule can be a viral vector, e.g., a lentiviral vector, an adenoviral vector, or a combination thereof. Some of the vectors are selected from the group consisting of vectors and retroviral vectors. In an embodiment, 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 an embodiment, the nucleic acid molecule in step (ii) is not on any vector. In this embodiment, step (ii) comprises: In some embodiments, the method further comprises transducing the subject with a viral vector comprising a nucleic acid molecule that encodes the nucleic acid. In some embodiments, step (ii) is performed together with step (i). Step (ii) is carried out within 20 hours after the initiation of step (i). Step (ii) is 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 4 In some embodiments, step (ii) is carried out within 17 or 18 hours. In some embodiments, step (iii) is performed within 18 hours after the initiation of step (i). In some embodiments, step (i) is performed within 26 hours after the initiation of step (i). Step (iii) is performed within 22, 23, 24 or 25 hours after the start of step (i). In some embodiments, step (iii) is performed 24 hours after the initiation of step (i). In some embodiments, step (iii) is carried out within a period of time following step (ii). In some embodiments, step (iii) is performed within 30 hours after initiation. 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (ii) This will be implemented within.

[0007] In some embodiments, the population of cells from step (iii) is not expanded. In embodiments, the population of cells from step (iii) may be, for example, The number of viable cells is assessed relative to the total number of cells at time of initiation. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or In some embodiments, the population of cells from step (iii) is expanded by no more than 40%. is assessed, for example, by the number of viable cells compared to the population of cells at the start of step (i). and multiplied by less than 10%.

[0008] In some embodiments, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3 In some embodiments, the agent that stimulates a costimulatory molecule is CD28, ICOS, CD 27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, C Agents that stimulate D30, TIM1, CD2, CD226, or any combination thereof In some embodiments, the agent that stimulates a costimulatory molecule is an agent that stimulates CD28. In some embodiments, the agent that stimulates the CD3 / TCR complex is an antibody (e.g., a monoclonal antibody). Domain antibodies (e.g., heavy chain variable domain antibodies), peptibodies, Fab fragments, etc. or scFv), small molecule or ligand (e.g., naturally occurring ligand, recombinant ligand, In some embodiments, the co-stimulatory molecule is selected from the group consisting of a stimulatory ligand, a chimeric ligand, and a co-stimulatory molecule. The agents include antibodies (e.g., single domain antibodies (e.g., heavy chain variable domain antibodies), peptides, and the like). tibody, Fab fragment or scFv), small molecule or ligand (e.g., natural Ligands present in the art, recombinant ligands or chimeric ligands. In embodiments, the agent that stimulates the CD3 / TCR complex does not include beads. In some embodiments, the agent that stimulates the costimulatory molecule does not include beads. The agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody. The agent that stimulates the molecule comprises an anti-CD28 antibody. The agent stimulating the aggregation was an anti-CD3 antibody covalently linked to a colloidal polymer nanomatrix. In some embodiments, the agent that stimulates a costimulatory molecule comprises a colloidal polymer nanoparticle. In some embodiments, the antibody comprises an anti-CD28 antibody covalently attached to a CD3 / TC Agents that stimulate the R complex and costimulatory molecules stimulate the T Cell TransA ct (trademark).

[0009] In some embodiments, the agent that stimulates the CD3 / TCR complex does not include a hydrogel. In some embodiments, the agent that stimulates costimulatory molecules does not include a hydrogel. In this embodiment, the agent that stimulates the CD3 / TCR complex does not include alginate. In some embodiments, the agent that stimulates costimulatory molecules does not include alginate.

[0010] In some embodiments, the agent that stimulates the CD3 / TCR complex comprises a hydrogel. In some embodiments, the agent that stimulates costimulatory molecules comprises a hydrogel. In some embodiments, the agent that stimulates the CD3 / TCR complex comprises alginate. In some embodiments, the agent that stimulates costimulatory molecules comprises alginate. / TCR complex stimulating agents or costimulatory molecule stimulating agents are Quad Techn and MagCloudz(TM) from Technologies.

[0011] In some embodiments, step (i) comprises determining whether or not C in the population of cells from step (iii) Increasing the percentage of AR-expressing cells, e.g., by concentrating the cells from step (iii). The group shows that, compared to cells produced by a similar method but without step (i), A higher percentage of CAR-expressing cells (e.g., at least 10, 20, 30, 40 , 50 or 60% higher).

[0012] In some embodiments, naive cells in the population of cells from step (iii), e.g. Percentage of naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells The naive cells in the population of cells at the start of step (i), e.g., naive T cells, For example, it is the same as the percentage of CD45RA+CD45RO-CCR7+ cells. In some embodiments, naive cells in the population of cells from step (iii), e.g., naive cells The percentage of isolated T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, is , naive cells, e.g., naive T cells, e.g., in the population of cells at the start of step (i) The percentage of CD45RA+CD45RO-CCR7+ cells and , 8, 9, 10, 11 or 12% or less. i) in a population of cells from naive cells, such as naive T cells, such as CD45RA+ The percentage of CD45RO-CCR7+ T cells is the percentage of cells at the start of step (i). Naive cells in the population, e.g., naive T cells, e.g., CD45RA+CD45RO-C The percentage of CR7+ cells differs by no more than 5 or 10%.

[0013] In some embodiments, the population of cells from step (iii) is , more than 26 hours after the start of step (i), e.g., 5, 6 , 7, 8, 9, 10, 11 or 12 days after the date of the test. naive cells, e.g., naive T cells, e.g., CD45RA A higher percentage of CD45RO-CCR7+ T cells (e.g., at least 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 In some embodiments, the population of cells from step (iii) exhibits: After step (ii) and before step (iii), a population of cells (e.g., T cells) is in vitro. The method further comprises growing the culture in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. In comparison to cells generated by otherwise similar methods, naive cells, e.g. A higher percentage of 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% higher).

[0014] In some embodiments, the central memory cells in the population of cells from step (iii) T cells, e.g., central memory T cells, e.g., CD95+ central memory T cells - percentage of central memory cells in the population of cells at the start of step (i), e.g. For example, the percentage of central memory T cells, e.g., CD95+ central memory T cells In some embodiments, the population of cells from step (iii) is central memory cells, e.g., central memory T cells, e.g., CD95+ central The percentage of memory T cells is determined by the percentage of central T cells in the population of cells at the start of step (i). central memory T cells, e.g., CD95+ central memory T cells. - The percentage of T cells is less than 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12% In some embodiments, the central melanoma in the population of cells from step (iii) is different. memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells The percentage of cells that are central memory in the population of cells at the start of step (i) cells, e.g., central memory T cells, e.g., CD95+ central memory T cells The percentages differ by no more than 5 or 10%.

[0015] In some embodiments, the population of cells from step (iii) is , more than 26 hours after the start of step (i), e.g., 5, 6 , 7, 8, 9, 10, 11 or 12 days after the date of the test. Compared to cells generated by For example, a lower percentage of CD95+ central memory T cells (e.g., At least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 3 In some embodiments, the cells from step (iii) exhibit a cellular proliferation rate of 0, 35 or 40% lower. The population may be a population of cells (e.g., T cells) after step (ii) and before step (iii). Growing the population in vitro for more than 3 days, e.g., 5, 6, 7, 8 or 9 days. Compared to cells produced by a similar method but further containing cells, e.g., central memory T cells, e.g., CD95+ central memory T cells A lower percentage (e.g., at least 10, 11, 12, 13, 14, 15, 1 6, 17, 18, 19, 20, 25, 30, 35 or 40% lower).

[0016] In some embodiments, stem memory T cells in the population of cells from step (iii). , for example, CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells The percentage is the percentage of stem memory T cells in the population of cells at the start of step (i), e.g. For example, the proportion of CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells In some embodiments, the percentage of cells from step (iii) is increased. CAR-expressing stem memory T cells in the population, e.g., CAR-expressing CD45RA+CD95+ The percentage of IL-2 receptor β+CCR7+CD62L+ T cells is determined by step (i). CAR-expressing stem memory T cells, e.g., CAR-expressing CD45 The percentage of RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells and In some embodiments, the level of steatosis in the population of cells from step (iii) is increased compared to Memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD The percentage of 62L+ T cells is determined by step (iii) after the start of step (i): More than 26 hours, for example, 5, 6, 7, 8, 9, 10, 11 or 24 hours after the start of step (i). in a population of cells generated by a similar method, but performed after more than 12 days. Stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+ In some embodiments, step (iii) ) CAR-expressing stem memory T cells, e.g., CAR-expressing CD45R The percentage of A+CD95+IL-2 receptor β+CCR7+CD62L+T cells is Step (iii) is continued for more than 26 hours after the start of step (i), e.g., step ( i) is implemented more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of the The present invention also includes CAR-expressing stem memory T cells, e.g., C AR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells In some embodiments, the population of cells from step (iii) is stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7 The percentage of +CD62L+ T cells after step (ii) and after step (iii) The population of cells (e.g., T cells) may be cultured in vitro for more than 3 days, e.g., 5, 6, 7, 8 or 9 days prior to the initiation of the in vitro culture. Cells made by a similar method, but with the additional step of growing the cells for 1 day. Stem memory T cells in the cell population, e.g., CD45RA+CD95+IL-2 receptor β In some embodiments, the percentage of T cells that are (iii) a CAR-expressing stem memory T cell in a population of cells from the The percentage of current CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells The infusion may comprise the step of infusing the cells (e.g., T cells) after step (ii) and before step (iii). The population is expanded in vitro for more than 3 days, e.g., 5, 6, 7, 8 or 9 days. A CAR-expressing stem cell in a population of cells produced by a similar method, but further comprising Memory T cells, e.g., CAR expressing CD45RA+CD95+IL-2 receptor β+CCR 7+CD62L+ T cells.

[0017] In some embodiments, the GeneSetScor of the population of cells from step (iii) is e Median (Up TEM vs. Down TSCM) is the average of the fine grains at the start of step (i). Median GeneSetScore of Cell Populations (Up TEM vs. Down TSCM) ) or differ by no more than about 25, 50, 75, 100, or 125% (e.g., increased by no more than 1). In some embodiments, Median GeneSetScore of a population of cells (Up TEM vs. Down TSC M) Step (iii) is performed more than 26 hours after the start of step (i), e.g. (i) is carried out more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). The median GeneSetScore for a population of cells generated by an otherwise similar method was Lower than the value (Up TEM vs. Down TSCM) (e.g., at least about 100 , 150, 200, 250 or 300% lower). ) Median GeneSetScore of a population of cells (Up TEM vs. Down TEM) TSCM) is a method for treating a cell (e.g., a T cell) after step (ii) and before step (iii). ) is grown in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. A GeneSet of cell populations generated by a similar method but containing the additional step Lower than the median score (Up TEM vs. Down TSCM) (e.g., less In some embodiments, the s Median GeneSetScore of the population of cells from step (iii) (Up Treg vs. Down Teff) is the GeneSe of the population of cells at the start of step (i). Is the median tScore (Up Treg vs. Down Teff) similar? or differs by no more than about 25, 50, 100, 150, or 200% (e.g., In some embodiments, the Gen of the population of cells from step (iii) is increased by 1. Median eSetScore (Up Treg vs. Down Teff) (iii) is not administered for more than 26 hours after the start of step (i), e.g. The same applies except that the implementation is carried out more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of the program. The median GeneSetScore (UpTree) of the cell population generated by the method g vs. Down Teff) lower (e.g., at least about 50, 100, 125 In some embodiments, the collection of cells from step (iii) The median GeneSetScore (Up Treg vs. Down Teff) of the group was After step (ii) and before step (iii), a population of cells (e.g., T cells) is inoculated. The method further comprises growing the method in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. The median GeneSetScore for a population of cells generated using a similar method but containing Lower than the value (Up Treg vs. Down Teff) (e.g., at least about 50 , 100, 150 or 175% lower). The median GeneSetScore (Down stemness) of a population of cells is The median GeneSetScore (Down step) of the population of cells at the start of step (i) mness) or about 25, 50, 100, 150, 200 or 2 In some embodiments, the step differs by no more than 50% (e.g., is increased by no more than that). The median GeneSetScore (Down step) of the population of cells from step (iii) mness) is when 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). GeneSetSco is a population of cells generated by a similar method, but performed re median (Down stemness) (e.g., at least about 50, 10 In some embodiments, the G The median eneSetScore (Down stemness) after step (ii) and prior to step (iii), the population of cells (e.g., T cells) is cultured in vitro for more than 3 days, e.g., A similar method is described, but further comprising the step of growing the cells for, for example, 5, 6, 7, 8, or 9 days. The median GeneSetScore (Down step) of the cell population generated by the method mness) (e.g., at least about 50, 100, or 125% lower). In embodiments, the median GeneSetScore of the population of cells from step (iii) (Up hypoxia) is the GeneSetS of the population of cells at the start of step (i). Approximately the same as the core median (Up hypoxia) or approximately 125, 150, 1 Different by no more than 75 or 200% (e.g., increased by no more than that). For morphology, the median GeneSetScore (U p hypoxia) is the step (iii) is 26 hours after the start of step (i). For example, more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the initiation of step (i). GeneSeed of cell populations generated by a similar method but carried out from Lower than the median tScore (Up hypoxia) (e.g., at least about 40, 5 In some embodiments, the cells from step (iii) The GeneSetScore median (Up hypoxia) of the population is ) and prior to step (iii), the population of cells (e.g., T cells) is incubated in vitro for more than 3 days; Similar methods, but with the additional step of growing for, e.g., 5, 6, 7, 8, or 9 days. The median GeneSetScore of the cell population generated by the method (Up hyp oxia) (e.g., at least about 40, 50, 60, 70, or 80% lower). In some embodiments, the GeneSetScore of the population of cells from step (iii) The median (Up autophagy) is the Genome of the population of cells at the start of step (i). Approximately the same as the median eSetScore (Up autophagy) or approximately 18 Different by 0, 190, 200, or 210% (e.g., increased by In some embodiments, the population of cells from step (iii) is subjected to GeneSetSco The median of re(Up autophagy) is the mean value of step (iii) compared to step (i). More than 26 hours after the start of step (i), e.g., 5, 6, 7, 8, 9, 10 , 11 or 12 days after the start of the experiment. Lower than the median GeneSetScore (Up autophagy) of the population (e.g. , at least about 20, 30, or 40% lower). The median GeneSetScore (Up autophagy) of the population of cells from After step (ii) and before step (iii), a population of cells (e.g., T cells) is in vitro. The method further comprises growing the culture in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. The median GeneSetScore of a population of cells generated by the same method except that (Up autophagy) lower (e.g., at least about 20, 30, or 40% lower) stomach).

[0018] In some embodiments, the population of cells from step (iii) is a CAR-recognized After incubation with cells expressing the antigen of interest, e.g., as shown in Figures 29C-29D As assessed using the method described in Example 8, step (iii) More than 26 hours after the start of step (i), e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, In the same manner except that it is carried out more than 8, 9, 10, 11 or 12 days after the The cells generated or the cells (e.g., T cells) after step (ii) and before step (iii) The population of cells is expanded in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. The method further comprises the step of: (e.g., at least 2, 4, 6, 8, 10, 12, or 14 times higher) do.

[0019] In some embodiments, the population of cells from step (iii) is administered in vivo. Step (iii) is performed more than 26 hours after the start of step (i), e.g. (i) is conducted more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); Other than that, the cells persist longer or have higher ( For example, at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 7 0, 75, 80, 85 or 90% higher) (see, e.g., FIG. 4C). As assessed using the methods described in Example 1. The population of cells from step (iii) is administered in vivo after step (ii) and Prior to step (iii), the population of cells (e.g., T cells) may be cultured in vitro for more than 3 days, e.g., 5 The method is similar, but further comprising the step of growing the cells for 6, 7, 8 or 9 days. longer lasting or more potent (e.g., at least 2 0, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90% higher) levels (see, for example, FIG. 4C and described in Example 1). (as assessed using methods known to those skilled in the art).

[0020] In some embodiments, the population of cells from step (iii) is administered in vivo. Step (iii) is performed more than 26 hours after the start of step (i), e.g. (i) is conducted 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 cells are iii) The population of cells (e.g., T cells) has been incubated in vitro for more than 3 days, e.g., 5, 6, 7, By a similar method, but with the additional step of growing for 8 or 9 days. More potent antitumor activity than the cells produced (e.g., at low doses, e.g., 0.15 × 10 6 , 0 .2×10 6 , 0.25×10 6 or 0.3 x 10 6 More potent with 1 viable CAR-expressing cell It shows potent antitumor activity.

[0021] In some embodiments, the population of cells from step (iii) is compared to the original population of cells, as assessed, for example, by the number of viable cells. In embodiments, the population of cells from step (iii) is, for example, quantified by the number of viable cells. is evaluated and reduced from the number of viable cells in the population of cells at the start of step (i). In this embodiment, the population of cells from step (iii) is the same as the population of cells at the start of step (i). For example, the number of viable cells compared to the total cell population can be assessed. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or In some embodiments, the population of cells from step (iii) is expanded by no more than 40%. is not expanded relative to the population of cells at the start of step (i) or is expanded by 0.5, 1, 1. The cells are grown for less than 5 or 2 hours, for example for less than 1 or 1.5 hours.

[0022] In some embodiments, steps (i) and (ii) include administering to the patient an IL-2, IL-15 (e.g. , hetIL-15 (IL15 / sIL-15Ra)), IL-6 (e.g., IL-6 / The assay was performed in a cell culture medium containing sIL-6Ra), an LSD1 inhibitor, or a MALT1 inhibitor. In some embodiments, steps (i) and (ii) include administering to the subject an antibody against IL-7, IL-21 or an antibody thereof. The method is carried out in a cell culture medium (e.g., serum-free medium) that includes the combination of these. In some embodiments, steps (i) and (ii) include administering to the patient a therapeutically effective amount of IL-2, IL-15 (e.g., hetIL- 15 (IL15 / sIL-15Ra)), IL-21, IL-7, IL-6 (e.g., I L-6 / sIL-6Ra), LSD1 inhibitor, MALT1 inhibitor or combination thereof In some embodiments, step (i) is carried out in a cell culture medium comprising IL-2, I L-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-6 (e.g. For example, IL-6 / sIL-6Ra), LSD1 inhibitor or MALT1 inhibitor in a cell culture medium. In some embodiments, step (ii) is carried out in the soil. For example, hetIL-15 (IL15 / sIL-15Ra), IL-6 (e.g., IL -6 / sIL-6Ra), in cell culture medium containing an LSD1 inhibitor or a MALT1 inhibitor In some embodiments, step (i) comprises administering to the patient IL-7, IL-21, or a combination thereof. In some embodiments, the method is performed in a cell culture medium (e.g., serum-free medium) containing the combination. Step (ii) comprises administering to a cell culture medium (e.g., In some embodiments, step (i) is performed in a serum-free medium. , IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-2 1. IL-7, IL-6 (e.g., IL-6 / sIL-6Ra), LSD1 inhibitor, MA In some embodiments, the method is carried out in a cell culture medium containing an LT1 inhibitor or a combination thereof. In step (ii), 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 culture medium is a serum-free medium that includes a serum replacement. In some embodiments, the serum replacement is CTS™ Immune Cell Serum. um Replacement (ICSR).

[0023] In some embodiments, the method further comprises, prior to step (i), (iv) providing an entity, e.g., a research Fresh leukapheresis products (or fresh whole blood products) from a laboratory, hospital, or healthcare provider , fresh bone marrow product, or fresh tumor or organ biopsy or resection (e.g., from a thymus ablation) The method further comprises receiving an alternative source of hematopoietic tissue, such as a fresh supply of hematopoietic tissue from a donor.

[0024] In some embodiments, the method further comprises, prior to step (i), (v) adding fresh leukocyte antigens to the leukocyte antigen. Laser therapy products (or fresh whole blood products, fresh bone marrow products, or fresh tumor or organ biopsies) Alternative sources, such as biopsy or resection (e.g., fresh product from thymus removal) The cells contacted in step (i) (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) In some embodiments, step (iii) further comprises isolating the population of , within 35 hours after the initiation of step (v), e.g., 27, 28 hours after the initiation of step (v) , 29, 30, 31, 32, 33, 34 or 35 hours, e.g., within the start of step (v). In some embodiments, the method comprises the steps of: The population is assessed by the number of viable cells compared to the population of cells at the end of step (v). or not multiplied by more than 5, 10, 15, 20, 25, 30, 35 or 40%, e.g. For example, the number of cells multiplied is less than 10%.

[0025] In some embodiments, the method comprises the steps of: or from a health care provider, leukapheresis products (or whole blood, bone marrow, or tumor or Hematopoietic tissue such as cryopreserved T cells isolated from organ biopsies or resections (e.g., thymus removal) The method further comprises receiving cryopreserved T cells isolated from a patient (an alternative source of tissue).

[0026] In some embodiments, the method further comprises, prior to step (i), (iv) providing an entity, e.g., a research Cryopreserved leukapheresis products (or cryopreserved whole blood) from a laboratory, hospital, or healthcare provider products, cryopreserved bone marrow products, or cryopreserved tumor or organ biopsies or resections (e.g., thymus The method further includes receiving an alternative source of hematopoietic tissue, such as a cryopreserved product from aspiration. nothing.

[0027] In some embodiments, the method further comprises, prior to step (i), (v) adding a cryopreserved leukocyte apheresis buffer to the cryopreserved leukocyte apheresis buffer. erythrosis products (or cryopreserved whole blood products, cryopreserved bone marrow products, or cryopreserved tumor or Replacement of hematopoietic tissue such as organ biopsy or resection (e.g., cryopreserved products from thymus removal) source), the cells contacted in step (i) (e.g., T cells, e.g., CD8+ and In some embodiments, the method further comprises isolating a population of CD4+ T cells (e.g., CD4+ T cells) from the culture. Step (iii) may be carried out within 35 hours after the initiation of step (v), e.g., step (v) Within 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of In some embodiments, step (v) is performed within 30 hours after the initiation of step (v). The population of cells from step (iii) has a higher percentage of viable cells compared to the population of cells at the end of step (v). The number of cells that did not proliferate or were 5, 10, 15, 20, 25, 30, 35 or 40 years old was evaluated. or less than 40%, for example less than 10%.

[0028] In some embodiments, the present invention provides a method for the production of a chimeric antigen receptor (CAR)-expressing cell (e.g., The present invention features a method for producing a population of cells (e.g., T cells, A population of T cells (e.g., isolated from frozen leukapheresis products) was treated with IL-2, IL-1, -7, IL-15, IL-21, IL-6, or a combination thereof (2) contacting a population of cells (e.g., T cells) with a CAR-encoding kinase; and contacting the nucleic acid molecule (e.g., a DNA or RNA molecule) with the nucleic acid molecule. (3) providing a population of cells (e.g., T cells); and (4) storing (e.g., cryopreserving) the cells. Reformulating a population of cells in culture medium) or collecting cells (e.g., T cells) for administration (a) step (2) is performed in conjunction with or in combination with step (1), Within 5 hours after the start of step (1), for example, 1, 2, 3, 4 or step (3) is performed within 26 hours after the initiation of step (1); Within 22, 23, 24 or 25 hours after the initiation of step (1), e.g., within 22, 23, 24 or 25 hours after the initiation of step (2), e.g., within 22, 23, 24 or 25 hours after the initiation of step (3), e.g., within 22, 23, 24 or 25 hours after the initiation of step (4), e.g. (b) within 24 hours after the initiation of step (1); or (b) the cells from step (3). The population of cells may be, for example, increased by the number of viable cells compared to the population of cells at the start of step (1). and no growth or 5, 10, 15, 20, 25, 30, 35, or 40 % or less, for example 10% or less. In some embodiments, the amount of the cells in step (2) is increased by 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. a viral vector, such as a lentiviral vector, an adenoviral vector, or a retroviral vector In some embodiments, the step The nucleic acid molecule in step (2) is on a non-viral vector. The nucleic acid molecule in step (2) is on a plasmid. The nucleic acid molecule in 2) is not on any vector. (2) infecting a population of cells (e.g., T cells) with a virus that contains a nucleic acid molecule encoding a CAR. This includes transducing with a vector.

[0029] In some embodiments, step (2) is performed together with step (1). In embodiments, step (2) is carried out within 5 hours after the initiation of step (1). In some embodiments, step (2) is performed 1, 2, 3, 4 or 5 days after the initiation of step (1). In some embodiments, step (3) is carried out within 5 hours after the start of step (1). In some embodiments, step (3) is performed within 26 hours after the start of the step ( In some embodiments, the method is performed within 22, 23, 24, or 25 hours after the initiation of 1). Step (3) is carried out within 24 hours after the initiation of step (1).

[0030] In some embodiments, the population of cells from step (3) is, e.g., No proliferation, as assessed by the number of viable cells, compared to the starting population of cells. In an embodiment, the population of cells from step (3) is, for example, the same as the population of cells at the start of step (1). As assessed by the number of viable cells compared to the total cell population, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40% In some embodiments, the population of cells from step (3) is expanded by, e.g., 10 , as assessed by the number of viable cells compared to the population of cells at the start of step (1). It will be multiplied by less than %.

[0031] In some embodiments, step (1) comprises treating a population of cells (e.g., T cells) with IL-2. In some embodiments, step (1) comprises contacting a cell (e.g., a T cell). In some embodiments, step (1) comprises contacting a population of: A population of cells (e.g., T cells) is transfected with IL-15 (e.g., hetIL-15 (IL15 / s In some embodiments, step (1) comprises contacting the antibody with an IL-15Ra). In some embodiments, the method includes contacting a population of cells (e.g., T cells) with IL-21. In the step (1), a population of cells (e.g., T cells) is treated with IL-6 (e.g., IL-6 / In some embodiments, step (1) comprises contacting the cells with an IL-6Ra. The method includes contacting a population of cells (e.g., T cells) with IL-2 and IL-7. In an embodiment, step (1) comprises treating a population of cells (e.g., T cells) with IL-2 and IL15. (e.g., hetIL-15 (IL15 / sIL-15Ra)) In some embodiments, step (1) comprises treating a population of cells (e.g., T cells) with IL-2 and In some embodiments, step (1) comprises contacting the cells ( A population of T cells (e.g., T cells) is treated with IL-2 and IL-6 (e.g., IL-6 / sIL-6Ra) In some embodiments, step (1) comprises contacting a cell (e.g., a T cell) with A population of cells expressing IL-7 and IL-15 (e.g., hetIL-15 (IL15 / sIL- In some embodiments, step (1) comprises contacting a cell ( Some embodiments include contacting a population of T cells (e.g., T cells) with IL-7 and IL-21. In the morphology, step (1) comprises inducing a population of cells (e.g., T cells) to express IL-7 and IL-6 ( In some embodiments, the method includes contacting the IL-6 / sIL-6Ra with a sIL-6 / sIL-6. TEP (1) is a method to induce a population of cells (e.g., T cells) to express IL-15 (e.g., hetIL-1 5 (IL-15 / sIL-15Ra) and IL-21. In an embodiment, step (1) comprises treating 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 / In some embodiments, step (1) comprises contacting the cells with an IL-6Ra. A population of cells (e.g., T cells) is treated with IL-21 and IL-6 (e.g., IL-6 / sIL-6 In some embodiments, step (1) comprises contacting a cell (e.g., , T cells) populations are expressed in IL-7, IL15 (e.g., hetIL-15 (IL15 / sIL -15Ra) and IL-21.

[0032] In some embodiments, the population of cells from step (3) can be treated with, for example, an anti-CD40 antibody. 3. Cells produced by a similar method, but further including the step of contacting with an antibody. Compared to CAR-expressing cells, a higher percentage of naive cells (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 , 35 or 40% higher).

[0033] In some embodiments, naive cells in the population of cells from step (3), e.g., naive cells The percentage of isolated T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, is , naive cells in the population of cells at the start of step (1), e.g., naive T cells, e.g. This is the same as the percentage of CD45RA+CD45RO-CCR7+ cells. In embodiments, naive cells, such as naive T cells, in the population of cells from step (3) are The percentage of cells, e.g., CD45RA+CD45RO-CCR7+ T cells, is Naive cells, e.g., naive T cells, e.g., CD4 The percentage of 5RA+CD45RO-CCR7+ cells and 3, 4, 5, 6, 7, 8, 9 In some embodiments, the fine particles from step (3) differ by no more than 10, 11, or 12%. Naive cells in a population of cells, e.g., naive T cells, e.g., CD45RA+CD45RO - The percentage of CCR7+ T cells is the percentage of Ni-CCR7+ T cells in the population of cells at the start of step (1). naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells In some embodiments, the percentage of step (3) differs from the percentage of step (2) by no more than 5 or 10%. Naive cells, e.g., naive T cells, e.g., CD45RA+CD The percentage of 45RO-CCR7+ T cells is the population of cells at the start of step (1). naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR In some embodiments, the percentage of 7+ cells is increased from step (3). Naive cells in a population of these cells, e.g., naive T cells, e.g., CD45RA+CD4 The percentage of 5RO-CCR7+ T cells in the population of cells at the start of step (1) naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7 + at least 10, 11, 12, 13, 14, 15, 16 compared to the percentage of cells In some embodiments, the step (3) is increased by 17, 18, 19, or 20%. Naive cells in a population of these cells, e.g., naive T cells, e.g., CD45RA+CD4 The percentage of 5RO-CCR7+ T cells in the population of cells at the start of step (1) naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7 + cells is increased by at least 10 or 20% compared to the percentage of + cells.

[0034] In some embodiments, the population of cells from step (3) is More than 26 hours after the start of step (1), e.g., 5, 6, 7, 8 , 9, 10, 11, or 12 days after the first exposure. Compared to cells that have been treated with CD45RA+CD4 5RO- A higher percentage of CCR7+ T cells (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40% In some embodiments, the population of cells from step (3) is ) and prior to step (3), the population of cells (e.g., T cells) may be cultured in vitro for more than 3 days, e.g., A similar method is described, but further comprising the step of growing the cells for, for example, 5, 6, 7, 8, or 9 days. naive cells, e.g., naive T cells, e.g., CD A higher percentage of 45RA+CD45RO-CCR7+ T cells (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 , 35 or 40% higher).

[0035] In some embodiments, central memory cells in the population of cells from step (3), For example, the percentage of central memory T cells, e.g., CD95+ central memory T cells The concentration is determined by the number of central memory cells in the population of cells at the start of step (i), e.g. Percentage of central memory T cells, e.g., CD95+ central memory T cells In some embodiments, the central region of the population of cells from step (3) is the same as the central region of the population of cells from step (3). Memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells The percentage of T cells in the population of cells at the start of step (i) is determined as central memory T cells. -cells, e.g., central memory T cells, e.g., CD95+ central memory T cells differs by no more than 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12% from the percentage of In some embodiments, the central memory cells in the population of cells from step (3), For example, the percentage of central memory T cells, e.g., CD95+ central memory T cells The concentration is determined by the number of central memory cells in the population of cells at the start of step (i), e.g. Percentage of central memory T cells, e.g., CD95+ central memory T cells In some embodiments, the collection of cells from step (3) differs from the above by no more than 5 or 10%. Central memory cells in the population, e.g., central memory T cells, e.g., CD95+ cells. The percentage of central memory T cells in the population of cells at the start of step (1) is Central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells. In some embodiments, the percentage of all memory T cells is reduced relative to the percentage of all memory T cells. (3) A central memory cell in the population of cells, e.g., a central memory T cell. For example, the percentage of CD95+ central memory T cells can be determined by the initiation of step (1). Central memory cells in the initial cell population, such as central memory T cells, at least 10 or 2% higher than the percentage of CD95+ central memory T cells In some embodiments, the centrioles in the population of cells from step (3) are reduced by 0%. central memory T cells, e.g., CD95+ central memory T cells The percentage of central nervous system T cells in the population of cells at the start of step (1) is memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells At least 10, 11, 12, 13, 14, 15, 16, compared with the percentage of cells It is reduced by 17, 18, 19 or 20%.

[0036] In some embodiments, the population of cells from step (3) is More than 26 hours after the start of step (1), e.g., 5, 6, 7, 8 , 9, 10, 11, or 12 days after the first exposure. Compared to cells that have been immunized, central memory cells, e.g., central memory T cells, e.g. A lower percentage of CD95+ central memory T cells (e.g., at least 1 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 In some embodiments, the population of cells from step (3) exhibits a serotonin level of 100 or 40% lower. After step (2) and before step (3), a population of cells (e.g., T cells) is cultured in vitro for 3 h. The method further comprises the step of growing the compound for more than one day, for example, 5, 6, 7, 8 or 9 days. Compared with cells generated by similar methods, central memory cells, e.g. A lower percentage of primary memory T cells, e.g., CD95+ central memory T cells page (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 1 9, 20, 25, 30, 35 or 40% lower).

[0037] In some embodiments, the population of cells from step (3) is administered in vivo, followed by: Step (3) is performed more than 26 hours after the start of step (1), e.g., step (1) Unless it is implemented more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of the longer lasting or higher (e.g., , at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 7 5, 80, 85, or 90% higher) (see, e.g., Example 4C with respect to FIG. 4C). In some embodiments, step (3) After administration in vivo, the population of cells from after step (2) and before step (3) The population of cells (e.g., T cells) is incubated in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. Cells produced by a similar method, but further comprising the step of growing the cells for longer lasting or higher (e.g., at least 20, 25, 30, 3 5, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90% higher ) levels (e.g., as assessed using the method described in Example 1 with respect to FIG. 4C). (so that it can be done).

[0038] In some embodiments, the population of cells from step (3) is The cells are not expanded relative to the population of cells, e.g., as assessed by the number of viable cells. In terms of morphology, the population of cells from step (3) is similar to the population of cells at the start of step (1). In comparison, as assessed, for example, by the number of viable cells, , 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40% or less In some embodiments, the population of cells from step (3) is expanded by 1) 10% or less of the starting population of cells, as assessed, for example, by the number of viable cells In some embodiments, the viable cells in the population of cells from step (3) are expanded by The number may be, for example, assessed by the number of viable cells, and may be the viability of the cells at the start of step (1). The number of surviving cells is reduced.

[0039] In some embodiments, the population of cells from step (3) is The cells are not expanded relative to the population of cells, e.g., as assessed by the number of viable cells. In terms of morphology, the population of cells from step (3) is similar to the population of cells at the start of step (1). In comparison, the cells are grown for less than 0.5, 1, 1.5 or 2 hours, e.g., for less than 1 or 1.5 hours. do.

[0040] In some embodiments, the population of cells is treated with an agent that stimulates the CD3 / TCR complex and / or not contacted in vitro with an agent that stimulates costimulatory molecules on the surface of those cells, or If contacted, the contacting step is for less than 2 hours, e.g., 1 or 1.5 hours or less. In some embodiments, the agent that stimulates the CD3 / TCR complex is a CD3 (e.g. , anti-CD3 antibodies). In some embodiments, an agent that stimulates a costimulatory molecule. The agents are CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226 or any of them In some embodiments, the agent stimulates a combination of co-stimulatory molecules. is an agent that stimulates CD28. In some embodiments, it stimulates the CD3 / TCR complex. The agents include antibodies (e.g., single domain antibodies (e.g., heavy chain variable domain antibodies), peptides, and the like). tibody, Fab fragment or scFv), small molecule or ligand (e.g., natural The ligand is selected from the group consisting of ligands present in the host, recombinant ligands, or chimeric ligands.

[0041] In some embodiments, steps (1) and / or (2) are 5, 4, 3, 2, 1 or 0 In some embodiments, steps (1) and (2) are carried out in a cell culture medium containing no more than 10% serum. and / or (2) is carried out in a cell culture medium containing 2% or less serum. Steps (1) and / or (2) are carried out in a cell culture medium containing about 2% serum. In this embodiment, steps (1) and / or (2) include administering an LSD1 inhibitor or a MALT1 inhibitor. In some embodiments, step (1) is carried out in a cell culture medium containing a cytotoxic agent. In some embodiments, the culture is performed in a cell culture medium containing less than 3, 2, 1, or 0% serum. Step (1) is carried out in a cell culture medium containing 2% or less serum. Step (1) is carried out in a cell culture medium containing about 2% serum. Step (2) is carried out in a cell culture medium containing 5, 4, 3, 2, 1 or 0% or less serum. In some embodiments, step (2) is carried out in a cell culture medium containing 2% or less serum. In some embodiments, step (2) is carried out in a cell culture medium containing about 2% serum. In some embodiments, step (1) comprises administering an LSD1 inhibitor or a MALT1 inhibitor to a patient. In some embodiments, step (2) is carried out in a cell culture medium containing an LSD1 inhibitor. Or, it is carried out in a cell culture medium containing a MALT1 inhibitor.

[0042] In some embodiments, the method further comprises, prior to step (i), (iv) providing an entity, e.g., a research Fresh leukapheresis products (or fresh whole blood products) from a laboratory, hospital, or healthcare provider , fresh bone marrow product, or fresh tumor or organ biopsy or resection (e.g., from a thymus ablation) The method further comprises receiving an alternative source of hematopoietic tissue, such as a fresh supply of hematopoietic tissue from a donor.

[0043] In some embodiments, the method further comprises, prior to step (i), (v) adding fresh leukocyte antigens to the leukocyte antigen. Laser therapy products (or fresh whole blood products, fresh bone marrow products, or fresh tumor or organ biopsies) Alternative sources of hematopoietic tissue, such as biopsy or resection (e.g., fresh product from thymus removal) and the cells contacted in step (i) (e.g., T cells, e.g., CD8+ and / or CD In some embodiments, the method further comprises the step of isolating a population of 4+ T cells. iii) is within 35 hours after the initiation of step (v), e.g., Within 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours, e.g., step ( In some embodiments, the step (iii) is performed within 30 hours after the initiation of step (v). These populations of cells are increased in number by the number of viable cells compared to the population of cells at the end of step (v). and no growth or 5, 10, 15, 20, 25, 30, 35, or 40 %, for example, 10% or less.

[0044] In some embodiments, the method comprises the steps of: or from a health care provider, leukapheresis products (or whole blood, bone marrow, or tumor or Alternative sources, such as cryopreserved T cells isolated from organ biopsies or resections (e.g., thymus removal) The method further comprises receiving cryopreserved T cells isolated from a source.

[0045] In some embodiments, the method further comprises, prior to step (i), (iv) providing an entity, e.g., a research Cryopreserved leukapheresis products (or cryopreserved whole blood) from a laboratory, hospital, or healthcare provider products, cryopreserved bone marrow products, or cryopreserved tumor or organ biopsies or resections (e.g., thymus The method further includes receiving an alternative source of hematopoietic tissue, such as a cryopreserved product from aspiration. nothing.

[0046] In some embodiments, the method further comprises, prior to step (i), (v) adding a cryopreserved leukocyte apheresis buffer to the cryopreserved leukocyte apheresis buffer. erythrosis products (or cryopreserved whole blood products, cryopreserved bone marrow products, or cryopreserved tumor or Replacement of hematopoietic tissue such as organ biopsy or resection (e.g., cryopreserved products from thymus removal) source), the cells contacted in step (i) (e.g., T cells, e.g., CD8+ and In some embodiments, the method further comprises isolating a population of CD4+ T cells (e.g., CD4+ T cells) from the culture. Step (iii) may be carried out within 35 hours after the initiation of step (v), e.g., step (v) Within 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of In some embodiments, step (v) is performed within 30 hours after the initiation of step (v). The population of cells from step (iii) has a higher percentage of viable cells compared to the population of cells at the end of step (v). The number of cells that did not proliferate or were 5, 10, 15, 20, 25, 30, 35 or 40 years old was evaluated. or less than 40%, for example less than 10%.

[0047] In some embodiments, the population of cells at the start of step (i) or step (1) comprises I Regarding IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ), In some embodiments, at the start of step (i) or step (1), The population of cells has 40, 45, 50, 55, 60, 65 or 70% or more IL6R expression The present invention includes cells (e.g., cells positive for IL6Rα and / or IL6Rβ).

[0048] In some embodiments, steps (i) and (ii) or steps (1) and (2) comprise: Cell culture containing IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)) In some embodiments, IL-15 is administered to a population of cells in a culture medium, for example, at 10, 15 In some embodiments, IL-15 increases the ability of the cells to proliferate after 20 or 25 days. The percentage of IL6Rβ expressing cells in a population of cells is increased.

[0049] In some embodiments of the above-described methods, the methods are carried out in a closed system. In an embodiment, T cell separation, activation, transduction, incubation and washing are all performed in a closed system. In some embodiments of the above-described methods, the method is carried out in a separate device. In some embodiments, T cell separation, activation and transduction, incubation and The cleaning and washing are carried out in separate equipment.

[0050] In some embodiments of the aforementioned methods, the method further comprises the step of using ajuva to enhance transduction efficiency. In some embodiments, the method further comprises adding a transduction enhancing agent or a transduction enhancing reagent to the cell culture medium. In some embodiments, the adjuvant or transduction reagent comprises a cationic polymer. The adjuvant or transduction enhancing reagent is LentiBOOST™ (Sirio n Biotech), vectofusin-1, F108, Hexane bromide Xadimethrine (Polybrene), PEA, Pluronic F68, Plur Choose from Synperonic F127, Synperonic or LentiTrans™ In some embodiments, the adjuvant is LentiBOOST™ (Sir ion Biotech).

[0051] In some embodiments of the methods described above, a population of cells (e.g., T cells) is transfected with a viral vector. The step of transducing a population of cells with a β-actin is carried out under conditions that enhance transduction efficiency. In one embodiment, the cells are spinocula. The gene is transduced by ion.

[0052] In some embodiments of the above-described methods, the cells (e.g., T cells) are plated with a gas-permeable membrane at the bottom. The cells are activated and transduced in a cell culture flask containing a gas-permeable membrane that provides gas exchange. Supports large medium volumes without compromising quality. In some embodiments, cell growth is convection This is achieved by providing access to nutrients through, for example, uninterrupted access.

[0053] In some embodiments of the aforementioned methods, the CAR comprises an antigen-binding domain, a transmembrane domain, and and an intracellular signaling domain.

[0054] In some embodiments, the antigen binding domain is CD19, CD20, CD22, BCMA , mesothelin, EGFRvIII, GD2, Tn antigen, sTn antigen, Tn-O-glycope peptide, sTn-O-glycopeptide, PSMA, CD97, TAG72, CD44v6 , CEA, EPCAM, KIT, IL-13Ra2, Legman, GD3, CD171, I L-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, Lewis Y, CD24, PDGFR-β, SSEA-4, folate receptor α, ERBB (e.g., ER BB2), Her2 / neu, MUC1, EGFR, NCAM, EphrinB2, CAIX , LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, FAP, legumain, HPV E6 or E7, ML-IA P, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, Neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, β-human chorionic gona Dotropin, AFP, Thyroglobulin, PLAC1, Globo H, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, mut hsp 7 0-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX 3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4 binds to an antigen selected from any peptide of these antigens presented on MHC In some embodiments, the antigen binding domain comprises the CDRs, VH, VL and VL sequences disclosed herein. In some embodiments, the antigen binding domain comprises a VH and a scFv or CAR sequence. VL, wherein the VH and VL are linked by a linker, and optionally the linker is Contains the amino acid sequence of sequence number 63 or 104.

[0055] In some embodiments, the transmembrane domain is an α, β or ζ chain of a 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 comprises a transmembrane domain of a protein selected from the group consisting of: In some embodiments, the transmembrane domain is represented by SEQ ID NO: 6 or a sequence at least about 85%, 90%, 95%, or 99% thereof In some embodiments, the nucleic acid molecule comprises an amino acid sequence having identity to the transmembrane domain. The nucleic acid sequence comprises a nucleic acid sequence encoding the nucleic acid sequence of SEQ ID NO: 17 or at least Also included are nucleic acid sequences having about 85%, 90%, 95% or 99% sequence identity.

[0056] In some embodiments, the antigen binding domain is linked to the transmembrane domain by a hinge region. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 2, 3, or 4. or an antigen having at least about 85%, 90%, 95% or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a hinge region. and the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 13, 14 or 15 or at least These include nucleic acid sequences having about 85%, 90%, 95% or 99% sequence identity.

[0057] In some embodiments, the intracellular signaling domain comprises a primary signaling domain. In some embodiments, the primary signaling domain is selected from the group consisting of CD3ζ, TCRζ, FcRγ , FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79 b, CD278 (ICOS), FcεRI, DAP10, DAP12, or CD66d In some embodiments, the primary signaling domain comprises a functional signaling domain that In some embodiments, the in comprises a functional signaling domain derived from CD3ζ. The primary signaling domain may comprise the amino acid sequence of SEQ ID NO: 9 or 10, or at least The amino acid sequence has about 85%, 90%, 95% or 99% sequence identity. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a primary signaling domain. The nucleic acid sequence is a nucleic acid sequence of SEQ ID NO: 20 or 21, or at least about 85%, 9 It includes nucleic acid sequences having 0%, 95% or 99% sequence identity.

[0058] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain comprises an MHC class I molecule, TN F receptor protein, immunoglobulin-like protein, cytokine receptor, integrin , signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, B TLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, C D30, CD40, CDS, ICAM-1, 4-1BB(CD137), B7-H3, I COS(CD278), GITR, BAFFR, LIGHT, HVEM(LIGHTR) , KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7 Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA 6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, IT GAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(C D244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SE MA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAM F1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD1) 62), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, C D28-OX40, CD28-4-1BB or CD83-specific ligand binding In some embodiments, the costimulatory signaling domain comprises a functional domain derived from 4-1BB. In some embodiments, the costimulatory signaling domain comprises a sequence The amino acid sequence of sequence number 7 or a sequence identical to at least about 85%, 90%, 95% or 99% thereof In some embodiments, the nucleic acid molecule comprises an amino acid sequence having sequence identity to a costimulatory signaling molecule. The nucleic acid sequence comprises a nucleic acid sequence encoding a signal transduction domain, the nucleic acid sequence being the nucleic acid sequence of SEQ ID NO: 18. or a nucleic acid having at least about 85%, 90%, 95% or 99% sequence identity thereto. Contains the acid sequence.

[0059] In some embodiments, the intracellular signaling domain comprises a functional sequence derived from 4-1BB. It contains a signal transduction domain and a functional signal transduction domain derived from CD3ζ. In one embodiment, 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 with 9 or 10 (or at least about 85%, 90%, or more thereof) %, 95% or 99% sequence identity). In one embodiment, 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 and 10 amino acid sequences.

[0060] In some embodiments, the CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO:1. Included.

[0061] In some embodiments, the present invention relates to any of the methods described above or disclosed herein. CAR-expressing cells generated by any other method (e.g., autologous or allogeneic CAR-expressing cells) In some embodiments, the present invention is characterized by a population of C cells (current T cells or NK cells) as disclosed herein. A pharmaceutical composition comprising a population of AR-expressing cells and a pharma- ceutically acceptable carrier is provided herein. Provided.

[0062] In some embodiments, in the final CAR cell product produced using the methods described herein, In this case, beads (e.g., CD4 beads, CD8 beads and / or TransACT beads) ) are 0.04, 0.05, and 0.06 of the total amount of beads added during the manufacturing process. , 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4 or 0.5% or less be.

[0063] In some embodiments, the present invention provides a CAR-expressing cell (e.g., , autologous or allogeneic CAR-expressing T cells or NK cells): (a) CAR Naive cells, e.g., naive T cells, in the same population of cells prior to being engineered to express the Compared with the percentage of CD45RO-CCR7+ cells, for example, naive cells at this stage, e.g., naive T cells, e.g., CD45RO-CCR7+ cells; (b) Naïve cells in the same population of cells before they are engineered to express a CAR, e.g. Compared with the percentage of naive T cells, e.g., CD45RO-CCR7+ cells, About 5% to about 1% of naive T cells, such as CD45RO-CCR7+ cells (c) Change within 0% in the same population of cells before they were engineered to express the CAR. Percentage of naive T cells, e.g., CD45RO-CCR7+ cells Increased, for example, by at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8 or 3-fold increased percentage of naive cells, e.g. (d) naive T cells, e.g., CD45RO-CCR7+ cells; (e) CAR-expressing T cells, e.g., CD45RO-CCR7+ cells; Central memory cells in the same population of cells before they were manipulated, e.g., The percentage of CCR7+CD45RO+ T cells was almost the same as that of CCR7+CD45RO+ T cells. The same percentage of central memory cells, e.g., central memory T cells, e.g. CCR7+CD45RO+ T cells; (e) The same cells before being engineered to express CAR. Central memory cells in a population of cells, e.g., central memory T cells, e.g., CCR 7+CD45RO+ T cell percentages, compared with central memory cells, e.g. Central memory T cells, e.g., about 5% to about 10% of CCR7+CD45RO+ T cells (f) Changes within the centra in the same population of cells before they were engineered to express the CAR. central memory T cells, e.g., CCR7+CD45RO+ T cells Reduced, e.g., by at least 20, 25, 30, 35, , 40, 45 or 50% reduced percentage of central memory cells, e.g. (g) central memory T cells, e.g., CCR7+CD45RO+ T cells; Stem memory T cells in the same population of cells before they were engineered to Percentage and ratio of A+CD95+IL-2 receptor β+CCR7+CD62L+T cells All showed roughly the same percentage of stem memory T cells, e.g., CD45RA+CD95 + IL-2 receptor β+ CCR7+ CD62L+ T cells; (h) engineered to express CAR Stem memory T cells in the same population of cells before generation, 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+CD6 (i) a change in about 5% to about 10% of 2L+ T cells; or (ii) an alteration in about 5% to about 10% of 2L+ T cells engineered to express a CAR. Stem memory T cells in the same population of cells before they were transplanted, e.g., CD45RA+CD95+I Increased percentage of L-2 receptor beta+CCR7+CD62L+T cells Percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor Body β+CCR7+CD62L+T cells.

[0064] In some embodiments, the present invention relates to a CAR-expressing cell (e.g., an autologous or allogeneic CAR-expressing cell). (a) characterizing a population of cells (current T cells or NK cells), Median score (Up TEM vs. Down TSCM) was 1.0, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.1, 1.2 ... The median GeneSetScore (Up TEM) of the same population of cells before they were manipulated Down TSCM) or approximately 25, 50, 75, 100 or less (b) the population of cells differs (e.g., is increased by less than) 125%; Median GeneSetScore (Up Treg vs. Down Teff) Median GeneSetScore (U p Treg vs. Down Teff) or about 25, 50, 10 Different by (e.g., increased by) 0, 150, or 200% or less; c) The median GeneSetScore (Down stemness) of a population of cells is Median GeneSetScore ( Down stemness) or approximately 25, 50, 100, 150, (d) differs (e.g., is increased by less than) 200 or 250%; The median GeneSetScore (Up hypoxia) of the population of cells expressing CAR The median GeneSetScore (Up hy) of a population of cells before they were engineered to express poxia) or about 125, 150, 175, or 200% less (e) the GeneSe of the population of cells is different (e.g., increased by less); Median tScore (Up autophagy) was 100% in mice engineered to express CAR The median GeneSetScore (Up autophagy) of the population of cells before autophagy was almost the same as that of the control group. are approximately the same or differ by no more than about 180, 190, 200, or 210% (e.g. , or less).

[0065] In some embodiments, the invention features a method of increasing an immune response in a subject, comprising: a population of CAR-expressing cells disclosed herein or a pharmaceutical composition disclosed herein The method includes administering the compound to an elephant, thereby increasing an immune response in the subject.

[0066] In some embodiments, a method of treating cancer in a subject is disclosed, which comprises administering to a subject a therapeutically effective amount of the compounds disclosed herein. A population of CAR-expressing cells or a pharmaceutical composition disclosed herein is administered to a subject, 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 Cancer, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colon cancer, prostate cancer, cervical cancer, skin cancer, melanoma, Kidney cancer, liver cancer, brain tumor, thymoma, sarcoma, carcinoma, uterine cancer, kidney cancer, gastrointestinal cancer, urothelial cancer, Solid cancer selected from one or more of pharyngeal cancer, head and neck cancer, rectal cancer, esophageal cancer, or bladder cancer Cancer or its metastasis. In some embodiments, the cancer is, for example, chronic lymphocytic leukemia (CL). L), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoblastic leukemia (ALL) , Hodgkin's lymphoma, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia TALL, small lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blastic morphology Plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma (DL BCL), DLBCL with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasms, follicular lymphoma follicular lymphoma, childhood follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma Lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (mucosa-associated lymphoid tissue type, extranodal follicular Marginal zone lymphoma, myelodysplastic syndrome, myelodysplastic syndrome, non-Hodgkin's lymphoma Plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macrophage Globulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse small red pulp cell type B Hairy cell lymphoma, Hairy cell leukemia-variant, Lymphoplasmacytic lymphoma, Heavy chain disease, Plasma Zuma cell myeloma, isolated bone plasmacytoma, extraosseous 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 arising in alveolar lymphoma and HHV8-associated multicentric Castleman disease , primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML) or unclassified It is a liquid cancer selected from lymphoma.

[0067] 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 chemotherapy agent, radiation therapy or is an immunomodulatory therapy. In some embodiments, the second therapeutic agent is IL-15 (e.g., h etIL-15 (IL15 / sIL-15Ra)).

[0068] Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. Suitable methods and materials are described below. Publications, patent applications, patents, and other references (e.g., sequence database reference numbers) are incorporated herein by reference in their entirety. The entirety of the present invention is incorporated by reference herein. All GenBank, Unigene and Entrez sequences mentioned (in the table) are The sequence is incorporated by reference. A gene or protein may have multiple sequence accession numbers. When referring to a sequence, all sequence variants are encompassed.

[0069] In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. However, subheadings or numbered or lettered elements, such as (a), (b), (i), etc. Headings or numbered or lettered elements herein are provided solely for ease of reading. Does the use of elements not require that steps or elements be performed in alphabetical order? Nor are the steps or elements necessarily distinct from one another. , objects and advantages will become apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0070] [Figure 1-1]Figure 1: When purified T cells were incubated with cytokines, naïve cells were the predominant population transduced. Figure 1A is a graph showing an exemplary cytokine process. Figure 1B is a set of graphs showing the percentage of CD3+CAR+ cells at each indicated time point after transduction. Figure 1C is a series of graphs showing transduction within the CD3+CCR7+CD45RO- population in a CD3 / CD28 bead stimulated population (left) compared to a cytokine only population (right) in two independent donors. For the sample designated "Short stim IL7+IL15" in Figure 1C, cells were stimulated with beads for 2 days before they were removed in the presence of IL7 and IL15. Figures 1D, 1E, and 1F are a series of flow cytometry graphs showing the transduction of T cell subsets cultured daily with IL2 (Figure 1D), IL15 (Figure 1E), and IL7+IL15 (Figure 1F) for 3 days. Figure 1G is a series of flow cytometry graphs showing T cell differentiation on day 0 (left) and day 1 (right) for CCR7 and CD45RO after stimulation with IL2 (top right panel) or IL-15 (bottom right panel). Figures 1H and 1I are a series of graphs showing the percentages of CD3+CCR7+RO-, CD3+CCR7+RO+, CD3+CCR7-RO+, and CD3+CCR7-RO- cells on day 0 or after 24 hours of incubation with the indicated cytokines. [Figure 1-2] (As stated above.) [Figure 1-3] (As stated above.) [Figure 1-4] (As stated above.) [Figure 1-5] (As stated above.) [Figure 1-6] (As stated above.) [Figure 1-7] (As stated above.) [Figure 1-8] (As stated above.) [Figure 1-9] (As stated above.) [Figure 1-10] (As stated above.) [Figure 1-11] (As stated above.) [Figure 1-12] (As stated above.) [Figure 2-1] Figure 2: CARTs generated with 1 day of cytokine stimulation were functional. Figure 2A: Purified T cells were transduced at an MOI of 1 and in all cytokine conditions tested, and the percentage of CAR-expressing cells observed on days 1 and 10 were comparable. CARTs were generated within 1 day and then harvested and expanded with CD3 / CD28 beads for 9 days 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 cytotoxic potential of day 1 CARTs after expansion was measured using Nalm6 as target cells. Figure 2B is a graph showing the % killing of CD19-positive Nalm6 cells by CARTs from each condition. Day 10 CARTs expanded with CD3 / CD28 beads were marked as "day 10". All other samples were day 1 CARTs. FIG. 2C: Proliferating day 1 CARTs in response to Nalm6 target cells were assayed for secretion of IFNg. FIG. 2C is a graph showing the amount of IFN-γ secretion by CARTs from each condition in the presence of CD19 positive or CD19 negative target cells. FIG. 2D: Proliferative capacity of day 1 CARTs was assayed by measuring EDU incorporation. FIG. 2D is a graph showing the average percentage of EDU positive cells for each condition. As in FIG. 2B, day 10 CARTs are labeled "day 10" and all other samples were day 1 CARTs. [Figure 2-2] (As stated above.) [Figure 2-3] (As stated above.) [Figure 2-4] (As stated above.) [Figure 3-1]Figure 3: Effect of MOI and media composition at day 0 transduction. 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 set of graphs plotting the percentage of CD3+CAR+ cells versus the MOI for each condition tested. Figure 3B: Media composition influenced the transduction with cytokine process. Figure 3B is a set of graphs showing the percentage of CD3+CAR+ cells at day 1 (left) and day 8 (right) for each condition tested. "2.50" indicates an MOI of 2.50. "5.00" indicates an MOI of 5.00. [Figure 3-2] (As stated above.) [Figure 3-3] (As stated above.) [Figure 4-1] Figure 4: CAR T cells generated within 24 hours can eliminate tumors. Figure 4A: Purified T cells were transduced with CAR19 and harvested 24 hours later. Figure 4A is a series of flow cytometry plots showing the transduction of T cells with CAR19 cultured with IL2, IL15, and IL7+IL15, showing transduction with each cytokine condition. Figure 4B: Graph showing the average viability of over 80% in all conditions tested. Figure 4C: Expansion 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 indicated time points after infusion for each condition tested. Day 10 CART are labeled "D10 1e6" or "D10 5e6", other samples were all day 1 CART. FIG. 4D: Day 1 CART was able to eliminate tumors in vivo, albeit with delayed kinetics compared to day 10 CART. FIG. 4D is a graph showing total flux at the indicated time points after tumor inoculation for each condition tested. 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. [Figure 4-2] (As stated above.) [Figure 4-3] (As stated above.) [Figure 4-4] (As stated above.) [Figure 4-5] (As stated above.) [Figure 4-6] (As stated above.) [Figure 4-7] (As stated above.) [Figure 4-8] (As stated above.) [Figure 5-1] Figure 5: The cytokine process was scalable. Figure 5A: T cells were enriched with CliniMACS® Prodigy® and the B cell compartment was reduced to less than 1%. Figure 5A is a series of flow cytometry plots showing staining of cells with anti-CD3 (left) or anti-CD19 and anti-CD14 (right) antibodies on leukopack cells (top) or CD4+CD8+ enriched cells (bottom). Figure 5B: Purified T cells from frozen apheresis were transduced with CAR19 either in 24-well plates or in PL30 bags after enrichment. CARs were harvested 24 hours later. Figure 5B is a series of flow cytometry plots showing staining of CD3 and CAR on cells produced in the presence of either IL2 or hetIL-15 (IL15 / sIL-15Ra). [Figure 5-2] (As stated above.) [Figure 6-1] Figure 6: CART produced by the activation process showed superior antitumor efficacy in vivo. Figures 6A and 6B are graphs plotting tumor burden against the indicated time points after tumor implantation. "d.1" represents CART produced using the activation process. "d.9" represents CART produced with 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. [Figure 6-2] (As stated above.) [Figure 6-3] (As stated above.) [Figure 7-1]Figure 7: IL6Rα- and IL6Rβ-expressing cells were enriched in less differentiated T cell populations. Fresh T cells were stained for the indicated surface antigens to assay the expression levels of IL6Rα and IL6Rβ on CD4 (Figure 7A) and CD8 (Figure 7B) T cell subsets. [Figure 7-2] (As stated above.) [Figure 8-1] Figure 8: Both IL6Rα and IL6Rβ expressing cells were enriched in less differentiated T cell populations. Fresh T cells were stained for the indicated surface antigens and assayed for expression levels of the indicated surface antigens on CD4 (Figure 8A) and CD8 (Figure 8B) T cell subsets. [Figure 8-2] (As stated above.) [Figure 9] IL6Rα-expressing cells expressed surface markers of less differentiated T cells. Fresh T cells were stained for the indicated surface antigens to examine the expression levels of various surface antigens in IL6Rα high, intermediate, and low expressing cell subsets. [Figure 10] IL6Rβ-expressing cells expressed surface markers of less differentiated T cells. Fresh T cells were stained for the indicated surface antigens to examine the expression levels of various surface antigens in IL6Rβ high, intermediate, and low expressing cell subsets. [Figure 11] IL6Rα, but not IL6Rβ, expression was downregulated following TCR engagement. T cells were activated with αCD3αCD28 beads on day 0 and then assayed for IL6Rα and IL6Rβ expression levels at the indicated time points. [Figure 12] Fold expansion of cytokine-treated T cells after TCR engagement. T cells were activated with αCD3αCD28 beads in the presence of the indicated cytokines on day 0, and cell numbers were monitored at the indicated time points. [Figure 13-1] Figure 13: IL2, IL7 and IL15 treatment did not affect cell size and viability following TCR engagement. T cells were activated with αCD3αCD28 beads in the presence of the indicated cytokines on day 0, and then cell size (Figure 13A) and viability (Figure 13B) were monitored at the indicated time points. [Figure 13-2] (As stated above.) [Figure 14-1] Figure 14: Kinetics of expression of various surface molecules on CD4 T cells after cytokine treatment. T cells were activated with αCD3αCD28 beads in the presence of the indicated cytokines on day 0, and then the expression of various surface molecules was assayed by flow cytometry at the indicated time points. [Figure 14-2] (As stated above.) [Figure 14-3] (As stated above.) [Figure 14-4] (As stated above.) [Figure 15-1] Figure 15: Kinetics of expression of various surface molecules on CD8 T cells after cytokine treatment. T cells were activated with αCD3αCD28 beads in the presence of the indicated cytokines on day 0, and then the expression of various surface molecules was assayed by flow cytometry at the indicated time points. [Figure 15-2] (As stated above.) [Figure 15-3] (As stated above.) [Figure 15-4] (As stated above.) [Figure 16] Following TCR engagement, IL6Rβ expression was restricted primarily to CD27-expressing T cell subsets. T cells were activated with αCD3αCD28 beads in the presence of the indicated cytokines on day 0, followed by assay of IL6Rβ expression by flow cytometry on day 15. [Figure 17] Following TCR engagement, IL6Rβ expression was suppressed primarily in CD57 non-expressing T cell subsets. T cells were activated with αCD3αCD28 beads in the presence of the indicated cytokines on day 0, followed by assay of IL6Rβ expression by flow cytometry on day 25. [Figure 18] Common gamma chain cytokine-treated T cells produced functional cytokines on day 25. T cells were activated with αCD3αCD28 beads in the presence of the indicated cytokines on day 0 and then assayed by flow cytometry on day 25 for the percentage of IL2-, IFN-γ-, and TNFα-producing T cells. [Figure 19-1]Figure 19: BCMA CAR expression on day 1 with ARM at 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. [Figure 19-2] (As stated above.) [Figure 20-1] Figure 20: In vitro CAR expression kinetics from days 1 to 4 for cells manufactured 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 points as measured by flow cytometry. Figures 20B and 20C are graphs showing CAR+% and MFI values ​​over time, respectively. [Figure 20-2] (As stated above.) [Figure 20-3] (As stated above.) [Figure 21-1] Figure 21: In vivo triage in a KMS-11-luc multiple myeloma xenograft mouse model. Each mouse received 1.5E6 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) following CART treatment. [Figure 21-2] (As stated above.) [Figure 22-1] Figure 22: In vivo triage of BCMA CAR with dose titration in KMS-11-luc multiple myeloma xenograft mouse model. Figure 22A is a panel of histograms showing CAR expression on days 1 and 3. Figure 22B is a graph showing tumor uptake kinetics after CART treatment with 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 kinetics over time during this study. [Figure 22-2] (As stated above.) [Figure 22-3] (As stated above.) [Figure 23-1]Figure 23: 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) (Figure 23B) of CD3+CAR+ cells observed over time (replicate 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. Numbers in the plots indicate CAR positivity (%). [Figure 23-2] (As stated above.) [Figure 23-3] (As stated above.) [Figure 23-4] (As stated above.) [Figure 24-1] FIG. 24: FIG. 24A is a graph showing the end-to-end composition of the starting material (Prodigy® product) and at harvest at various time points from the initiation of culture. Native (n), central memory (cm), effector memory (em) and effector (eff) subsets were defined by CD4, CD8, CCR7 and CD45RO surface expression or lack thereof. CD4 composition is shown. At each time point, the left bar shows the cellular composition of the entire CD3+ population (bulk) and the right bar shows the cellular composition of the CAR+ fraction. FIG. 24B is a panel of flow cytometry plots showing the gating strategy applied to viable CD3+ events to determine the overall transduction efficiency (top row), CD4 / CD8 composition (middle row) and memory subsets (bottom row) within the entire CD3+ population (bulk) and the CAR+ fraction. [Figure 24-2] (As stated above.) [Figure 24-3] (As stated above.) [Figure 24-4] (As stated above.) [Figure 24-5] (As stated above.) [Figure 24-6] (As stated above.) [Figure 24-7] (As stated above.) [Diagram 25]Dynamics of T cell subsets expressing surface CAR over time, represented as the number of viable cells in each subset. [Figure 26] Viable cell recovery 12–24 h after initiation of culture (number of viable cells recovered at harvest relative to number of viable cells inoculated) as determined from prewash counts. [Figure 27] Viability of fast CART harvested 12–24 h after initiation of culture, as determined pre- and post-wash at the time of harvest. [Figure 28-1]Figure 28: Figure 28A is a graph showing the composition of the starting material (healthy donor leukopac: LKPK) and the T cell enriched product analyzed by flow cytometry. Numbers indicate % of 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 rates (forward scatter FSC vs. CAR) and T cell subsets (CD4 vs. CD8 and CCR7 vs. CD45RO). For ARM CD19CAR (CD19C ART cells manufactured using the Activated Rapid Manufacturing (ARM) process) and TM-CD19CAR (CD19C ART cells manufactured using the Traditional Manufacturing (TM) process), the bottom left panel represents bulk culture and the right panel represents CAR+ T cells. "ARM-UTD" and "TM-UTD" refer to untransduced T cells (UTD) produced according to the ARM and TM processes, respectively. Numbers in quadrants indicate % of parental population. Boxes in TM-UTD and TM-CD19CAR plots indicate skew of the TM process towards TCM phenotype. Boxes in ARM-UTD and ARM-CD19CAR plots indicate maintenance of naïve-like cells by the ARM process. NA: Not applicable. Figure 28C is a graph showing end-to-end T cell composition of ARM-CD19CAR and TM-CD19CAR. Composition is shown for "bulk" and "CAR+" populations, where applicable. Percentages of each population refer to % of either parental, CD3+ or CAR+CD3+ as the case may be. % of CD4 cells of representative bulk or CAR+ populations are displayed. LKPK: Leukopack starting material; 4 and 8: CD4+ and CD8+, respectively; eff: effector; em: effector memory; cm: central memory; n: native-like. Data are representative of three full-scale studies with three different healthy donors (n=3) and several small-scale studies used to optimize the process. Figure 28D is a table showing the percentages shown in Figure 28C. [Figure 28-2] (As stated above.) [Figure 28-3] (As stated above.) [Figure 28-4] (As stated above.) [Figure 28-5] (As stated above.) [Figure 28-6] (As stated above.) [Figure 28-7] (As stated above.) [Figure 29-1] Figure 29: Cytokine concentrations in cell culture supernatants. IFN-γ (Figures 29A and 29B) and IL-2 (Figures 29C and 29D). Figures 29A and 29C: TM-CD19CAR, ARM-CD19CAR and representative UTD were co-cultured with NALM6-WT (ALL), TMD-8 (DLBCL) or cultured without cancer cells (T cells alone). After 48 h, the supernatants were collected. Figures 29B and 29D: ARM-CD19CAR was co-cultured with NALM6-WT, NALM6-19KO (CD19 negative) or cultured alone. After 24 h or 48 h, the supernatants were collected. To further evaluate antigen-specific cytokine secretion, ARM-CD19CAR was cultured alone for 24 h, washed and then co-cultured with target cells for 24 h. Data shown are from two healthy donor T cells and are representative of two experiments with a total of three donors. [Figure 29-2] (As stated above.) [Figure 29-3] (As stated above.) [Figure 29-4] (As stated above.) [Figure 30-1]Figure 30: Figure 30A is a graph showing an outline 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. ARM-CD19CAR cells were cultured for the periods indicated in the figure prior to flow cytometry analysis. Gating for 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. NGS mice were injected with the pre-B ALL line NALM6 expressing a luciferase reporter gene; tumor burden is expressed as total body luminescence (p / s) and shown as the mean tumor burden with 95% confidence interval. Mice were treated with each dose (number of viable CAR+T cells) of ARM-CD19CAR or TM-CD19CAR on the 7th day after tumor inoculation. The high dose ARM-CD19CAR group was stopped on day 33 due to the onset of X-GVHD. Vehicle (PBS) and untransduced T cells (UTD) were used as negative controls. n=5 mice for all groups except for n=4 for the ARM-UTD 1x106 dose and all TM-CD19CAR dose groups. Five xenograft studies were performed with CAR+T cells generated from five different healthy donors, three of which included comparisons with TM-CD19CAR. [Figure 30-2] (As stated above.) [Figure 30-3] (As stated above.) [Figure 30-4] (As stated above.) [Figure 30-5] (As stated above.) [Figure 30-6] (As stated above.) [Figure 30-7] (As stated above.) [Diagram 31]Plasma cytokine levels of NALM6 tumor-bearing mice treated with ARM-CD19CAR or TM-CD19CAR at each CAR-T cell dose. Mice were bled and plasma cytokines were measured by MSD assay. IFN-γ (Figures 31A and 31B) and IL-2 (Figures 31C and 31D) are shown for mice treated with CAR-T (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 (4th, 7th, 10th, 12th, 16th, 19th, 23rd, 26th days from the left). Horizontal bars and numbers indicate fold change comparison between ARM-CD19CAR (1x106 dose group) and TM-CD19CAR (0.5x106 dose group) represented by letters: 3-fold for IFN-γ; and 10-fold for IL-2. Groups withdrawn due to tumor burden or weight loss do not show the final time point. Plasma cytokine levels were measured for two trials. no tumour: no tumor. [Figure 32-1] 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 the indicated time points and are shown as mean cells with 95% confidence intervals. [Figure 32-2] (As stated above.) [Figure 32-3] (As stated above.) [Figure 32-4] (As stated above.) [Figure 33-1]Figure 33: IL-6 levels (pg / mL) in three-party co-culture supernatants. ARM-CD19CAR / K562 (Figure 33A) or TM-CD19CAR / K562 (Figure 33B) co-cultured cells, incubated for 6 or 24 hours at different ratios (1:1 and 1:2.5), were then added to PMA-differentiated THP-1 cells for another 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 designated ARM-CD19CAR only and TM-CD19CAR only represent CAR-T cell cultures without target cells (6h, 24h). Mean + SEM, n=1 (TM-CD19CAR) and n=3 (ARM-CD19CAR) of two replicates. [Figure 33-2] (As stated above.) [Diagram 34] The ARM process preserves BCMA CAR+ T cell stemness. RI61, R1G5 and BCMA10 CART cells produced using the ARM process were evaluated for CAR expression at thaw (Figure 34A) and 48h post-thaw (Figure 34B). CCR7 / CD45RO markers were also evaluated for the 48h post-thaw product (Figure 34C). Data shown are representative from two experiments performed with two donor T cells. [Diagram 35] The TM process yielded primarily central memory T cells (TCM) (CD45RO+ / CCR7+), while native-like T cell populations were largely lost in CAR+ T cells 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). CCR7 / CD45RO markers were also evaluated on the day 9 product after thawing (Figure 35B). Data shown are representative from two experiments performed with two donor T cells. [Diagram 36]ARM-treated BCMA CAR-T cells show BCMA-specific activation and secrete higher levels of IL2 and IFN-γ. IL2 and IFN-γ concentrations in cell supernatants. RI61, R1G5 and BCMA10 CART cells and their respective UTDs produced using the ARM or TM process were co-cultured with KMS-11 at a 2.5:1 ratio. Supernatants were collected after 20 h. For the ARM product, IFN-γ concentrations are shown in Figure 36A and IL-2 concentrations are shown in Figure 36B. For the TM product, IFN-γ concentrations are shown in Figure 36C and IL-2 concentrations are shown in Figure 36D. Data shown are representative from two experiments performed with two donor T cells. [Figure 37-1] Figure 37: 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 expressing cells per cell. The "nUMI" graph shows the number of unique molecular identifiers (UMIs) per cell. [Figure 37-2] (As stated above.) [Figure 37-3] (As stated above.) [Figure 38-1]Figure 38: T-distributed Stochastic Neighbor Embedding (TSNE) plots comparing input cells (Figure 38A), day 1 cells (Figure 38B), and day 9 cells (Figure 38C) for proliferation signatures, which were determined based on the expression of genes CCNB1, CCND1, CCNE1, PLK1, and MKI67. Each dot represents a cell in that sample. Cells shown as light grey 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 that include genes characteristic of resting versus activated T cell states for day 1, day 9, and input cells. In Figure 38D, higher gene set scores (Up resting vs. Down activated) indicate increased resting T cell phenotype, whereas lower gene set scores (Up resting vs. Down activated) indicate increased activated T cell phenotype. Input cells were overall more quiescent compared to day 9 and day 1 cells, with day 1 cells showing the highest activated gene set score. [Figure 38-2] (As stated above.) [Figure 38-3] (As stated above.) [Figure 38-4] (As stated above.) [Figure 39-1]Figure 39: Gene set analysis for input cells, day 1 cells and day 9 cells. In Figure 39A, a higher gene set score for the gene set "Up TEM vs. Down TSCM" indicates an increase in effector memory T cell (TEM) phenotype of cells in the sample, and a lower gene set score indicates an increase in stem cell memory T cell (TSCM) phenotype. In Figure 39B, a higher gene set score for the gene set "Up Treg vs. Down Teff" indicates an increase in regulatory T cell (Treg) phenotype, and a lower gene set score indicates an increase in effector T cell (Teff) phenotype. In Figure 39C, a lower gene set score for the gene set "Down stemness" indicates an increase in stemness phenotype. In Figure 39D, a higher gene set score for the gene set "Up hypoxia" indicates an increase in hypoxic phenotype. In Figure 39E, a higher gene set score for the gene set "Up autophagy" indicates an increase in autophagy phenotype. Day 1 cells appeared similar to input cells with respect to memory, stem-like and differentiation signatures, whereas day 9 cells showed high enrichment for metabolic stress. [Figure 39-2] (As stated above.) [Figure 39-3] (As stated above.) [Figure 39-4] (As stated above.) [Figure 39-5] (As stated above.) [Figure 40-1]Figure 40: Gene cluster analysis for input cells. Figures 40A-40C are violin plots showing gene set scores from gene set analysis of four clusters of input cells. Each dot overlaid on the violin plot in Figures 40A-40C represents the gene set score of a cell. In Figure 40A, a higher gene set score for the gene set "Up Treg vs. Down Teff" indicates an increase in Treg cell phenotype, and a lower gene set score for the gene set "Up Treg vs. Down Teff" indicates an increase in Teff cell phenotype. In Figure 40B, a higher gene set score for the gene set "Progressively up in memory differentiation" indicates an increase in late memory T cell phenotype, and a lower gene set score for the gene set "Progressively up in memory differentiation" indicates an increase in early memory T cell phenotype. In Figure 40C, a higher gene set score "Up TEM vs. Down TN" for the gene set indicates an increase in effector memory T cell phenotype, and a lower gene set score "Up TEM vs. Down TN" for the gene set indicates an increase in naive T cell phenotype. Cluster 3 cells are seen to be in a late memory, more differentiated T cell state compared to cells in cluster 1 and cluster 2, which are in an early memory, less differentiated T cell state. Cluster 0 is considered to be in an intermediate T cell state. Taken together, this data indicates a significant level of heterogeneity in the input cells. [Figure 40-2] (As stated above.) [Figure 40-3] (As stated above.) [Figure 41-1] Figure 41: TCR sequencing and measurement of clonotype diversity. Day 9 cells have a flatter distribution of clonotype frequencies (higher diversity). [Figure 41-2] (As stated above.) [Figure 41-3] (As stated above.) [Diagram 42]1 is a flow chart showing the design of a Phase I clinical trial testing BCMA CART cells manufactured using the ARM process in adult patients with relapsed and / or refractory multiple myeloma. [Diagram 43] Graph showing FACS analysis of ARM-BCMA CAR expression at various harvest time points after virus addition in the presence or absence of AZT at two different concentrations (30 μM and 100 μM). Lentiviral vector was added 1 hour late before AZT treatment at activation and cell inoculation. [Figure 44-1] Figure 44: Graph showing evaluation of ARM-BCMA CARs for CAR expression upon thawing (Figure 44A) and 48h post-thaw, as well as CCR7 / CD45RO markers of the 48h post-thaw product, and also evaluation of the TM-BCMA CAR at day 9 (Figure 44B). Data shown are representative from two experiments performed with T cells from two donors. [Figure 44-2] (As stated above.) [Figure 44-3] (As stated above.) [Diagram 45] Figure 1 shows cytokine concentrations in cell culture supernatants. ARM-BCMA and TM-BCMA CARs and representative UTDs were co-cultured with KMS-11. Supernatants were collected after 24 h. Data shown are representative from two experiments performed with T cells from two donors. [Figure 46] 1 is a graph outlining the xenograft efficacy study to test for ARM-BCMA. [Figure 47]Graph comparing efficacy of ARM-BCMA CAR with that of TM-BCMA CAR in xenograft model. NSG mice were injected with MM cell line KMS11 expressing luciferase reporter gene. Tumor burden is expressed as total body luminescence (p / s) and shown as mean tumor burden + SEM. Mice were treated with ARM-BCMA CAR or TM-BCMA CAR at respective doses (number of viable CAR+ T cells) on day 8 after tumor inoculation. Vehicle (PBS) and UTD T cells were used as negative controls. N=5 mice for all groups except N=4 for ARM-BCMA CAR (1e4 cells), PBS and UTD groups. [Figure 48-1] Figure 48: Graph showing plasma IFN-γ kinetics in mice treated with ARM-BCMA CAR or TM-BCMA CAR. Plasma IFN-γ levels in KMS11-luc tumor-bearing mice treated with the UTD of each CAR-T dose, ARM-BCMA CAR or TM-BCMA CAR. IFN-γ levels, all shown as mean ± SEM. Mice were bled and plasma cytokines were measured by Meso Scale Discovery (MSD) assay. [Figure 48-2] (As stated above.) [Figure 48-3] (As stated above.) [Figure 49] Graph showing cell kinetics of ARM-BCMA CAR and TM-BCMA CAR in vivo. Cell kinetics of peripheral blood of KMS11 tumor-bearing mice treated with TM UTD, ARM UTD, ARM-BCMA CAR and TM-BCMA CAR at different doses. Cell counts are expressed as mean cell count + SD. Mice were treated with ARM-BCMA CAR or TM-BCMA CAR at the respective doses (number of viable CAR+T cells) on day 8 after tumor inoculation. Vehicle (PBS) and UTD T cells were used as negative controls. Blood samples were taken 7, 14 and 21 days after CAR-T injection and analyzed by flow cytometry at the scheduled time points. N=5 mice for all groups except N=4 for ARM-BCMA CAR (1e4 cells), PBS and UTD groups. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] definition Unless otherwise defined, all technical and scientific terms used herein are intended to be understood as meaning those terms which are within the scope of the present invention. It has the same meaning as commonly understood by one of ordinary skill in the relevant technical field.

[0072] The terms "a" and "an" refer to one of the grammatical referents of the article. or more than one (i.e., at least one). By way of example, "an element" refers to one element. It means an element or more than one element.

[0073] The term "about" is used when referring to a measurable value, such as an amount, a duration in time, etc. ±20% from the value, or in some cases ±10%, or in some cases ±5%, or in some cases Variations of ±1%, or in some cases ±0.1%, are considered to be adequate for carrying out the methods of the present disclosure. is meant to be encompassed as being.

[0074] The compositions and methods of the invention include polypeptides and nucleic acids having designated sequences or sequences related thereto. Substantially identical or similar sequences, e.g., sequences that are at least 85%, 90% similar to the designated sequence In the context of amino acid sequences, the term "substantially identical" includes sequences that are 95% or more identical to one another. "of" as used herein means that the first and second amino acid sequences share a common structural domain and / or The amino acids in the second amino acid sequence may have a common functional activity. a sufficient or minimum number of amino acid residues that are i) identical to or ii) conservative substitutions thereof A first amino acid sequence containing amino acid residues, e.g., a reference sequence, e.g., a sequence described herein At least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 9 A gene that contains a common structural domain with 6%, 97%, 98% or 99% identity. It is used to refer to an amino acid sequence.

[0075] In reference to nucleotide sequences, the term "substantially identical" is used herein to refer to a first and and the second nucleotide sequence encode a polypeptide having a common functional activity or These polypeptides are designed to encode common structural polypeptide domains or common functional polypeptide activities. and a sufficient or maximum number of nucleotides identical to the aligned nucleotides in the second nucleic acid sequence. A first nucleic acid sequence, e.g., a reference sequence, e.g., a sequence described herein, that contains a small number of nucleotides. At least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, Nucleotides with 94%, 95%, 96%, 97%, 98% or 99% identity Used to point to an array.

[0076] The term "variant" refers to a variant that has an amino acid sequence that is substantially identical to a reference amino acid sequence, or It refers to a polypeptide encoded by substantially the same nucleotide sequence. In this embodiment, the variant is a functional variant.

[0077] The term "functional variant" refers to a variant that has substantially identical amino acid sequence to a reference amino acid sequence. or a polypeptide encoded by a substantially identical nucleotide sequence, It refers to a polypeptide that may have one or more activities of a sequence of amino acids.

[0078] The term cytokine (e.g., IL-2, IL-7, IL-15, IL-21 or IL- 6) can be used to express full-length naturally occurring cytokines, fragments or variants, e.g., functional variants ( At least 10%, 30% of the activity, e.g., immunomodulatory activity, of a naturally occurring cytokine; The present invention includes fragments and functional variants thereof having 50% or 80% of the original structure. In embodiments, the cytokine is substantially identical (e.g., at least At least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98% or 99% identity) or a naturally occurring polypeptide encoding a cytokine. nucleotide sequence substantially identical (e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9 9% identity) of the amino acid sequence encoded by the nucleotide sequence. In this embodiment, as will be understood from the context, the cytokine may be a receptor domain, e.g. It further comprises a cytokine receptor domain (eg, IL-15 / IL-15R).

[0079] The term "chimeric antigen receptor" or alternatively "CAR" refers to at least one chimeric antigen receptor, as defined below. Both have extracellular antigen-binding domains, transmembrane domains, and functional signal transduction from stimulatory molecules. A cytoplasmic signaling domain (referred to herein as an "intracellular signaling domain") that includes a In some embodiments, the CAR polypeptide refers to a recombinant polypeptide construct comprising a CAR polypeptide. The domains in the polypeptide construct may be arranged in the same polypeptide chain, e.g., as a chimeric fusion protein. In some embodiments, the domains in the CAR polypeptide construct are adjacent to each other. For example, in different polypeptide chains, e.g., RCAR as described herein. Provided within.

[0080] In some embodiments, the cytoplasmic signaling domain is a primary signaling domain. (e.g., the primary signaling domain of CD3ζ). The signaling domain comprises one or more domains derived from at least one costimulatory molecule as defined below. In some embodiments, the costimulatory molecule further comprises a functional signaling domain of 1BB (i.e., CD137), CD27, ICOS and / or CD28 In some embodiments, the CAR is a chimeric fusion protein comprising an extracellular antigen recognition domain. , an intracellular signaling domain that includes a transmembrane domain and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a CAR comprising an extracellular antigen recognition domain. Functional signaling domains derived from melanoma fusion proteins, transmembrane domains and costimulatory molecules The present invention provides an intracellular signaling domain that includes a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain. It contains two functional signaling domains derived from a protein, a transmembrane domain, and one or more costimulatory molecules. Intracellular signaling domains, including functional signaling domains derived from cytokines and stimulatory molecules In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain. The protein comprises a transmembrane domain and at least two functional signals from one or more costimulatory molecules. Intracellular signaling domains containing a null transduction domain and a functional signal transduction domain derived from a stimulatory molecule In some embodiments, the CAR comprises a transduction domain at the amino terminus of the CAR fusion protein. In some embodiments, the CAR comprises an optional leader sequence at the N-terminus. The antigen recognition domain further comprises a leader sequence at the N-terminus thereof, which leader sequence is capable of binding to a cellular promoter. During cloning and localization of the CAR to the cell membrane, the antigen recognition domain (e.g., scFv) is released Optionally disconnected.

[0081] A particular tumor antigen X, such as those described herein (X is a tumor antigen as described herein). An antigen-binding domain (e.g., scFv, single domain) that targets a target gene (which may be a tumor marker) a TCRα-binding domain or a TCRβ-binding domain) A CAR comprising an antigen-binding domain that targets BCMA is also referred to as an XCAR. A CAR comprising the BCMA polypeptide is referred to as a BCMA CAR. A CAR can be used in any cell, for example, as described herein. can be expressed in immune effector cells (e.g., T cells or NK cells) described in .

[0082] The term "signaling domain" refers to a domain that acts by generating second messengers. or function as effectors by responding to such messengers. and delivers information intracellularly to regulate cellular activity via identified signaling pathways. It refers to the functional portion of a protein that acts by transmitting a signal.

[0083] The term "antibody" as used herein refers to an immunoglobulin that specifically binds to an antigen. Antibodies refer to a protein or polypeptide sequence derived from a molecule. The immunoglobulins may be monoclonal, multi-chain or single-chain or intact immunoglobulins, and may be derived from naturally occurring sources. The antibody may be a tetramer of immunoglobulin molecules.

[0084] The term "antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof. It refers to the body and provides recognition and specific binding of the antibody fragment to a target, e.g., an antigen. "Antibody" refers to an antigen-binding domain sufficient to bind an antigen, such as the antigenic variable region of an intact antibody. Examples of antibody fragments are Fab, Fab', F(ab')2 and Fv fragments, s cFv antibody fragments, linear antibodies, single domain antibodies such as sdAbs (VL or VH (either one) and the camelid VHH domain and the hinge region are linked by disulfide bridges. two or more linked together, e.g., two Fab fragments or two or more linked antibodies, e.g. Antibody fragments, such as bivalent fragments containing two isolated CDRs or other epitope fragments, These include, but are not limited to, multispecific antibodies formed from antibody fragments. The segments include single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, and bodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (e.g., Hollinger and Hudson, Nature (See Biotechnology 23:1126-1136, 2005) Antibody fragments are based on polypeptides such as fibronectin type III (Fn3). The fibronectin polypeptide minibody can also be grafted onto a scaffold that supports the (See U.S. Patent No. 6,703,199, which describes the term "scFv " refers to at least one antibody fragment containing the variable region of a light chain and at least one antibody fragment containing the variable region of a heavy chain. The term refers to a fusion protein comprising at least one antibody fragment, including a light chain and a heavy chain variable domain. The domains are closely linked by short flexible polypeptide linkers, forming a single polypeptide chain. The scFv can be expressed as a peptide and can be expressed as an intact peptide from which it is derived. Unless otherwise specified, as used herein, scFv The VL and VH variable regions are, for example, either at the N-terminal or C-terminal end of the polypeptide. and the scFv may comprise a VL-linker-VH or a VH-linker -VL.

[0085] In some embodiments, the scFv is 2 -V L -Linker-V H -COOH or NH 2 -V H -Linker-V L It may include the structure -COOH.

[0086] As used herein, the term "complementarity determining region" or "CDR" refers to a region that determines antigen specificity and The term refers to the sequence of amino acids within an antibody variable region that confers binding affinity and specificity to the antibody. For example, typically, each heavy chain There are three CDRs in the variable region (e.g., HCDR1, HCDR2, and HCDR3), Each light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3). The exact amino acid sequence boundaries of a given CDR are determined by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest”,5th Ed.Public Health Service,N ational Institutes of Health,Bethesda,MD ("Kabat" numbering scheme), Al-Lazikani et al.,(19 97) JMB 273,927-948 ("Chothia" numbering scheme) Determination using any of a number of well-known schemes, including those described or combinations thereof. Yes, some implementations of the combined Kabat and Chothia numbering scheme In some embodiments, the CDRs may be a portion of the Kabat CDRs, a portion of the Chothia CDRs, or corresponds to an amino acid residue that is both.

[0087] The portion of the CAR composition of the invention that comprises an antibody or antibody fragment thereof may exist in a variety of forms. For example, where the antigen binding domain is, for example, a single domain antibody fragment. (sdAb), single chain antibody (scFv) or polyclonal antibody, including, for example, human or humanized antibodies. It is expressed as part of a peptide chain (Harlow et al., 1999, Using Antibodies:A Laboratory Manual,Cold Spr. ing Harbor Laboratory Press, NY; Harlow et al. 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 242:423-42 6). In some embodiments, the antigen-binding domain of the CAR of the present invention comprises an antibody fragment. In some embodiments, the CAR comprises an antibody fragment comprising an scFv.

[0088] As used herein, the term "binding domain" or "antibody molecule" (as used herein as " The anti-target binding domain (also referred to as the "anti-target binding domain") is composed of at least one immunoglobulin variable domain. The term "binding site" refers to a protein comprising the binding site, such as an immunoglobulin chain or a fragment thereof. "Binding domain" or "antibody molecule" includes antibodies and antibody fragments. In some embodiments, the antibody molecule is a multispecific antibody molecule, e.g., it is an antibody molecule that binds multiple immunoglobulins. a first immunoglobulin variable domain of the plurality, The sequence has binding specificity for a first epitope and is associated with a second immunoglobulin of the plurality. The variable domain sequence has binding specificity for a second epitope. Thus, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody is a molecule that contains two or less Bispecific antibody molecules have binding specificity for a first epitope. A first immunoglobulin variable domain sequence having a different affinity and binding to a second epitope. The antibody is characterized by a second immunoglobulin variable domain sequence having specificity.

[0089] The terms "bispecific antibody" and "multiple bispecific antibodies" refer to the combination of two antibodies in a single molecule. A bispecific antibody is a molecule that binds two antigens simultaneously or sequentially. Bispecific antibodies can bind to different antibodies. Methods for making bispecific antibodies are known in the art. Various formats for binding two antibodies are also known in the art. As is well known to those skilled in the art, examples of bispecific antibody forms include diabodies, single chain antibodies, and the like. , Fab dimerization (Fab-Fab), Fab-scFv and tandem antibodies. But not limited to these.

[0090] The term "antibody heavy chain" refers to the two types of heavy chains present in an antibody molecule in their naturally occurring conformation. The term "antibody class" refers to the larger of the polypeptide chains, which usually determines to which class the antibody belongs.

[0091] The term "antibody light chain" refers to the two types of light chain present in an antibody molecule in its naturally occurring conformation. The smaller polypeptide chain of the IL-10 family. Kappa (κ) and lambda (λ) light chains are the two major This refers to the antibody light chain isotype.

[0092] The term "recombinant antibody" refers to an antibody produced, for example, by a bacteriophage or yeast expression system. The term refers to antibodies made using recombinant DNA technology, such as antibodies expressed A DNA molecule encoding the antibody protein or a DNA molecule encoding the antibody protein. It should also be construed to mean an antibody produced by synthesis of a specified amino acid sequence. The DNA or amino acid sequences may be derived from recombinant DNA or This was obtained using amino acid sequencing techniques.

[0093] The term "antigen" or "Ag" refers to a molecule that provokes an immune response. This may involve either or both antibody production or activation of specific immunologically competent cells. Any macromolecule can be an antigen, including virtually any protein or peptide. It will be understood that the antigens may be derived from recombinant or genomic DNA. The vendor therefore provides nucleotide sequences or fragments that code for proteins that generate an immune response. Any DNA containing a partial nucleotide sequence is referred to as such by the term as used herein. It will be understood that the term "antigen" may be used interchangeably with "antigen" to refer to a gene that encodes an antigen. It will be understood that the nucleic acid sequence need not be encoded solely by the full-length nucleotide sequence of the nucleic acid. The present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene. and that their nucleotide sequences are polypeptides that produce the desired immune response. It is readily apparent that the sequences may be arranged in various combinations to code for the sequences. However, those skilled in the art will appreciate that an antigen need not necessarily be encoded by a "gene." Antigens can be synthetically produced, obtained from biological samples, or can be polypeptides. It is readily apparent that the biological sample may contain macromolecules other than tides. These include, but are not limited to, tissue samples, tumor samples, cells or body fluids along with other biological components. obtain.

[0094] The terms "anti-tumor effect" and "anti-cancer effect" are used interchangeably and include, but are not limited to, for example, A reduction in tumor or cancer volume, a reduction in the number of tumor or cancer cells, a reduction in the number of metastases, Increased life expectancy, reduced tumor cell proliferation or cancer cell proliferation, tumor cell survival or cancer cell By a variety of means, including reducing survival or ameliorating various physiological symptoms associated with cancerous conditions. "Anti-tumor effect" or "anti-cancer effect" refers to a biological effect that may be exerted by the peptide of the present invention. The polynucleotides, cells and antibodies are also manifested by their ability to prevent the development of tumors or cancer. obtain.

[0095] The term "autologous" refers to any material derived from the same individual that is later reintroduced into that individual. This refers to the material that will be used.

[0096] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are considered identical to each other when the genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species is said to be allogeneic. , may be sufficiently genetically distinct to interact antigenically.

[0097] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0098] As used herein, the term "apheresis" refers to the process of transferring blood from a donor or patient to a donor or Blood is taken from the patient, passed through a device that separates selected components, and then used, for example, for reinfusion. This refers to an art-recognized ex vivo procedure in which the remainder is then returned to the donor or patient. Therefore, in reference to an "apheresis sample," a sample obtained using apheresis is Refers to a sample.

[0099] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of cancers are described herein, including but not limited to breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, These include cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer. In some embodiments, the cancer treated by the methods described herein is multiple myeloma, Hodgkin's Includes lymphoma or non-Hodgkin's lymphoma.

[0100] The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms refer to It includes solid and liquid, e.g., diffuse or circulating tumors. The term "cancer" or "tumor" includes pre-cancerous and malignant cancers and tumors.

[0101] "Derived from," as the term is used herein, refers to the association of a first molecule with a second molecule. This generally refers to the structural similarity between a first molecule and a second molecule. and limiting the manner or source by which the first molecule is derived from the second molecule. For example, the intracellular signaling domain derived from the CD3ζ molecule does not imply or encompass In the case of The CD3ζ structure is sufficient to generate CD3 nucleases. It does not imply or imply any limitation to a particular method of making the signaling domain, e.g., in cells. To provide the intracellular signaling domain, unnecessary sequences were deleted, beginning with the CD3ζ sequence. or mutations must be introduced to direct the expression of the intracellular signaling domain. Doesn't mean that.

[0102] The term "conservative sequence modifications" refers to the binding of an antibody or antibody fragment containing that amino acid sequence. Conservative modifications refer to amino acid modifications that do not significantly affect or change the properties of the amino acid. Such conservative modifications include Modifications include amino acid substitutions, additions and deletions. The antibodies or polypeptides of the invention can be synthesized by standard techniques known in the art, such as mediated mutagenesis. Conservative substitutions are those in which the amino acid residues share similar side chains. The amino acid residues having the same side chains are substituted with amino acid residues having the same side chains. Families are defined in the art. These families include those that contain basic side chains. (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine , threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, leucine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β Branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, The amino acids include those having the following amino acids: glycine, phenylalanine, tryptophan, and histidine. Thus, one or more amino acid residues in the CAR of the invention may be linked to other amino acid residues from the same side chain family. The amino acid residues can be replaced and altered in the CAR, and the altered CAR can have the functions described herein. The activity can be tested using a functional assay.

[0103] In the context of stimulation by stimulatory and / or costimulatory molecules, the term "stimulation" refers to the stimulation of a stimulatory molecule (e.g. For example, the TCR / CD3 complex) and / or costimulatory molecules (for example, C28 or 4-1BB Responses induced by binding of a ligand to its cognate ligand, such as a primary or secondary response. This refers to, but is not limited to, signal transduction via the TCR / CD3 complex. Stimuli mediate signaling events in the nervous system, resulting in changes in the expression of specific molecules and / or cytoskeletal structures. It can mediate reorganization of the body.

[0104] The term "stimulatory molecule" refers to a molecule that is expressed by a T cell and that mediates at least one of the T cell signaling pathways. For some embodiments, the primary cytoplasmic signal transduction pathway that regulates primary activation of the TCR complex is In some embodiments, the ITAM-containing domain in a CAR is In some embodiments, the TCR signaling pathway is recapitulated by the primary TCR independent of the endogenous TCR complex. So the primary signal is, for example, the TCR / CD3 complex and the peptide-loaded MHC molecule. The primary signaling pathway is initiated by binding to IL-1, which is involved in, but not limited to, proliferation, activation, and These primary cytoplasmic signals act in a stimulatory manner to mediate T cell responses, including differentiation, proliferation, and differentiation. The primary signaling domain (also called the primary transduction domain) is a tyrosine-based domain that mediates the expression of immunoreceptors. The present invention may include signal transduction motifs known as ITAMs or activation motifs for the transcription factor receptor. An example of an ITAM-containing primary cytoplasmic signaling sequence that is particularly useful in Rζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD7 9a, CD79b, CD278 (also known as "ICOS"), FcεRI and CD 66d, DAP10 and DAP12. In particular CARs of the invention, the cells of any one or more CARS of the invention The intracellular signaling domain is a domain that encodes an intracellular signaling sequence, e.g., the primary signaling domain of CD3-ζ. The term "antigen-presenting cell" or "APC" refers to a cell that contains major histocompatibility genes on its surface. The immune system, including accessory cells that present foreign antigens complexed with the major histocompatibility complex (MHC) T cells are cells that express their T cell receptor (TCR) APCs can recognize these complexes using the antigen receptor. Present to.

[0105] An "intracellular signaling domain," as that term is used herein, is a domain that is In embodiments, the intracellular signal domain is an effector function signal. They transduce the nucleic acid that instructs the cell to carry out a specific function. Although the entire chain can be used, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of a null transduction domain is used, such a truncated portion may be It may be used in place of the complete chain as long as it transmits the effector function signal. The intracellular signaling domain therefore provides sufficient intracellular signaling for effector function signal transduction. It is intended to include any truncated portion of the intracellular signaling domain.

[0106] The intracellular signaling domain controls the immune effector of the CAR-containing cell, e.g., the CART cell. For example, they generate signals that promote immune effector functions of CART cells. Examples include helper activities, including cytolytic activity and secretion of cytokines.

[0107] In some embodiments, the intracellular signaling domain is a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include a primary stimulus- or antigen-dependent domain. In some embodiments, the intracellular signaling pathway is mediated by a signal transduction pathway that is involved in sexual stimulation. The core transduction domain may include a costimulatory intracellular domain. The reach domain may be derived from a molecule involved in costimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain is the T cell receptor The co-receptor or co-stimulatory intracellular signaling domain may comprise a cytoplasmic sequence, It may include cytoplasmic sequences from a stimulatory molecule.

[0108] The primary intracellular signaling domain is an immunoreceptor tyrosine-based activation motif or ITAM. These include signaling motifs known as ITAM-containing primary cytoplasmic signaling motifs. Examples of null transfer sequences include, but are not limited to, CD3ζ, FcRγ, FcRβ, CD3γ, C D3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (“ICO S), derived from FcεRI, CD66d, DAP10 and DAP12. This includes:

[0109] The term "zeta" or alternatively "zeta chain", "CD3-zeta" or "TCR-zeta" refers to the 47. Swiss-Prot Accession No. P20963 refers to an exemplary human CD3 ζ amino acid sequence. The "zeta stimulatory domain" or alternatively the "CD3-zeta stimulatory domain" provides the amino acid sequence of Alternatively, a "TCR-ζ stimulatory domain" may refer to a CD3ζ or a variant thereof (e.g., a mutant, e.g., "Stimulatory domain" refers to a stimulatory domain of a polypeptide (e.g., a molecule having a point mutation, fragment, insertion or deletion). In some embodiments, the cytoplasmic domain of ζ is selected from the group consisting of those listed in GenBank Accession No. BAG36664. Residues 52 to 164 of .1 or a variant thereof (e.g., a mutation, e.g., a point mutation, a flag In some embodiments, the "zeta-stimulatory domain" includes a molecule having a "zeta-stimulatory domain" (a molecule having a "zeta-stimulatory domain", an insertion or deletion). The "CD3-ζ stimulatory domain" or "CD3-ζ stimulatory domain" is a sequence represented by the sequence provided as SEQ ID NO: 9 or 10. or variants thereof (e.g., mutations, such as point mutations, fragments, insertions or deletions) A molecule with a loss.

[0110] The term "costimulatory molecule" refers to a molecule that specifically binds to a costimulatory ligand, thereby inhibiting T cell proliferation and metabolism. cognate binding part on T cells that mediates costimulatory responses, including but not limited to proliferation Costimulatory molecules are molecules other than antigen receptors or their ligands that are necessary for an efficient immune response. Costimulatory molecules are cell surface molecules that act as a signaling pathway for immune cells, such as MHC class I molecules, TNF receptor proteins, and Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytes Sex activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligase androcyte receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, C DS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137) , B7-H3, CDS, ICAM-1, ICOS(CD278), GITR, BAFFR , LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80( KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, C D8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, I TGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, C D11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM , CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18 , LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RA NKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), C D160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAM F6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3) , BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GA DS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4 Ligands that specifically bind to CD83 and CD85 include, but are not limited to, CD85-1BB and ligands that specifically bind to CD83. There is none.

[0111] A costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule.

[0112] An intracellular signaling domain is the entire intracellular portion of the molecule from which it is derived or the naturally occurring It may include the entire intracellular signaling domain or a functional fragment thereof.

[0113] The term "4-1BB" refers to CDR137 or a member of the tumor necrosis factor receptor superfamily. Swiss-Prot accession number P20963 refers to an exemplary human 4-1BB The amino acid sequence of the 4-1BB costimulatory domain is provided. or variants thereof (e.g., mutations, such as point mutations, fragments, insertions or In some embodiments, a "4-1BB costimulatory domain" refers to a molecule having the sequence The sequence provided as sequence number 7 or a variant thereof (e.g., a mutation, e.g., a point mutation, a molecule having a sequence, fragment, insertion or deletion.

[0114] "Immune effector cells," as that term is used herein, are cells that mediate an immune response, e.g. For example, they refer to cells that are involved in promoting immune effector responses. Examples of immune effector cells include , T cells, such as α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells These include lymphocytes, natural killer T (NKT) cells, mast cells and myeloid-derived phagocytes.

[0115] "Immune effector function or immune effector response" refers to an immune response as that term is used herein. When administered, the antibody enhances or promotes immune attack of target cells, e.g., by immune effector cells. For example, an immune effector function or response may be the killing or proliferation of a target cell. It refers to the property of T cells or NK cells that promotes proliferation or inhibits proliferation. Stimulation and costimulation are examples of immune effector functions or responses.

[0116] The term "effector function" refers to the specialized function of a cell. Effector T cells The function can be, for example, cytolytic activity or helper activity, including secretion of cytokines.

[0117] The term "encodes" refers to a defined nucleotide sequence (e.g., rRNA, tRNA, A and mRNA) or in biological processes that have a defined amino acid sequence Genes, cDNA or other molecules that serve as templates for the synthesis of other polymers and macromolecules. The unique properties of specific nucleotide sequences within a polynucleotide, such as mRNA, and Thus, a gene, cDNA, or RNA refers to a biological property resulting from that Proteins are produced in cells or other living systems by transcription and translation of the mRNA corresponding to a gene. It encodes a protein when expressed, and its nucleotide sequence is identical to the mRNA sequence. and as a transcription template for the coding strand and gene or cDNA, which are usually provided in the sequence listing. Both the coding and non-coding strands of the cDNA for that gene encode the protein or other product. This can be referred to as loading.

[0118] Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" are degenerate versions of each other. A protein is a sequence of a nucleic acid that contains all of the nucleotide sequences that code for the same amino acid sequence. The phrase "a nucleotide sequence encoding a protein or RNA" includes the sequence that encodes that protein. To the extent that the nucleotide sequence may, in any version, contain one or more introns, Introns may also be included.

[0119] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein and refer to a therapeutically effective amount for a particular organism. The compounds, formulations, materials or compositions described herein are effective in achieving a biological result. Refers to the quantity of a product.

[0120] The term "endogenous" refers to a substance that is produced from or within an organism, cell, tissue, or system. This refers to any substance that has been

[0121] The term "exogenous" refers to something that is introduced from or produced outside an organism, cell, tissue, or system. The term refers to any material that is produced by

[0122] The term "expression" refers to the transcription and / or translation of a particular nucleotide sequence. In some embodiments, expression includes translation of mRNA that has been introduced into a cell.

[0123] The term "transfer vector" refers to a composition containing an isolated nucleic acid, which is used for the simple delivery of the nucleic acid to the interior of a cell. The term refers to compositions that can be used to deliver isolated nucleic acids. Polynucleotides, plasmids and related compounds, ionic or amphiphilic compounds Many vectors are known, including viruses. Hence the term "transfer vector." " includes autonomously replicating plasmids or viruses. The term includes, for example, polylysine-linked Non-plasmid and non-viral vectors that facilitate the transfer of nucleic acids into cells, such as compounds and liposomes. The term should be construed to further include viral compounds. Examples of vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, and vectors, retroviral vectors, lentiviral vectors, etc.

[0124] The term "expression vector" refers to an expression control vector operably linked to a nucleotide sequence to be expressed. An expression vector refers to a vector that contains a recombinant polynucleotide that contains a sequence sufficient for expression. The gene contains the necessary cis-acting elements, with other expression elements being supplied or not provided by the host cell. The expression vector can be used to carry a recombinant polynucleotide. Integrating cosmids, plasmids (e.g., naked or contained in liposomes) and Viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) These include all those known in the art, including fluorouracil, fluorouracil, fluororus, fluorouracil ...

[0125] The term "lentivirus" refers to a genus in the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells. Uniquely, lentiviruses are able to deliver large amounts of genetic information into the DNA of host cells. This makes it one of the most efficient gene delivery vectors available. , SIV and FIV are all examples of lentiviruses.

[0126] The term "lentiviral vector" refers in particular to the vectors described in Milone et al., Mol. Self-inactivation as provided in Her. 17(8):1453-1464 (2009) Vectors derived from at least a portion of a lentiviral genome, including lentiviral vectors. Other examples of lentiviral vectors that can be used clinically include, but are not limited to, However, for example, LENTIVECTOR (registered trademark) from Oxford BioMedica Gene delivery technology, LENTIMAX™ Vectors from Lentigen Preclinical lentiviral vectors are also available. It will be known to those skilled in the art.

[0127] The terms "homology" or "identity" refer to the degree of similarity between two polymeric molecules, e.g., between two DNA molecules or Subunits between two nucleic acid molecules, such as two RNA molecules, or between two polypeptide molecules. The term refers to unit sequence identity. For example, when each position of two DNA molecules is occupied by an adenine, If two sequences are occupied by the same amino acid sequence, then they are homologous or identical at that position. The sex is a direct function of the number of matching or homologous positions in two sequences. Half of the positions (e.g., 5 positions in a polymer 10 subunits long) are homologous. If 90% of the positions (e.g., 9 out of 10) are identical, then the two sequences are 50% identical. If two sequences (one or more) are matched or homologous, then the two sequences are 90% homologous.

[0128] "Humanized" forms of non-human (e.g., murine) antibodies are derived from non-human immunoglobulins. Chimeric immunoglobulins, immunoglobulin chains or fragments thereof (antibody Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences. In most cases, humanized antibodies and antibody fragments thereof are produced by combining human immunoglobulins (recipients) Residues from the recipient's complementarity determining regions (CDRs) in the recipient antibody or antibody fragment The group may be cultured in a non-human species such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, the CDRs of a human antibody are replaced with residues from the CDRs of the donor antibody. Fv framework region (FR) residues of the immunoglobulin are replaced by corresponding non-human residues Furthermore, the humanized antibody / antibody fragment can be transferred to the recipient antibody. These modifications may include residues that are not found in the CDR or framework sequences. The performance of the antibody or antibody fragment may be further refined and optimized. Generally, humanized antibodies or an antibody fragment thereof, which comprises at least one, and typically two, variable domains. and all or substantially all of the CDR regions are non-human immunoglobulins. and all or most of the FR regions are from human immunoglobulin sequences. Humanized antibodies or antibody fragments are made by deleting the immunoglobulin constant region (Fc), typically human. For further details, the antibody may also include at least a portion of the Fc of a human immunoglobulin. Jones et al., Nature, 321:522-525, 1986; eichmann et al.,Nature,332:323-329,1988; Presta,Curr.Op.Struct.Biol.,2:593-596,19 Please see reference 92.

[0129] "Fully human" refers to an antibody or immunoglobulin molecule in which the entire molecule is of human origin or in a human form. It refers to immunoglobulins such as antibodies or antibody fragments that have the same amino acid sequence as vinegar.

[0130] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally occurring in a living animal is referred to as "isolated." The same is not "alive" but is partially or completely separated from the coexisting materials in its natural state. The same nucleic acid or peptide is "isolated." An isolated nucleic acid or protein is substantially purified. The vector may exist in a modified form or may exist in a non-native environment, such as a host cell. It is possible.

[0131] In the context of the present invention, the following abbreviations are used for the commonly occurring nucleic acid bases: "A" stands for adenosine, "C" stands for cytosine, and "G" stands for guanosine. where "T" refers to thymidine and "U" refers to uridine.

[0132] The terms "operably linked" or "transcriptional control" refer to a regulatory sequence and a heterologous nucleic acid sequence. For example, a first nucleic acid sequence may be linked to a second nucleic acid sequence to provide for expression of the latter in the context of a gene that is expressed ... A first nucleic acid sequence functions in a functional relationship with a second nucleic acid sequence when placed in a functional relationship with the second nucleic acid sequence. For example, a promoter can affect the transcription or expression of a coding sequence. A promoter is operably linked to a coding sequence if it has an effect on the transcription of the gene. The DNA sequences to be cloned may be contiguous with one another, for example to join two protein coding regions. If necessary, they should be in the same reading frame.

[0133] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous ( iv), intramuscular (im), or intrasternal injection, intratumoral, or infusion techniques.

[0134] The terms "nucleic acid," "nucleic acid molecule," "polypeptide," or "polynucleotide molecule" refer to a Deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single-stranded or double-stranded form and polymers thereof. Unless specifically limited, the term includes the same as the reference nucleic acid. Natural nucleotides that have the same binding properties as naturally occurring nucleotides and are metabolized in the same way as naturally occurring nucleotides. In some embodiments, the term "nucleic acid" refers to a nucleic acid that contains a known analog of a nucleic acid. "Polynucleotide molecule", "polypeptide" or "polynucleotide molecule" refers to a nucleotide / nucleoside molecule. Unless otherwise indicated, a particular nucleic acid sequence implicitly includes any derivative or analog thereof. Conservatively modified variants (e.g., degenerate codon substitutions, e.g., conservative substitutions), alleles, It also includes allologs, SNPs and complementary sequences as well as sequences explicitly indicated. Degenerate codon substitutions, e.g., conservative substitutions, are substitutions of the 3′-nucleotide sequence of one or more selected (or all) codons. To generate sequences in which the 10th position is substituted with a mixed base and / or a deoxyinosine residue. This can be achieved by Res.19:5081(1991);Ohtsuka et al., J.Biol. Chem. 260:2605-2608 (1985); and Rossolini et al. al., Mol. Cell. Probes 8:91-98 (1994)).

[0135] The terms "peptide," "polypeptide," and "protein" are used interchangeably. A compound consisting of amino acid residues covalently linked by tethered bonds. A protein or peptide must contain at least two amino acids and be a part of a protein. There is no limit to the maximum number of amino acids that a sequence or peptide can contain. A peptide is any peptide that contains two or more amino acids joined together by peptide bonds. As used herein, this term includes peptides or proteins. Short chains, also commonly referred to as peptides, oligopeptides and oligomers, and those of skill in the art The term "polysaccharide" refers to both the many different types of long chains commonly referred to as proteins. "Peptide" includes, for example, biologically active fragments, substantially homologous polypeptides, , oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides Polypeptides include, among others, naturally occurring peptides, derivatives, analogs, and fusion proteins. , recombinant peptides, or combinations thereof.

[0136] The term "promoter" refers to a molecule required to initiate the specific transcription of a polynucleotide sequence. The term "antigen-binding domain" refers to a DNA sequence that is recognized by the synthetic machinery of a cell or that has been introduced into the cell.

[0137] The term "promoter / regulatory sequence" refers to a sequence that is operably linked to the promoter / regulatory sequence. This refers to a nucleic acid sequence necessary for expression of a gene product derived from a core promoter. In other instances, this sequence may be an enhancer sequence and a promoter sequence that regulates expression of the gene product. The promoter / regulatory sequence may also contain other regulatory elements necessary for the production of the gene. The object may be expressed in a tissue-specific manner.

[0138] The term "constitutive" promoter refers to a polynucleotide that encodes or specifies a gene product. When operably linked to a nucleotide, it is capable of functioning in cells under most or all physiological conditions of the cell. It refers to a nucleotide sequence that results in the production of a gene product in the cell.

[0139] The term "inducible" promoter refers to a polynucleotide that encodes or specifies a gene product. An inducer that substantially corresponds to a promoter when operably linked to a nucleotide. A nucleotide sequence that causes the production of a gene product in a cell only if the nucleotide sequence is present in the cell. Point to the column.

[0140] The term "tissue-specific" promoter refers to a promoter that encodes or is specified by a gene. In effect, the cell responds to the promoter when the promoter is operably linked to a polynucleotide that A nucleotide that causes the production of a gene product in cells only if they are cells of the corresponding tissue type. Refers to the octide sequence.

[0141] The terms "cancer associated antigen," "tumor antigen," "hyperproliferative disorder antigen," and "hyperproliferative disorder antigen" are used interchangeably herein. "Associated antigen" refers interchangeably to an antigen common to a particular hyperproliferative disorder. In certain embodiments, these terms refer to the expression of a cellular marker, either in whole or in fragments (e.g., MHC A molecule (typically a protein, carbohydrate or lipid) that is expressed as a peptide (or peptides). and refers to molecules useful for preferential targeting of pharmacological agents to cancer cells. In some embodiments, tumor antigens are markers that are expressed by both normal and cancer cells, such as lineage In some embodiments, the tumor antigen is a marker, such as CD19 on B cells. Overexpression in cancer cells compared to normal cells (e.g., 1-fold overexpression, 2-fold overexpression compared to normal cells) In some embodiments, the tumor antigen is a cell surface molecule that is overexpressed (currently overexpressed 3-fold or more). Cell surface molecules that are inappropriately synthesized in cancer cells, e.g., deleted compared to molecules expressed on normal cells In some embodiments, the tumor antigen is a molecule that contains a nucleotide sequence, an addition, or a mutation on the cell surface of a cancer cell. It is expected that the MHC / peptide fragments will be expressed only in the In some embodiments, the methods of the present invention are directed to a method for the preparation of a cellular endothelial cell line comprising: Hyperproliferative disorder antigens include primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, Liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, Kidney cancer, breast cancer, prostate cancer (e.g., castration-resistant or treatment-resistant prostate cancer or metastasis) prostate cancer), ovarian cancer, pancreatic cancer, etc., or plasma cell proliferative disorders, such as asymptomatic myeloma ( Smoldering multiple myeloma or indolent myeloma), monoclonal gamma-gamma of undetermined significance macroglobulinemia (MGUS), Waldenström's macroglobulinemia, plasmacytoma (e.g. For example, plasmacytosis, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multilineage. Plasmacytoma), systemic light-chain amyloidosis and POEMS syndrome (also known as Crow- Also known as Fukase syndrome, Takatsuki disease and PEP syndrome In some embodiments, the CAR of the present invention is derived from an MHC-presented peptide. The CAR comprises an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to the CAR. Normally, peptides derived from endogenous proteins are bound to major histocompatibility complex (MHC) clones. It fits into a pocket on the RasI molecule and is activated by the T cell receptor (TCR) on CD8+ T lymphocytes. The MHC class I complex is constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes are used as immunotherapy targets. A unique class of cell surface targets. Human leukocyte antigen (HLA)-A1 or HLA- TCR-like antibodies targeting peptides derived from viral or tumor antigens in the context of A2 As described (e.g., Sastry et al., J Virol. 2011 8 5(5):1935-1942;Sergeeva et al.,Blood,201 1 117(16):4262-4272;Verma et al.,J Immun ol 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;T assev et al.,Cancer Gene Ther 2012 19(2) For example, TCR-like antibodies can be produced using human scFv phage dimers. They can be identified by screening libraries, such as spray libraries. .

[0142] The terms "tumor-supporting antigen" or "cancer-supporting antigen" refer interchangeably to the antigens of cells that are not themselves cancerous. surface expression, which may affect, for example, their proliferation or survival, e.g., resistance to immune cells. The molecules that support cancer cells by promoting Exemplary cells of this type include stromal cells and bone marrow-derived immunosuppressive cells ( Tumor-supporting antigens themselves are present on cells that support cancer cells. As long as the cellular components are intact, they do not need to play a supporting role in tumor cells.

[0143] The term "flexible polypeptide linker" or "linker" is used in the context of scFv. When used alone or in combination to link together the variable heavy and light chain regions, This refers to a peptide linker consisting of amino acids such as glycine and / or serine residues that is used In some embodiments, the flexible polypeptide linker is a Gly / Ser linker. and an amino acid sequence (Gly-Gly-Gly-Ser) n , where n is a positive integer of 1 or more. Integers, e.g., n=1, n=2, n=3, n=4, n=5, and n=6, n=7, n=8, n In some embodiments, the mobile polypeptide comprises a polypeptide sequence similar to that of SEQ ID NO: 41, where n=9 and n=10. The peptide linker may include, but is not limited to, (Gly4 Ser)4 (SEQ ID NO:27) or In some embodiments, the phospholipase C comprises (Gly4 Ser)3 (SEQ ID NO: 28). The car is (Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO: 25 ) are repeated multiple times. Also included within the scope of the present invention are linkers described in

[0144] As used herein, 5' cap (RNA cap, RNA 7-methylguanosine cap, Syncap or RNA m 7 The G-cap (also called the G-cap) is a part of the eukaryotic membrane immediately after transcription initiation. It is a modified guanine nucleotide added to the "front" or 5' end of a messenger RNA. The 5' cap consists of a terminal group attached to the first transcribed nucleotide. Its presence Capping is important for recognition by the ribosome and protection from RNases. They are linked and occur transcriptionally co-dependently, with each influencing the other. Shortly after initiation, a cap associated with RNA polymerase is added to the 5' end of the mRNA being synthesized. The synthetic complex binds to the mRNA. This enzyme complex catalyzes the chemical reactions required for mRNA capping. The synthesis proceeds as a multi-step biochemical reaction. The capping portion is located at the base of the mRNA. The polypeptide can be modified to adjust its function, such as its stability or translation efficiency.

[0145] As used herein, "in vitro transcribed RNA" refers to RNA synthesized in vitro. In some embodiments, the RNA is mRNA. NA is produced from an in vitro transcription vector. Contains the template used to make transcribed RNA.

[0146] As used herein, "poly(A)" refers to a molecule that is attached to an mRNA by polyadenylation. In some embodiments of the construct for transient expression, In some embodiments, poly(A) is greater than 64. In some embodiments, the poly(A) is greater than 100. In some embodiments, poly(A) is greater than 300. In some embodiments, poly(A) is greater than 400. ) sequences can be chemically or chemically modified to modulate mRNA function such as localization, stability, or translation efficiency. It can be enzymatically modified.

[0147] As used herein, "polyadenylation" refers to the addition of polyadenylation to a messenger RNA molecule. It refers to the covalent attachment of a liadenylyl moiety or a modified variant thereof. In eukaryotes, most messenger RNAs The messenger RNA (mRNA) molecule is polyadenylated at the 3' end. 3' poly(A) The tail is added to the pre-mRNA by the action of the enzyme polyadenylate polymerase A long sequence (often several hundred) of adenine nucleotides that form a chain of adenine nucleotides. In higher eukaryotes, Poly(A) tails are added to transcripts that contain a specific sequence, a polyadenylation signal. The poly(A) tail and the proteins bound to it act to digest mRNA by exonuclease. Polyadenylation is involved in the termination of transcription, the export of mRNA from the nucleus, and It is also important for translation: polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA. However, it can also occur later in the cytoplasm. After transcription is terminated, The mRNA strand is cleaved by the action of an endonuclease complex that cleaves the mRNA. The cleavage site is usually It is characterized by the presence of the base sequence AAUAAA near the cleavage site. After this, an adenosine residue is added to the free 3' end of the cleavage site.

[0148] As used herein, "transient" refers to a period of time lasting a few hours, days, or weeks. This refers to the expression of a transgene that is not integrated and that does not interact with the genome in the host cell. Expression of genes when integrated into or contained in stable plasmid replicons shorter than the period.

[0149] As used herein, the terms "treat," "treatment," and "treating" refer to or more therapies (e.g., one or more therapeutic agents, such as a CAR of the invention). The present invention relates to a method for treating a proliferative disorder, comprising the steps of: It refers to the amelioration of one or more symptoms (preferably one or more discernible symptoms) of a disorder. In embodiments, the terms "treat", "treatment" and "treating" refer to At least one measurable physical manifestation of a proliferative disorder, such as tumor growth, that is not recognizable In another embodiment, the terms "treat," "treatment," and "treatment" refer to an improvement in a patient's ability to "Placing" refers to the inhibition of the progression of a proliferative disorder physically, e.g., by stabilization of a discernible symptom. refers to either or both of the following: In another embodiment, the terms "treat", "treatment" and "treating" refer to It refers to a reduction or stabilization of the size or number of cancerous cells.

[0150] The term "signal transduction pathway" refers to the transmission of a signal from one part of a cell to another part of a cell. The term "cell surface" refers to the biochemical relationships between various signaling molecules that play a role in A "receptor" has the ability to receive a signal and transmit the signal across the membrane of a cell. Includes molecules and molecular complexes.

[0151] The term "subject" refers to a living organism in which an immune response can be generated (e.g., a mammal). , e.g., humans).

[0152] The term "substantially purified" cells refers to cells that are essentially free of other cell types. A qualitatively purified cell is one that is distinct from other cells with which it is normally associated in its naturally occurring state. A substantially purified cell population also refers to cells that have been separated into different cell types. In other instances, the term refers simply to a population of cells that are naturally associated with the It refers to cells that have been separated from the cells to which they are attached. In some embodiments, these cells In some embodiments, the cells are cultured in vitro. Not being nourished.

[0153] As used herein, the term "therapeutic" refers to treatment. A therapeutic effect is a reduction in a pathological condition. This is achieved by suppression, remission or eradication.

[0154] As used herein, the term "prophylaxis" refers to the prevention or prophylactic treatment of a disease or condition. Taste.

[0155] The terms "transfected" or "transformed" or "transduced" "Transfection" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A cell that has been "infected" or "transformed" or "transduced" has an exogenous nucleic acid. Transfected, transformed or transduced. Cells include primary subject cells and This includes their descendants.

[0156] The term "specifically binds" refers to a binding partner that is specifically linked to a cognate binding partner (e.g., They recognize and bind to stimulatory and / or costimulatory molecule (T cell) proteins present on T cells. The antibody or ligand is a ligand that recognizes and binds to the target molecule but does not substantially recognize or bind to other molecules in the sample. This refers to the .

[0157] As used herein, the term "regulatable chimeric antigen receptor (RCAR)" refers to an immunoreactive When present in an immune effector cell, it has specificity for target cells, typically cancer cells, and A set of polypeptides that endow a cell with internal signal generation, typically in its simplest embodiment In some embodiments, RCAR refers to two polypeptides. As defined herein in relation to the extracellular antigen binding domain, the transmembrane domain and the CAR molecule. and / or co-stimulatory molecules, such as stimulatory and / or costimulatory molecules. signal transduction domain (also referred to herein as "intracellular signaling domain" In some embodiments, the series of polypeptides in the RCAR include In some embodiments, the RCAR is A dimerization switch, which, in the presence of a dimerization molecule, links the polypeptides together. For example, an antigen-binding domain can be linked to an intracellular signaling domain. In some embodiments, the RCAR can be a cell described herein (e.g., , immune effector cells), such as RCAR-expressing cells (also referred to as "RCARX cells" In some embodiments, the RCARX cell is a T cell and is expressed in an RCARX T cell. In some embodiments, RCARX is a NK cell and RCARN is a NK cell. RCAR induces RCAR-expressing cells to target cells, typically cancer cells. These can provide specificity and regulatable intracellular signaling or proliferation. may optimize the immune effector properties of RCAR-expressing cells. CAR cells acquire specificity for target cells that contain an antigen bound to the antigen-binding domain. The binding domain is dependent, at least in part, on the antigen-binding domain to bind the antigen.

[0158] A "membrane anchor" or "membrane tethering domain" as that term is used herein. and a polypeptide or polypeptides sufficient to bind the extracellular or intracellular domain to the plasma membrane. It refers to a moiety such as the myristoyl group.

[0159] "Switch domain" is used herein to refer to, for example, RCAR. When the switch domain is dimerized, an entity, typically a polymorphism, that binds to another switch domain in the presence of the dimerization molecule is selected. This binding is a peptide-based entity that links to the first switch domain, e.g. and a second switch domain, e.g., a fused first entity and a linked second entity. The first and second switch domains collectively dimerize, resulting in functional coupling of the first and second switch domains. In some embodiments, the first and second switch domains are are identical to each other, e.g., they are both polypeptides having the same primary amino acid sequence, In some embodiments, the first and second switches are The switch domains are distinct from one another, e.g., they have different primary amino acid sequences. In some embodiments, the heterodimerization switch is a polypeptide, collectively referred to as a heterodimerization switch. In some embodiments, the switch is intracellular. In some embodiments, the switch is extracellular. In some embodiments, the switch domain is a polypeptide-based entity, such as FKB The dimerization molecule is a small molecule, e.g., a rapalog. In embodiments, the switch domain is a polypeptide-based entity, such as a myc peptide. The dimerization molecule is a polypeptide, a fragment thereof, or a polypeptide. multimers of myc ligands, e.g., myc ligands or myc scFvs that bind to one or more myc scFvs. In some embodiments, the switch domain is a multimer of a polypeptide. The dimerization molecule is an antibody or a fragment thereof, e.g. For example, a myc antibody.

[0160] The term "dimerization molecule" as that term is used herein to refer to, for example, RCAR. When used, the molecule promotes binding of the first switch domain to the second switch domain. In some embodiments, the dimerization molecule is not naturally occurring or is a naturally occurring molecule in a subject. In some embodiments, the dimerization molecule is not present in a concentration that would cause significant dimerization. An antagonist is an antagonist, such as rapamycin or a rapalog, such as RAD001.

[0161] The dose "low immune enhancing dose" is a dose of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor , e.g., RAD001 or rapamycin or a catalytic mTOR inhibitor. When activated, it partially inhibits mTOR activity, as measured, for example, by inhibition of P70 S6 kinase activity. This refers to a dose of an mTOR inhibitor that partially, but not completely, inhibits P70 Methods for assessing mTOR activity by inhibition of S6 kinase activity are described in detail herein. This dose is insufficient to produce complete immunosuppression, but sufficient to enhance the immune response. In some embodiments, the low immune enhancing dose of the mTOR inhibitor is sufficient to treat PD-1 positive A decrease in the number of T cells and / or an increase in the number of PD-1 negative T cells or In some embodiments, the administration of an mTOR inhibitor results in an increased ratio of mTOR-positive T cells to PD-1-positive T cells. A low immune enhancing dose results in an increase in the number of naive T cells. Low immune-boosting doses of R inhibitors For example, the following markers on memory T cells, e.g., memory T cell precursors: CD62L high , CD127 high , CD27 + and increased expression of one or more of BCL2; For example, reduced expression of KLRG1 in memory T cells, e.g., memory T cell precursors; and For example, the following features: CD62L high Increased CD127 high Increase in CD2 7 + Increase in KLRG1, decrease in KLRG1, and increase in BCL2, or any combination thereof Increased numbers of memory T cell precursors with where any of the aforementioned changes are, for example, compared to a non-treated control. And so, at least temporarily.

[0162] "Refractory," as used herein, refers to a disease, such as cancer, that does not respond to treatment. In some embodiments, the refractory cancer is resistant to treatment prior to or at the time of initiation of treatment. In other embodiments, the refractory cancer may become resistant during treatment. It is also called anticancer.

[0163] As used herein, "relapsed" or "recurrence" refers to a condition that occurs after a period of improvement or response, e.g., of a disease (e.g., cancer) after previous treatment with a certain therapy (e.g., cancer therapy) or The initial response period refers to the recurrence or reappearance of signs and symptoms of the disease. Below a certain threshold, e.g., 20%, 1%, 10%, 5%, 4%, 3%, 2% or 1% Reappearance can include a decrease in the level of cancer cells above a certain threshold, e.g., 20%, 1% , 10%, 5%, 4%, 3%, 2%, or 1% increase. For example, re-emergence may be , e.g., in relation to B-ALL, after a complete response, e.g., blood, bone marrow (>5%) or any In this context, a complete response may include the reappearance of blasts at extramedullary sites in the peritoneal cavity or in the peritoneal cavity. More generally, in some embodiments, a response (e.g., a complete response) may be observed. or partial response) may include the absence of detectable MRD (minimal residual disease). wherein the initial period of responsiveness is at least 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10, or 12 months; or Lasts for at least 1, 2, 3, 4 or 5 years.

[0164] Ranges: Throughout this disclosure, various embodiments of this invention may be presented in a range format. The description in brackets format is merely for convenience and brevity and is intended to provide a general understanding of the scope of the invention. It should be understood that the scope of the present invention should not be construed as an inflexible limitation. The disclosure includes all the possible subranges specifically disclosed as well as individual numerical values ​​within those ranges. For example, a range such as 1-6 should be interpreted as 1-3 or 1-4. , 1-5, 2-4, 2-6, 3-6, etc., specifically disclosed subranges, as well as within those ranges 1, 2, 2.7, 3, 4, 5, 5.3 and 6. As another example, a range such as 95-99% identity may be expressed as 95%, 96%, %, 97%, 98% or 99% identity, and 96-99%, 96- 98%, 96-97%, 97-99%, 97-98% and 98-99% identity etc. This applies regardless of the width of the range.

[0165] "Gene editing system" as that term is used herein means a gene editing system A modification, e.g., deletion, of one or more nucleic acids at or near the site of the genomic DNA targeted by the target gene. Gene editing refers to a system, e.g., one or more molecules, that directs and executes the deletion. This is known in the art and is described in more detail below.

[0166] Administered in combination with, as used herein, is intended to describe a method for treating a subject's suffering from a disease. It means that two (or more) different treatments are delivered to a subject in a continuous course, e.g. For example, after a subject has been diagnosed with a disease and the disease is cured or eliminated, or treatment is Two or more treatments are delivered before being discontinued for other reasons. The delivery of the treatment is still ongoing when the delivery of the second treatment begins, thereby There is an overlap in the administration of the drugs. This is referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, delivery of one treatment may occur prior to the start of delivery of the other treatment. In some embodiments in either case, treatment is more effectively achieved by co-administration. For example, the second treatment is more effective, e.g., fewer second treatments are needed to achieve the same results. or the second treatment is administered without the first treatment. alleviates symptoms to a degree greater than that seen with the first treatment or produces a similar effect to that of the first treatment In some embodiments, delivery may result in relief of symptoms or other parameters related to the disorder. the cost of delivery of the goods to the buyer is higher than would be allowed if one were delivered without the other. The effect of the two treatments may be partially additive, fully additive, or greater than additive. The delivery is such that the effect of the first treatment delivered is still detectable when the second treatment is delivered. It is possible to make it possible to output the

[0167] "Depletion" or "depleting" are used interchangeably herein. The term refers to a process, e.g., after a selection step, e.g., negative selection, has been performed. refers to a decrease or reduction in the level or amount of cells, proteins or macromolecules in a sample. The depletion may be a complete or partial depletion of cells, proteins or macromolecules. In an embodiment, depletion is achieved by removing cells, proteins or other substances from the sample before the process is performed. The level or amount of a cell, protein or macromolecule compared to the level or amount of a 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% reduction or decrease.

[0168] As used herein, "naive T cells" refer to antigen-naive T cells. In one embodiment, the antigen-naive T cell has encountered its cognate antigen in the thymus, In some embodiments, naive T cells are not encountered in the periphery. In some embodiments, naive T cells are precursors of both CD45RA and CCR7. In some embodiments, the naive T cells express C Expression of D62L, CD27, CCR7, CD45RA, CD28 and CD127, and C In some embodiments, the tumor necrosis factor may be characterized by the absence of D95 or CD45RO isoforms. Naive T cells express CD62L, IL-7 receptor α, IL-6 receptor, and CD132. In some embodiments, the IL-16 expression vector expresses CD25, CD44, CD69, or CD45RO. In this state, naive T cells express CD45RA, CCR7, and CD62L, but not CD In some embodiments, the surface expression levels of the markers The activity is assessed using flow cytometry.

[0169] The term "central memory T cells" refers to T cells that are CD45RO positive in humans and have CCR In some embodiments, the term refers to a subset of T cells that express central memory T cells. In some embodiments, the central memory T cells express IL- In some embodiments, the cells express central IL-2R, IL-7R, and / or IL-15R. Memory T cells express CD45RO, CD95, IL-2 receptor β, CCR7, and CD62 In some embodiments, the surface expression level of the marker is determined by flow cytometry. It is evaluated using

[0170] The terms "stem memory T cells," "stem cell memory T cells," and "stem cell-like memory T cells" are used interchangeably. "memory stem T cell", "T memory stem cell", "T stem cell memory cell " or "TSCM cells" refer to cells that possess stem cell-like capabilities, such as the ability to self-renew and / or memory. Multipotent memory and / or effector T cell subsets This refers to a subset of T cells. In some embodiments, stem memory T cells are CD45R A, expresses CD95, IL-2 receptor beta, CCR7, and CD62L. In some cases, the surface expression levels of markers are assessed using flow cytometry. In embodiments, exemplary stem memory T cells are those described in Gattinoni et al., Disclosed in Nat Med. 2017 January 06;23(1):18-27 No. 6,399,413, the entire contents of which are incorporated herein by reference.

[0171] For purposes of clarity, unless otherwise noted, a particular marker may be designated as "not expressing" or "not expressing" Classify a cell or population of cells as "absent" or "negative for" Similarity does not necessarily mean the absence of a marker. comparing the cells to positive and / or negative controls and / or setting a predefined threshold; and Using conventional detection methods, e.g., flow cytometry, e.g., the methods described herein As described in the Examples, the cells may be characterized as having an expression level below a predetermined threshold, or ... as described in the Examples. When a population has an overexpression level below a predefined threshold, the population does not express the marker. or classifying a cell or population of cells as negative for the marker. 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 FIG. 1G.

[0172] As used herein, the “GeneSetScore(Up TEM vs The term "T cell downregulation" refers to the expression of T cell downregulation in the stem cell memory (TSCM) subpopulation. The score indicates the degree to which the patient exhibits the effector memory T cell (TEM) phenotype relative to the current type. Higher GeneSetScore (Up TEM vs. Down TSCM) , indicating an increase in the TEM phenotype, whereas a lower GeneSetScore (Up TE M vs. Down TSCM) indicates an increase in the TSCM phenotype. GeneSetScore (Up TEM vs. Down TSCM) is One or more genes that are upregulated in the cells and / or downregulated in the TSCM, e.g., MX RA7, CLIC1, NAT13, TBC1D2B, GLCCI1, DUSP10, AP OBEC3D, CACNB3, ANXA2P2, TPRG1, EOMES, MATK, A RHGAP10, ADAM8, MAN1A1, SLFN12L, SH2D2A, EIF2 C4, CD58, MYO1F, RAB27B, ERN1, NPC1, NBEAL2, AP OBEC3G, SYTL2, SLC4A4, PIK3AP1, PTGDR, MAF, PL EKHA5, ADRB2, PLXND1, GNAO1, THBS1, PPP2R2B, C YTH3, KLRF1, FLJ16686, AUTS2, PTPRM, GNLY and GF PT2. In some embodiments, GeneSetScore (Up vs. Down TSCM) can be used to express RNA- For each cell, use RNA sequencing (RNA-seq), e.g., single-cell RNA-seq (scRNA-seq). In some embodiments, GeneSetScore(UpTEM vs. Down TSCM) is the average log-normalized gene expression value of all genes in the gene set It is calculated by obtaining

[0173] As used herein, the “GeneSetScore(Up Treg v The term "effector T cell (Teff)" refers to cells that are downregulated by T cell receptors. This refers to a score that indicates the degree to which regulatory T cells (Treg) are present in relation to the present type. SetScore (Up Treg vs. Down Teff) is a measure of the Treg phenotype. Up Treg vs. Do In some embodiments, the GeneSe tScore (Up Treg vs. Down Teff) is upregulated in Treg cells. One or more genes that are regulated and / or downregulated in Teff cells, e.g., C12or f75, SELPLG, SWAP70, RGS1, PRR11, SPATS2L, SPA TS2L, TSHR, C14orf145, CASP8, SYT11, ACTN4, AN XA5, GLRX, HLA-DMB, PMCH, RAB11FIP1, IL32, FAM 160B1, SHMT2, FRMD4B, CCR3, TNFRSF13B, NTNG2, CLDND1, BARD1, FCER1G, TYMS, ATP1B1, GJB6, FGL 2, TK1, SLC2A8, CDKN2A, SKAP2, GPR55, CDCA7, S1 00A4, GDPD5, PMAIP1, ACOT9, CEP55, SGMS1, ADPR H, AKAP2, HDAC9, IKZF4, CARD17, VAV3, OBFC2A, I TGB1, CIITA, SETD7, HLA-DMA, CCR10, KIAA0101, SLC14A1, PTTG3P, DUSP10, FAM164A, PYHIN1, MYO 1F, SLC1A4, MYBL2, PTTG1, RRM2, TP53INP1, CCR5 , ST8SIA6, TOX, BFSP2, ITPRIPL1, NCAPH, HLA-DP B2, SYT4, NINJ2, FAM46C, CCR4, GBP5, C15orf53, LMCD1, MKI67, NUSAP1, PDE4A, E2F2, CD58, ARHGE F12, LOC100188949, FAS, HLA-DPB1, SELP, WEE1, HLA-DPA1, FCRL1, ICA1, CNTNAP1, OAS1, METTL7A , CCR6, HLA-DRB4, ANXA2P3, STAM, HLA-DQB2, LGA LS1, ANXA2, PI16, DUSP4, LAYN, ANXA2P2, PTPLA, ANXA2P1, ZNF365, LAIR2, LOC541471, RASGRP4, B CAS1, UTS2, MIAT, PRDM1, SEMA3G, FAM129A, HPGD , NCF4, LGALS3, CEACAM4, JAKMIP1, TIGIT, HLA-D RA, IKZF2, HLA-DRB1, FANK1, RTKN2, TRIB1, FCRL 3 and FOXP3. In some embodiments, GeneSetScore(Up Treg vs. Down Teff) are illustrated in Example 10 with reference to, for example, FIG. 39B. As mentioned above, RNA-seq, e.g., single-cell RNA-seq (scRNA-seq), is used to In some embodiments, the GeneSetScore (Up Treg vs .Down Teff) is the average log-normalized gene expression of all genes in the gene set It is calculated by taking the value.

[0174] As used herein, the “GeneSetScore (Down stem The term "stemness" refers to a score that represents the degree to which a cell exhibits a stemness phenotype. A lower GeneSetScore(Down stemness) indicates a stemness phenotype. In some embodiments, GeneSetScore(Down stemness) indicates an increase in ss) is downregulated in hematopoietic stem cells, whereas it is upregulated in differentiating stem cells. One or more genes, e.g. ACE, BATF, CDK6, CHD2, ERCC2, HOXB 4. from the group consisting of MEOX1, SFRP1, SP7, SRF, TAL1 and XRCC5 In some embodiments, the expression of one or more selected genes is measured. For example, GeneSetScore(Down stemness) is As exemplified by reference Example 10, RNA-seq, e.g., single-cell RNA-seq, In some embodiments, the gene expression level is determined using GeneSetSq(scRNA-seq). core(Down stemness) is the average log It is calculated by taking the normalized gene expression value.

[0175] As used herein, the "GeneSetScore(UP hypoxia)" of a cell The term "a)" refers to a score that represents the extent to which a cell exhibits a hypoxic phenotype. neSetScore(UP hypoxia) indicates an increase in the hypoxic phenotype. In an embodiment, GeneSetScore(UP hypoxia) is the score for the number of subjects who have suffered hypoxia. One or more genes that are upregulated in cells that are DM, ADORA2B, AK2, AK3, ALDH1A1, ALDH1A3, ALDOA , ALDOC, ANGPT2, ANGPTL4, ANXA1, ANXA2, ANXA5, ARHGAP5, ARSE, ART1, BACE2, BATF3, BCL2L1, BCL 2L2, BHLHE40, BHLHE41, BIK, BIRC2, BNIP3, BNIP 3L, BPI, BTG1, C11orf2, C7orf68, CA12, CA9, CAL D1, CCNG2, CCT6A, CD99, CDK1, CDKN1A, CDKN1B, C ITED2, CLK1, CNOT7, COL4A5, COL5A1, COL5A2, CO L5A3, CP, CTSD, CXCR4, D4S234E, DDIT3, DDIT4, 1 -Dec, DKC1, DR1, EDN1, EDN2, EFNA1, EGF, EGR1, E IF4A3, ELF3, ELL2, ENG, ENO1, ENO3, ENPEP, EPO, ERRFI1, ETS1, F3, FABP5, FGF3, FKBP4, FLT1, FN1 , FOS, FTL, GAPDH, GBE1, GLRX, GPI, GPRC5A, HAP1 , HBP1, HDAC1, HDAC9, HERC3, HERPUD1, HGF, HIF1 <h2 style=";text-align:left;direction:ltr">A、HK1、HK2、HLA-DQB1、HMOX1、HMOX2、HSPA5、HSP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D1、HSPH1、HYOU1、ICAM1、ID2、IFI27、IGF2、IGFB<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> P1、IGFBP2、IGFBP3、IGFBP5、IL6、IL8、INSIG1、I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> RF6, ITGA5, JUN, KDR, KRT14, KRT18, KRT19, LDHA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、LDHB、LEP、LGALS1、LONP1、LOX、LRP1、MAP4、MET<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、MIF、MMP13、MMP2、MMP7、MPI、MT1L、MTL3P、MUC1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、MXI1、NDRG1、NFIL3、NFKB1、NFKB2、NOS1、NOS2、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> NOS2P1、NOS2P2、NOS3、NR3C1、NR4A1、NT5E、ODC1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、P4HA1、P4HA2、PAICS、PDGFB、PDK3、PFKFB1、PFK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> FB3、PFKFB4、PFKL、PGAM1、PGF、PGK1、PGK2、PGM1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、PIM1、PIM2、PKM2、PLAU、PLAUR、PLIN2、PLOD2、P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> NN、PNP、POLM、PPARA、PPAT、PROK1、PSMA3、PSMD9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、PTGS1、PTGS2、QSOX1、RBPJ、RELA、RIOK3、RNASE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L、RPL36A、RRP9、SAT1、SERPINB2、SERPINE1、SGS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M2, SIAH2, SIN3A, SIRPA, SLC16A1, SLC16A2, SLC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20A1, SLC2A1, SLC2A3, SLC3A2, SLC6A10P, SLC6A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 16、SLC6A6、SLC6A8、SORL1、SPP1、SRSF6、SSSCA1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、STC2、STRA13、SYT7、TBPL1、TCEAL1、TEK、TF、TF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F3, TFRC, TGFA, TGFB1, TGFB3, TGFBI, TGM2, TH, T HBS1, THBS2, TIMM17A, TNFAIP3, TP53, TPBG, TPD 52, TPI1, TXN, TXNIP, UMPS, VEGFA, VEGFB, VEGFC Expression of one or more genes selected from the group consisting of VIM, VPS11 and XRCC6 In some embodiments, the GeneSetScore(U P hypoxia) can be, for example, as illustrated in Example 10 with reference to FIG. Determined using RNA-seq, e.g. single-cell RNA-seq (scRNA-seq) In some embodiments, GeneSetScore(UP hypoxia) is Calculated by taking the average log-normalized gene expression value of all genes in the gene set. can be.

[0176] As used herein, the "GeneSetScore(UP autoph The term “agy” denotes a score that represents the extent to which a cell displays an autophagic phenotype. The higher the GeneSetScore (UP autophagy), the higher the autophagy index. In some embodiments, GeneSetScore(UP autop hagy) is a gene that expresses one or more genes that are upregulated in cells undergoing autophagy. Children, e.g. ABL1, ACBD5, ACIN1, ACTRT1, ADAMTS7, AKR 1E2, ALKBH5, ALPK1, AMBRA1, ANXA5, ANXA7, ARSB , ASB2, ATG10, ATG12, ATG13, ATG14, ATG16L1, AT G16L2, ATG2A, ATG2B, ATG3, ATG4A, ATG4B, ATG4C , ATG4D, ATG5, ATG7, ATG9A, ATG9B, ATP13A2, ATP 1B1、ATPAF1-AS1、ATPIF1、BECN1、BECN1P1、BLOC 1S1、BMP2KL、BNIP1、BNIP3、BOC、C11orf2、C11or f41、C12orf44、C12orf5、C14orf133、C1orf210、 C5、C6orf106、C7orf59、C7orf68、C8orf59、C9or f72、CA7、CALCB、CALCOCO2、CAPS、CCDC36、CD163 L1、CD93、CDC37、CDKN2A、CHAF1B、CHMP2A、CHMP2 B、CHMP3、CHMP4A、CHMP4B、CHMP4C、CHMP6、CHST3 、CISD2、CLDN7、CLEC16A、CLN3、CLVS1、COX8A、CP A3、CRNKL1、CSPG5、CTSA、CTSB、CTSD、CXCR7、DAP 、DKKL1、DNAAF2、DPF3、DRAM1、DRAM2、DYNLL1、DY NLL2、DZANK1、EI24、EIF2S1、EPG5、EPM2A、FABP1 、FAM125A、FAM131B、FAM134B、FAM13B、FAM176A、 FAM176B、FAM48A、FANCC、FANCF、FANCL、FBXO7、F CGR3B、FGF14、FGF7、FGFBP1、FIS1、FNBP1L、FOXO 1、FUNDC1、FUNDC2、FXR2、GABARAP、GABARAPL1、G ABARAPL2、GABARAPL3、GABRA5、GDF5、GMIP、HAP1 、HAPLN1、HBXIP、HCAR1、HDAC6、HGS、HIST1H3A、H IST1H3B、HIST1H3C、HIST1H3D、HIST1H3E、HIST1 H3F、HIST1H3G、HIST1H3H、HIST1H3I、HIST1H3J、 HK2、HMGB1、HPR、HSF2BP、HSP90AA1、HSPA8、IFI1 6、IPPK、IRGM、IST1、ITGB4、ITPKC、KCNK3、KCNQ1 、KIAA0226、KIAA1324、KRCC1、KRT15、KRT73、LAM P1、LAMP2、LAMTOR1、LAMTOR2、LAMTOR3、LARP1B、 LENG9、LGALS8、LIX1、LIX1L、LMCD1、LRRK2、LRSA M1、LSM4、MAP1A、MAP1LC3A、MAP1LC3B、MAP1LC3B 2、MAP1LC3C、MAP1S、MAP2K1、MAP3K12、MARK2、MB D5、MDH1、MEX3C、MFN1、MFN2、MLST8、MRPS10、MRP S2、MSTN、MTERFD1、MTMR14、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、PLEKH M1、PLOD2、PNPO、PPARGC1A、PPY、PRKAA1、PRKAA2 、PRKAB1、PRKAB2、PRKAG1、PRKAG2、PRKAG3、PRKD 2、PRKG1、PSEN1、PTPN22、RAB12、RAB1A、RAB1B、R AB23、RAB24、RAB33B、RAB39、RAB7A、RB1CC1、RBM<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 18、REEP2、REP15、RFWD3、RGS19、RHEB、RIMS3、RN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F185、RNF41、RPS27A、RPTOR、RRAGA、RRAGB、RRAG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C、RRAGD、S100A8、S100A9、SCN1A、SERPINB10、SE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SN2、SFRP4、SH3GLB1、SIRT2、SLC1A3、SLC1A4、SL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C22A3, SLC25A19, SLC35B3, SLC35C1, SLC37A4, S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LC6A1、SLCO1A2、SMURF1、SNAP29、SNAPIN、SNF8、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SNRPB、SNRPB2、SNRPD1、SNRPF、SNTG1、SNX14、SP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ATA18、SQSTM1、SRPX、STAM、STAM2、STAT2、STBD1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、STK11、STK32A、STOM、STX12、STX17、SUPT3H、TB<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C1D17、TBC1D25、TBC1D5、TCIRG1、TEAD4、TECPR1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、TECPR2、TFEB、TM9SF1、TMBIM6、TMEM203、TMEM2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 08, TMEM39A, TMEM39B, TMEM59, TMEM74, TMEM93,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TNIK、TOLLIP、TOMM20、TOMM22、TOMM40、TOMM5、T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> OMM6、TOMM7、TOMM70A、TP53INP1、TP53INP2、TRA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> PPC8、TREM1、TRIM17、TRIM5、TSG101、TXLNA、UBA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 52、UBB、UBC、UBQLN1、UBQLN2、UBQLN4、ULK1、ULK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2、ULK3、USP10、USP13、USP30、UVRAG、VAMP7、VAM<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> P8、VDAC1、VMP1、VPS11、VPS16、VPS18、VPS25、VP<h2 style=";text-align:left;direction:ltr"> S28, VPS33A, VPS33B, VPS36, VPS37A, VPS37B, VP S37C, VPS37D, VPS39, VPS41, VPS4A, VPS4B, VTA1 , VTI1A, VTI1B, WDFY3, WDR45, WDR45L, WIPI1, WI PI2, XBP1, YIPF1, ZCCHC17, ZFYVE1, ZKSCAN3, ZN One or more genes selected from the group consisting of F189, ZNF593 and ZNF681 In some embodiments, the expression of the gene is determined by measuring the expression of the gene. (UP autophagy) is illustrated in Example 10, for example with reference to FIG. 39E. As mentioned above, RNA-seq, e.g., single-cell RNA-seq (scRNA-seq), is used to In some embodiments, the GeneSetScore(UP autophagy gy) is to obtain the average log-normalized gene expression value of all genes in the gene set. It is calculated as follows:

[0177] As used herein, the "GeneSetScore (Uprestin Score)" of a cell The term "activated vs. down-activated" refers to the cells that express activated T cells. This refers to a score that represents the degree to which the type exhibits a resting T cell phenotype. core (Up resting vs. Down activated) is the resting T cell The lower GeneSetScore (Up resti ng vs. Down activated) indicates an increase in the activated T cell phenotype. In some embodiments, GeneSetScore (Up resting vs. Down resting) activated) is upregulated in resting T cells and / or downregulated in activated T cells ABCA7, ABCF3, ACAP2, AMT, and A.S NKH, ATF7IP2, ATG14, ATP1A1, ATXN7, ATXN7L3B. BCL7A, BEX4, BSDC1, BTG1, BTG2, BTN3A1, C11orf 21, C19orf22, C21orf2, CAMK2G, CARS2, CCNL2, C.S D248, CD5, CD55, CEP164, CHKB, CLK1, CLK4, CTSL 1, DBP, DCUN1D2, DENND1C, DGKD, DLG1, DUSP1, EA PP, ECE1, ECHDC2, ERBB2IP, FAM117A, FAM134B, F AM134C, FAM169A, FAM190B, FAU, FLJ10038, FOXJ 2. FOXJ3, FOXL1, FOXO1, FXYD5, FYB, HLA-E, HSPA 1L, HYAL2, ICAM2, IFIT5, IFITM1, IKBKB, IQSEC1 、IRS4、KIAA0664L3、KIAA0748、KLF3、KLF9、KRT1 8. LEF1, LINC00342, LIPA, LIPT1, LLGL2, LMBR1L 、LPAR2、LTBP3、LYPD3、LZTFL1、MANBA、MAP2K6、M AP3K1, MARCH8, MAU2, MGEA5, MMP8, MPO, MSL1, MS L3, MYH3, MYLIP, NAGPA, NDST2, NISCH, NKTR, NLR P1, NOSIP, NPIP, NUMA1, PAIP2B, PAPD7, PBXIP1 PCIF1, PI4KA, PLCL2, PLEKHA1, PLEKHF2, PNISR. PPFIBP2, PRKCA, PRKCZ, PRKD3, PRMT2, PTP4A3, P.S XN, RASA2, RASA3, RASGRP2, RBM38, REPIN1, RNF3 8, RNF44, ROR1, RPL30, RPL32, RPLP1, RPS20, RPS 24, RPS27, RPS6, RPS9, RXRA, RYK, SCAND2, SEMA4 C, SETD1B, SETD6, SETX, SF3B1, SH2B1, SLC2A4RG , SLC35E2B, SLC46A3, SMAGP, SMARCE1, SMPD1, SN PH, SP140L, SPATA6, SPG7, SREK1IP1, SRSF5, STA T5B, SVIL, SYF2, SYNJ2BP, TAF1C, TBC1D4, TCF20 , TECTA, TES, TMEM127, TMEM159, TMEM30B, TMEM6 6, TMEM8B, TP53TG1, TPCN1, TRIM22, TRIM44, TSC 1, TSC22D1, TSC22D3, TSPYL2, TTC9, TTN, UBE2G2 , USP33, USP34, VAMP1, VILL, VIPR1, VPS13C, ZBE D5, ZBTB25, ZBTB40, ZC3H3, ZFP161, ZFP36L1, ZF P36L2, ZHX2, ZMYM5, ZNF136, ZNF148, ZNF318, ZN F350, ZNF512B, ZNF609, ZNF652, ZNF83, ZNF862 and and ZNF91. In some embodiments, GeneSetScore(Up resting value) is set. s.Down activated) is, for example, as shown in Example 10 with reference to FIG. 38D. As indicated, RNA-seq, e.g., single-cell RNA-seq (scRNA-seq), In some embodiments, the GeneSetScore(Up rest Down activated vs. Down activated) is the average of all genes in the gene set It is calculated by taking the log normalized gene expression values.

[0178] As used herein, the “GeneSetScore(ProgressionScore)” of a cell "Very Up in Memory Differentiation" is a - Refers to the score that represents the stage of differentiation of cells. The higher the GeneSetScore (P regressively up in memory differentiation n) show an increase in late memory T cell phenotype, whereas lower GeneSetSc ore(Progressively up in memory different In some embodiments, Ge iation indicates an increase in the early memory T cell phenotype. neSetScore(UP autophagy) is upregulated during memory differentiation One or more genes that may be involved in the progression of glaucoma, such as MTCH2, RAB6C, KIAA0195, SETD2, C2orf24, NRD1, GNA13, COPA, SELT, TNIP1, CBFA2 T2, LRP10, PRKCI, BRE, ANKS1A, PNPLA6, ARL6IP1 , WDFY1, MAPK1, GPR153, SHKBP1, MAP1LC3B2, PIP 4K2A, HCN3, GTPBP1, TLN1, C4orf34, KIF3B, TCIR G1, PPP3CA, ATG4D, TYMP, TRAF6, C17orf76, WIPF 1, FAM108A1, MYL6, NRM, SPCS2, GGT3P, GALK1, CL IP4, ARL4C, YWHAQ, LPCAT4, ATG2A, IDS, TBC1D5, DMPK、ST6GALNAC6、REEP5、ABHD6、KIAA0247、EMB 、TSEN54、SPIRE2、PIWIL4、ZSCAN22、ICAM1、CHD9 、LPIN2、SETD8、ZC3H12A、ULBP3、IL15RA、HLA-DQ B2、LCP1、CHP、RUNX3、TMEM43、REEP4、MEF2D、ABL 1、TMEM39A、PCBP4、PLCD1、CHST12、RASGRP1、C1o rf58、C11orf63、C6orf129、FHOD1、DKFZp434F14 2、PIK3CG、ITPR3、BTG3、C4orf50、CNNM3、IFI16、 AK1、CDK2AP1、REL、BCL2L1、MVD、TTC39C、PLEKHA 2、FKBP11、EML4、FANCA、CDCA4、FUCA2、MFSD10、T BCD、CAPN2、IQGAP1、CHST11、PIK3R1、MYO5A、KIR 2DL3、DLG3、MXD4、RALGDS、S1PR5、WSB2、CCR3、TI PARP、SP140、CD151、SOX13、KRTAP5-2、NF1、PEA1 5、PARP8、RNF166、UEVLD、LIMK1、CACNB1、TMX4、S LC6A6、LBA1、SV2A、LLGL2、IRF1、PPP2R5C、CD99、 RAPGEF1、PPP4R1、OSBPL7、FOXP4、SLA2、TBC1D2B 、ST7、JAZF1、GGA2、PI4K2A、CD68、LPGAT1、STX11 、ZAK、FAM160B1、RORA、C8orf80、APOBEC3F、TGFB I、DNAJC1、GPR114、LRP8、CD69、CMIP、NAT13、TGF B1、FLJ00049、ANTXR2、NR4A3、IL12RB1、NTNG2、R<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> DX、MLLT4、GPRIN3、ADCY9、CD300A、SCD5、ABI3、P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TPN22、LGALS1、SYTL3、BMPR1A、TBK1、PMAIP1、RA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SGEF1A、GCNT1、GABARAPL1、STOM、CALHM2、ABCA2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、PPP1R16B、SYNE2、PAM、C12orf75、CLCF1、MXRA7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、APOBEC3C、CLSTN3、ACOT9、HIP1、LAG3、TNFAIP3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> DCBLD1, KLF6, CACNB3, RNF19A, RAB27A, FADS3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> DLG5、APOBEC3D、TNFRSF1B、ACTN4、TBKBP1、ATXN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1、ARAP2、ARHGEF12、FAM53B、MAN1A1、FAM38A、PL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> XNC1、GRLF1、SRGN、HLA-DRB5、B4GALT5、WIPI1、P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TPRJ、SLFN11、DUSP2、ANXA5、AHNAK、NEO1、CLIC1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、EIF2C4、MAP3K5、IL2RB、PLEKHG1、MYO6、GTDC1、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> EDARADD、GALM、TARP、ADAM8、MSC、HNRPLL、SYT11<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、ATP2B4、NHSL2、MATK、ARHGAP18、SLFN12L、SPAT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S2L、RAB27B、PIK3R3、TP53INP1、MBOAT1、GYG1、K<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ATNAL1、FAM46C、ZC3HAV1L、ANXA2P2、CTNNA1、NP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C1、C3AR1、CRIM1、SH2D2A、ERN1、YPEL1、TBX21、S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LC1A4、FASLG、PHACTR2、GALNT3、ADRB2、PIK3AP1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、TLR3、PLEKHA5、DUSP10、GNAO1、PTGDR、FRMD4B、<h2 style=";text-align:left;direction:ltr"> ANXA2, EOMES, CADM1, MAF, TPRG1, NBEAL2, PPP2R 2B, PELO, SLC4A4, KLRF1, FOSL2, RGS2, TGFBR3, P RF1, MYO1F, GAB3, C17orf66, MICAL2, CYTH3, TOX , HLA-DRA, SYNE1, WEE1, PYHIN1, F2R, PLD1, THBS 1, CD58, FAS, NETO2, CXCR6, ST6GALNAC2, DUSP4, AUTS2, C1orf21, KLRG1, TNIP3, GZMA, PRR5L, PRD M1, ST8SIA6, PLXND1, PTPRM, GFPT2, MYBL1, SLAM F7, FLJ16686, GNLY, ZEB2, CST7, IL18RAP, CCL5, The expression of one or more genes selected from the group consisting of KLRD1 and KLRB1 is measured. In some embodiments, the GeneSetScore(ProgresScore) is used. For example, For example, as illustrated in Example 10 with reference to FIG. 40B, RNA-seq, e.g., single Determined using cellular RNA-seq (scRNA-seq). GeneSetScore(Progressively up in memory differentiation) is the average log-normalized mean of all genes in the gene set It is calculated by taking gene expression values.

[0179] As used herein, the “GeneSetScore(Up TEM vs The term "T cell downregulation (TN)" refers to the expression of T cells that have evolved to a naive T cell (TN) phenotype. This refers to a score that indicates the degree to which the effector memory T cell (TEM) phenotype is exhibited. eneSetScore (Up TEM vs. Down TN) is the increase in the TEM phenotype. Up vs. Down TN) indicates an increase in the TN phenotype. (Up TEM vs. Down TN) is upregulated in TEM cells and / or T One or more genes that are downregulated in N cells, e.g., MYO5A, MXD4, STK3, S 1PR5, GLCCI1, CCR3, SOX13, KRTAP5-2, PEA15, PA RP8, RNF166, UEVLD, LIMK1, SLC6A6, SV2A, KPNA2 , OSBPL7, ST7, GGA2, PI4K2A, CD68, ZAK, RORA, TG FBI, DNAJC1, JOSD1, ZFYVE28, LRP8, OSBPL3, CMI P, NAT13, TGFB1, ANTXR2, NR4A3, RDX, ADCY9, CHN 1, CD300A, SCD5, PTPN22, LGALS1, RASGEF1A, GCN T1, GLUL, ABCA2, CLDND1, PAM, CLCF1, MXRA7, CLS TN3, ACOT9, METRNL, BMPR1A, LRIG1, APOBEC3G, C ACNB3, RNF19A, RAB27A, FADS3, ACTN4, TBKBP1, F AM53B, MAN1A1, FAM38A, GRLF1, B4GALT5, WIPI1, DUSP2, ANXA5, AHNAK, CLIC1, MAP3K5, ST8SIA1, T ARP, ADAM8, MATK, SLFN12L, PIK3R3, FAM46C, ANX A2P2, CTNNA1, NPC1, SH2D2A, ERN1, YPEL1, TBX21 , STOM, PHACTR2, GBP5, ADRB2, PIK3AP1, DUSP10, PTGDR, EOMES, MAF, TPRG1, NBEAL2, NCAPH, SLC4A 4, FOSL2, RGS2, TGFBR3, MYO1F, C17orf66, CYTH3 , WEE1, PYHIN1, F2R, THBS1, CD58, AUTS2, FAM129 A, TNIP3, GZMA, PRR5L, PRDM1, PLXND1, PTPRM, GF PT2, MYBL1, SLAMF7, ZEB2, CST7, CCL5, GZMK and KL RB1, In some embodiments, GeneSetScore (Up TEM vs. Down TEM) TN) can be used in RNA-seq, e.g., as illustrated in Example 10 with reference to FIG. 40C. For example, this is determined using single-cell RNA-seq (scRNA-seq). In the embodiment, GeneSetScore (Up TEM vs. Down TN) is Calculated by taking the average log-normalized gene expression value of all genes in the gene set. can be.

[0180] In relation to the GeneSetScore value (e.g., median GeneSetScore) When the positive GeneSetScore is reduced by 100%, this value becomes 0. When the neSetScore increases by 100%, this value becomes 0. For example, in Figure 39A, The median GeneSetScore for day 1 samples was -0.084; The median GeneSetScore for the 9-day samples was 0.035; The median GeneSetScore is −0.1. In FIG. A 100% increase in the median GeneSetScore of re value; a 200% increase in the median GeneSetScore of the input samples This results in a GeneSetScore value of 0.1. A 100% decrease in the median GeneSetScore of a ore value obtained; 200% decrease in median GeneSetScore for day 9 samples gives a GeneSetScore value of -0.035.

[0181] As used herein, the term "beads" refers to beads having a diameter of about 0.1 μm to several millimeters. Beads refer to discrete particles with a solid surface of a certain size. ) or may have an irregular shape. The beads may be made of, but are not limited to, paramagnetic materials. , ceramic, plastic, glass, polystyrene, methylstyrene, acrylic polymer Includes titanium, latex, Sepharose, cellulose, nylon, etc. In some embodiments, the beads are relatively uniform and have a diameter of about 4. 5 μm, spherical, superparamagnetic polystyrene beads, such as CD3 (e.g., CD3ε ) and an antibody against CD28, e.g., In some embodiments, the beads are Dynabeads®. In an embodiment, both anti-CD3 and anti-CD28 antibodies are bound to the same beads to facilitate antigen presentation. Mimicking the stimulation of T cells by cells. Characteristics of Dynabeads® and cells The use of Dynabeads® for isolation and propagation is known in the art. , e.g., Neurauter et al., Cell isolation and expansion using Dynabeads,Adv Biochem En g Biotechnol 2007;106:41-73, the entire contents of which are incorporated herein by reference. The body is incorporated herein by reference.

[0182] As used herein, the term "nanomatrix" refers to a matrix of mobile polymer chains. Nanomatrix refers to a nanostructure containing a matrix. In some embodiments, the matrix of mobile polymer chains is T-shaped. One or more agonists that provide an activation signal to the cells, e.g., agonist anti-CD3 and / or or is bound to an anti-CD28 antibody. In some embodiments, the nanomatrix comprises one or more agonists of one or more co-stimulatory molecules and / or agonists of one or more co-stimulatory molecules, e.g. In some embodiments, the one or more The agonist of the stimulatory molecule is a CD3 agonist (e.g., an anti-CD3 agonist antibody). In some embodiments, the agonist of one or more stimulatory molecules is a CD28 agonist (e.g., In some embodiments, the nanomatrix is, for example, an anti-CD28 agonist antibody. For example, the use of solid surface ligands as attachment points for agonists such as anti-CD3 and / or anti-CD28 antibodies. In some embodiments, the nanomatrix is ​​characterized by the absence of Nanomatrix or Miltenyi disclosed in brochure 048920A1 MACS® GMP T Cell TransA from Biotcc GmbH Nanomatrix as provided in the ct™ kit, the entire contents of which are incorporated by reference. MACS® GMP T Cell Trans Act™ is a humanized recombinant agonistic antibody directed against human CD3 and CD28. It is composed of a bound colloidal polymer nanomatrix.

[0183] Various embodiments of the compositions and methods described herein are described in further detail below. Additional definitions are set forth throughout the specification.

[0184] Provided herein are immunoglobulins engineered to express a CAR, e.g., a CAR described herein. Methods for producing immune effector cells (e.g., T cells or NK cells), such cells and methods of using such cells to treat a disease, such as cancer, in a subject are provided. In some embodiments, the methods disclosed herein produce CAR in less than 24 hours. It is possible to produce immune effector cells engineered to express the Although not intended to be limiting, the methods described herein may involve the use of naive T cells, etc. during the manufacturing process. These CAR-expressing cells with an undifferentiated phenotype are In some embodiments, the cells may persist longer and / or grow better in vivo after implantation. The CART cells produced by the production method described herein are, for example, scRNA- seq (see, e.g., FIG. 39A and described in Example 10). (as measured using the method) compared with CART cells produced by traditional manufacturing processes. In some embodiments, the cells contain a higher percentage of stem cell memory T cells compared to cells in the control group. CART cells produced by the methods described herein can be, for example, scRNA-seq. q (e.g., as described in Example 10 with reference to FIG. 39B). (as measured using In some embodiments, the present invention provides a method for the production of T cells that are highly effective against T cell-associated leukemias. CART cells produced by the manufacturing method described in the document can be analyzed, for example, by scRNA-seq. As measured by (e.g., using the method described in Example 10 with reference to FIG. 39C) ) compared to CART cells produced by traditional manufacturing processes. In some embodiments, the methods described herein provide for the preparation of cells that better preserve stemness. CART cells produced by the For example, as measured using the method described in Example 10 with reference to FIG. 39D ), Compared to CART cells produced by traditional manufacturing processes, they exhibited lower levels of hypoxia. In some embodiments, the CART cells produced by the methods described herein include For example, as measured using scRNA-seq (see, e.g., FIG. 39E). (as measured using the method described in Example 10) produced by traditional manufacturing processes. These cells show lower levels of autophagy compared to control CART cells.

[0185] In some embodiments, the methods disclosed herein include the use of Dynabeads® This includes the use of beads such as CD3 / CD28 Dynabeads®. First, there is no de-beading step. In some embodiments, the methods disclosed herein The CART cells produced may be cultured using minimal ex vivo expansion, e.g., less than 1 day, less than 12 hours, or less than 8 hours. Less than 1 hour, less than 6 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour or ex vivo The method described herein can be administered to a subject without enlargement. The present invention provides a rapid manufacturing process for producing improved CAR-expressing cell products for use in the treatment of Provide.

[0186] Cytokine Process In some embodiments, the present disclosure provides a method for the production of a chimeric antigen receptor (CAR)-expressing cell (e.g., The present invention provides a method for producing a population of human T cells, comprising: (1) treating the population of cells with IL-2, I IL-7, IL-15, IL-21, IL-6, or a combination thereof (2) contacting a population of cells (e.g., T cells) with a CAR-coding inhibitor; contacting the nucleic acid molecule (e.g., a DNA or RNA molecule) that contains the nucleic acid molecule, (3) obtaining a population of cells (e.g., T cells) that contain the T cells; and (4) storing (e.g., cryopreserving) the T cells. (e.g., reformulating a population of cells in a culture medium) or preserving cells (e.g., T cells) for administration. (a) step (2) is performed in conjunction with or in conjunction with step (1), Within 5 hours after the start of step (1), e.g., 1, 2, 3, 4 hours after the start of step (1) or within 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 initiation of step (1), for example, (b) the population of cells from step (3) is expanded or cultured within 24 hours after initiation. or, for example, the number of surviving cells compared to the population of cells at the start of step (1). 5, 10, 15, 20, 25, 30, 35 or 40% or less, e.g., 1 In some embodiments, the nucleic acid molecule in step (2) is amplified by D 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 a viral vector, For example, lentiviral vectors, adenoviral vectors, or retroviral vectors In some embodiments, the viral vector in step (2) is selected from the group consisting of The nucleic acid molecule is on a non-viral vector. In some embodiments, in step (2), In some embodiments, the nucleic acid molecule in step (2) is on a plasmid. In some embodiments, the molecule is not on any vector. A population of human T cells (e.g., T cells) is transduced with a viral vector containing a nucleic acid molecule encoding a CAR. This includes introducing

[0187] In some embodiments, the population of cells (e.g., T cells) is obtained by apheresis from a subject. The blood is collected from a sample (e.g., a leukapheresis sample).

[0188] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) (e.g., cryopreserved samples) are collected from the subject and stored at a cell manufacturing facility as frozen samples (e.g., cryopreserved samples). The frozen apheresis samples are then thawed and transported to a using a machine (e.g., CliniMACS® Prodigy® device) To identify T cells (e.g., CD4+ T cells and / or CD8+ T cells) from the apheresis sample, Then, the selected T cells (e.g., CD4+ T cells and / or CD 8+ T cells) for CART production using the cytokine process described herein. In some embodiments, at the end of the cytokine process, the CART cells are cryopreserved. In some embodiments, the selected T cells (e.g., , CD4+ T cells and / or CD8+ T cells) prior to inoculation for CART production. It is subjected to one or more rounds of freeze-thawing steps.

[0189] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) (e.g., non-frozen product) from the subject and delivered to a cell manufacturing facility as a fresh product (e.g., non-frozen product). For example, the cells are transported to a cell sorting machine (e.g., CliniMACS® P Using a rodigy® device, T cells (e.g. , CD4+ T cells and / or CD8+ T cells). Then, the selected T cells ( For example, CD4+ T cells and / or CD8+ T cells are treated with cytokines as described herein. In some embodiments, selected T cells are inoculated for CART production using the process. Cells (e.g., CD4+ T cells and / or CD8+ T cells) are inoculated for CART production. Prior to this, the cells are subjected to one or more rounds of freeze-thaw steps.

[0190] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) For example, a cell sorting machine (e.g., CliniMACS ( The Prodigy® device is used to extract T from the apheresis sample. The cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected. The T cells (e.g., CD4+ T cells and / or CD8+ T cells) are frozen samples (e.g., The selected T cells (e.g., C and / or CD4+ T cells) are subsequently thawed and treated with cytokines as described herein. The process is used to inoculate CART production.

[0191] In some embodiments, cells (e.g., T cells) are inoculated and administered one or more cytokines (e.g., For example, IL-2, IL-7, IL-15 (e.g., hetIL-15 (IL15 / sIL -15Ra), IL-21 or IL-6 (e.g., IL-6 / sIL-6R) one or more cytokines of choice) and a vector encoding a CAR (e.g., After 20-24 hours of incubation, the cells is washed and then formulated for storage or administration.

[0192] Unlike traditional CART manufacturing approaches, the cytokine process provided herein The therapy does not involve CD3 and / or CD28 stimulation or ex vivo T cell expansion. Extensively expanded ex vivo T cells contacted with anti-CD28 antibodies were identified as central nervous system T cells. Without wishing to be bound by theory, The cytokine process provided herein preserves the undifferentiated phenotype of T cells during CART production. and generating a CART product that can persist for an extended period of time after being injected into a subject. .

[0193] In some embodiments, the population of cells is treated with one or more cytokines (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20 -7, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL -21 or IL-6 (e.g., IL-6 / sIL-6Ra) The mixture is brought into contact with the

[0194] In some embodiments, the population of cells is contacted with IL-2. In some embodiments, the population of cells is contacted with IL-7. , hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, the population of cells is contacted with IL-21. (e.g., IL-6 / sIL-6Ra). In some embodiments, the population of cells is contacted with In some embodiments, the population of cells is contacted with IL-2 and IL-7. Contact with IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)) In some embodiments, the population of cells is contacted with IL-2 and IL-21. In morphology, a population of cells is differentiated into IL-2 and IL-6 (e.g., IL-6 / sIL-6Ra) In some embodiments, the population of cells is contacted with IL-7 and IL-15 (e.g., he In some embodiments, the cells are contacted with tIL-15 (IL15 / sIL-15Ra). In some embodiments, the population of cells is contacted with IL-7 and IL-21. In some embodiments, the antibody is contacted with IL-7 and IL-6 (e.g., IL-6 / sIL-6Ra). In this embodiment, a population of cells is treated with IL-15 (e.g., hetIL-15 (IL15 / sIL-15 In some embodiments, the population of cells is contacted with IL-15 ( For example, hetIL-15 (IL15 / sIL-15Ra) and IL-6 (e.g., I In some embodiments, the population of cells is contacted with IL-21. and IL-6 (e.g., IL-6 / sIL-6Ra). Then, the cell population is transfected with IL-7, IL-15 (e.g., hetIL-15 (IL15 / sIL- 15Ra) and IL-21. In some embodiments, the population of cells is contacted with LSD1 In some embodiments, the population of cells is further contacted with a MALT1 inhibitor. Contact with.

[0195] In some embodiments, the population of cells is diluted by 20, 30, 40, 50, 60, 70, 80, 90 , 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 , 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 U / ml of IL-2. , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 In some embodiments, the population of cells is contacted with 8, 19, or 20 ng / ml of IL-7. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, The cells are contacted with 17, 18, 19 or 20 ng / ml of IL-15.

[0196] In some embodiments, the population of cells is contacted with a nucleic acid molecule encoding a CAR. In this embodiment, a population of cells is transduced with a DNA molecule encoding a CAR.

[0197] In some embodiments, the population of cells is contacted with a nucleic acid molecule encoding a CAR. The step includes contacting the population of cells with one or more cytokines as described above. In some embodiments, a population of cells is contacted with a nucleic acid molecule encoding a CAR. The step includes contacting the population of cells with one or more cytokines as described above. 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7 In some embodiments, the concentration of cells is increased within 10 hours. The step of contacting the population of cells with a nucleic acid molecule encoding a CAR can include contacting the population of cells with one of the aforementioned In some embodiments, the cytokine contacting step is performed within 5 hours after the start of the cytokine contacting step. In the method, the step of contacting the population of cells with a nucleic acid molecule encoding a CAR comprises contacting the population of cells with a nucleic acid molecule encoding a CAR. is performed within 4 hours after initiation of the step of contacting with one or more cytokines as described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR. The method includes the steps of: (a) contacting the population of cells with one or more cytokines as described above; and (b) determining whether the population of cells is in a state where the cytokines are present; In some embodiments, the population of cells is contacted with a nucleic acid molecule encoding a CAR. The step of contacting the population of cells with one or more cytokines as described above. In some embodiments, the population of cells is subjected to a CAR-encoding The step of contacting with the nucleic acid molecule may further comprise contacting the population of cells with one or more cytokines as described above. This is done within one hour after the start of the step of allowing the

[0198] In some embodiments, the population of cells is harvested for storage or administration.

[0199] In some embodiments, the population of cells is contacted with one or more of the cytokines described above. 72, 60, 48, 36, 32, 31, 30, 29, 28, 27, 26, The population of cells was stored or injected within 25, 24, 23, 22, 21, 20, 19, or 18 hours. In some embodiments, the population of cells is harvested for administration to one or more of the above-mentioned cytokines. The population of cells is harvested for storage or administration within 26 hours after the initiation of the step of contacting with the cytochrome P450. In some embodiments, the population of cells is contacted with one or more cytokines as described above. The population of cells is harvested for storage or administration within 25 hours after the initiation of the step of In an embodiment of the present invention, the step of contacting the population of cells with one or more cytokines as described above. Within 24 hours after initiation, the population of cells is harvested for storage or administration. The method includes the steps of: 23 after initiation of the step of contacting the population of cells with one or more cytokines as described above; Within hours, the population of cells is harvested for storage or administration. In some embodiments, the collection of cells The cells are then subjected to a 22-hour incubation at 4° C. for 22 hours after the initiation of the step of contacting the cells with one or more of the cytokines described above. A population of cells is harvested for storage or administration.

[0200] In some embodiments, the population of cells is not expanded ex vivo.

[0201] In some embodiments, the population of cells is contacted with, for example, one or more cytokines as described above. The number of viable cells is assessed by comparing the number of viable cells to the population of cells before and after the incubation period. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 3 In some embodiments, the cells are expanded by no more than 5, 40, 45, 50, 55, or 60%. The population of cells may be, for example, more abundant than the population of cells prior to contact with one or more of the cytokines described above. In some embodiments, the cells are expanded by 5% or less as assessed by the number of viable cells. The population of cells may be, for example, 0.1 to 0.5% of the total cells, as compared to the population of cells prior to contact with one or more of the cytokines described above. All of the cultures are expanded by no more than 10% as assessed by the number of viable cells. The population of cells may be, for example, a population of cells prior to contact with one or more of the cytokines described above. In some embodiments, the number of viable cells is increased by 15% or less compared to the number of viable cells. In the present invention, a population of cells may be, for example, a population of cells prior to contact with one or more of the cytokines described above. The population is expanded by less than 20% as assessed by the number of viable cells. In some embodiments, the population of cells may be, for example, a population of cells prior to contact with one or more of the cytokines described above. The number of viable cells compared to the total cell population is estimated by 25% or less. In embodiments, the population of cells may be, for example, cultured prior to contact with one or more of the cytokines described above. Compared to the population of cells in the original culture, the number of viable cells is estimated by expanding by less than 30%. In some embodiments, the population of cells is contacted with one or more cytokines, e.g., as described above. The population of cells that was expanded by 35% or less compared to the original population of cells, as assessed by the number of viable cells. In some embodiments, the population of cells is contacted with one or more cytokines, e.g., as described above. The population of cells expanded by 40% or less compared to the original population of cells, as assessed by the number of viable cells. can be.

[0202] In some embodiments, the population of cells is treated with, for example, one or more of the cytokines described above. The number of viable cells was assessed as 1, 1.5, 2, or 3, compared to the population of cells before exposure. 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20 , grown for no more than 24, 36 or 48 hours.

[0203] In some embodiments, the population of cells is a population of cells that expresses a CD3 / TCR complex (e.g., an anti-CD3 antibody). The antibody can be used to stimulate the immune system (the immune system) and / or a costimulatory molecule on the surface of the cell (e.g., anti-CD28 antibody). No in vitro contact with a stimulating agent or, if contacted, the contacting step is less than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 hours.

[0204] In some embodiments, the population of cells is a population of cells that expresses a CD3 / TCR complex (e.g., an anti-CD3 antibody). The antibody can be used to stimulate the immune system (the immune system) and / or a costimulatory molecule on the surface of the cell (e.g., anti-CD28 antibody). Stimulating agents and in vitro immunization for 20, 21, 22, 23, 24, 25, 26, 27, or 28 hours Contact is made by phone.

[0205] In some embodiments, the cytokines are produced using the cytokine processes described herein. The population of cells may be subjected to, for example, a CD3 / TCR complex (e.g., an anti-CD3 antibody) to induce cell proliferation and cell motility. Binding agents and / or costimulatory molecules (e.g., anti-CD28 antibodies) on the surface of cells. and cells produced by a similar method, but further comprising the step of contacting the cells with an agent that Compared to CAR-expressing cells, a higher percentage of naive cells (e.g., Tomo 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1 9, 20, 25, 30, 35, 40, 45, 50, 55 or 60% higher).

[0206] In some embodiments, the cytokine processes described herein are at least 0, 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 In some embodiments, the method is performed in a cell culture medium containing 8% or less serum. The cytokine process described above can be inhibited by an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof. The method is carried out in a cell culture medium containing the combination.

[0207] Activation Process In some embodiments, the present disclosure provides a method for the production of a chimeric antigen receptor (CAR)-expressing cell (e.g., The present invention provides a method for producing a population of cells (e.g., T cells), comprising: (i) selecting a cell (e.g., T cells, A population of T cells (e.g., isolated from frozen or fresh leukapheresis products) , an agent that stimulates the CD3 / TCR complex and / or a costimulatory molecule on the surface of the cell (ii) contacting a population of cells (e.g., T cells) with an agent encoding a CAR; and contacting the nucleic acid molecule (e.g., a DNA or RNA molecule) with the nucleic acid molecule. (iii) providing a population of cells (e.g., T cells); and (iv) storing (e.g., freezing) the cells. Reformulating a population of cells in a storage medium) or cells (e.g., T cells) for administration (a) step (ii) is combined with step (i) to obtain a population of or within 20 hours after the initiation of step (i), e.g., within 12, 1 Within 3, 14, 15, 16, 17 or 18 hours, for example 1 hour after the initiation of step (i). and step (iii) is carried out within 26 hours after the start of step (i). Within 22, 23 or 24 hours after the start of step (i), for example within ... (b) step (ii) is performed within 24 hours after the initiation of step (i); ) or within 20 hours after the start of step (i), e.g. Within 12, 13, 14, 15, 16, 17 or 18 hours of, for example, step (i) and step (iii) is carried out 18 hours after the initiation of step (ii). Within 30 hours, for example, 22, 23, 24, 25, 26, 2 (c) within 7, 28, 29 or 30 hours from step (iii); The population of cells may, for example, be increased in number of viable cells compared to the population of cells at the start of step (i). Therefore, it is evaluated and not grown or 5, 10, 15, 20, 25, 30, 35 or In some embodiments, the cells are expanded by no more than 40%, for example no more than 10%. 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. The molecule may be a viral vector, such as a lentiviral vector, an adenoviral vector, or is on a viral vector selected from a 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 this embodiment, step (ii) comprises transfecting a population of cells (e.g., T cells) with a nuclease encoding a CAR. This involves transducing with a viral vector that contains the acid molecule.

[0208] In some embodiments, the population of cells (e.g., T cells) is obtained by apheresis from a subject. The antibody is collected from a blood sample (e.g., a leukapheresis sample).

[0209] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) (e.g., cryopreserved samples) are collected from the subject and stored at a cell manufacturing facility as frozen samples (e.g., cryopreserved samples). The frozen apheresis samples are then thawed and transported to a using a machine (e.g., CliniMACS® Prodigy® device) To identify T cells (e.g., CD4+ T cells and / or CD8+ T cells) from the apheresis sample, Then, the selected T cells (e.g., CD4+ T cells and / or CD 8+ T cells) are inoculated for CART production using the activation process described herein. In some embodiments, selected T cells (e.g., CD4+ T cells and / or CD8 + T cells) were subjected to one or more rounds of freeze-thaw steps before inoculation for CART production. Attached.

[0210] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) (e.g., non-frozen product) from the subject and delivered to a cell manufacturing facility as a fresh product (e.g., non-frozen product). For example, the cells are transported to a cell sorting machine (e.g., CliniMACS® P Using a rodigy® device, T cells (e.g. , CD4+ T cells and / or CD8+ T cells). Then, the selected T cells ( For example, CD4+ T cells and / or CD8+ T cells are subjected to the activation process described herein. In some embodiments, selected T cells (e.g., For example, CD4+ T cells and / or CD8+ T cells) may be used prior to inoculation for CART production. The mixture is then subjected to one or more rounds of freeze-thaw steps.

[0211] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) For example, a cell sorting machine (e.g., CliniMACS ( The Prodigy® device is used to extract T from the apheresis sample. The cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected. The T cells (e.g., CD4+ T cells and / or CD8+ T cells) are frozen samples (e.g., The selected T cells (e.g., C The cells are then subsequently thawed and subjected to the activation process described herein. The cells are inoculated for CART production using the .

[0212] In some embodiments, cells (e.g., T cells) are treated with anti-CD3 and anti-CD28 antibodies, e.g., After 12 hours of contact, the vector encoding the CAR (e.g., lentiviral vector After 24 hours of incubation, the cells are washed and stored or The formulation is made for this purpose.

[0213] Without intending to be bound by theory, it has been shown that short-term CD3 and anti-CD28 stimulation can induce autologous Efficient transduction of regenerative T cells can be promoted compared with traditional CART manufacturing approaches Thus, the activation process provided herein does not involve extended ex vivo proliferation. Similar to the itokine process, the activation process provided herein also involves the production of CART. , preserving undifferentiated T cells.

[0214] In some embodiments, the population of cells is treated with an agent that stimulates the CD3 / TCR complex and / or The cells are contacted with an agent that stimulates costimulatory molecules on their surface.

[0215] In some embodiments, the agent that stimulates the CD3 / TCR complex stimulates CD3 In some embodiments, the agent that stimulates a costimulatory molecule is CD28, ICO S, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GI Stimulating TR, CD30, TIM1, CD2, CD226, or any combination thereof In some embodiments, the agent that stimulates a costimulatory molecule stimulates CD28. In some embodiments, the agent that stimulates the CD3 / TCR complex is an antibody (e.g., For example, single domain antibodies (e.g., heavy chain variable domain antibodies), peptibodies, Fab flags, ment or scFv), small molecule or ligand (e.g., naturally occurring ligand, In some embodiments, the CD3 / The agent that stimulates the TCR complex is an antibody. In some embodiments, the CD3 / TCR complex The agent that stimulates the body is an anti-CD3 antibody. In some embodiments, the agent stimulates costimulatory molecules. The agent may be an antibody (e.g., a single domain antibody (e.g., a heavy chain variable domain antibody), a peptibody, a dimer, a Fab fragment or scFv), a small molecule or a ligand (e.g., a naturally occurring In some embodiments, the ligand is selected from the group consisting of a naturally occurring ligand, a recombinant ligand, or a chimeric ligand. In some embodiments, the agent that stimulates a costimulatory molecule is an antibody. In some embodiments, the agent that stimulates the CD3 / TCR complex is an anti-CD28 antibody. In some embodiments, the agent that stimulates a co-stimulatory molecule does not include beads. The CD3 / TCR complex-stimulating drug is covalently attached to the colloidal polymer nanomatrix. In some embodiments, the agent that stimulates a costimulatory molecule comprises an anti-CD3 antibody attached to the In some embodiments, the antibody comprises an anti-CD28 antibody covalently attached to a loid polymer nanomatrix. In this state, agents that stimulate the CD3 / TCR complex and agents that stimulate costimulatory molecules stimulate T cells. Includes TransAct(trademark).

[0216] In some embodiments, the matrix can be, for example, a polymer that is non-toxic to cells. The support may comprise or consist of, for example, a biodegradable or biocompatible inert material. In some embodiments, the matrix is ​​composed of hydrophilic polymer chains, which means that the water in the chains In some embodiments, the mobile matrix is The cox may be collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans or The composition may be made of an extracellular matrix composition. Examples of polysaccharides include cellulose. Ether, starch, gum arabic, agarose, dextran, chitosan, hyaluron Examples of suitable polysaccharides include polysaccharides, pectins, xanthan gum, guar gum, and alginates. Polymers include polyesters, polyethers, polyacrylates, and polyacrylamides. , polyamine, polyethyleneimine, polyquaternium polymer, polyphosphazene, poly polyvinyl alcohol, polyvinyl acetate, polyvinylpyrrolidone, block copolymers or In some embodiments, the flexible matrix may be a dextrose. It is a polymer of orchid.

[0217] In some embodiments, the population of cells is contacted with a nucleic acid molecule encoding a CAR. In this embodiment, the population of cells is transduced with DNA encoding the CAR.

[0218] In some embodiments, the population of cells is contacted with a nucleic acid molecule encoding a CAR. The step comprises treating a population of cells with an agent that stimulates the CD3 / TCR complex and / or the expression of said cells. In some embodiments, this is done simultaneously with the step of contacting the costimulatory molecule on the surface with the agent that stimulates the costimulatory molecule. In one embodiment, contacting the population of cells with a nucleic acid molecule encoding a CAR comprises contacting the population of cells with a nucleic acid molecule encoding a CAR. The group is then coupled to an agent that stimulates the CD3 / TCR complex and / or a costimulatory molecule on the surface of the cell. 30, 29, 28, 27, 26, 25, 2 after the start of the step of contacting with the stimulating agent 4, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0.5 hours. In embodiments, the step of contacting the population of cells with a nucleic acid molecule encoding a CAR comprises: A population of cells is treated with an agent that stimulates the CD3 / TCR complex and / or a co-stimulatory agent on the surface of the cells. The step of contacting the excitatory molecule with the agent that stimulates the excitatory molecule is performed within 20 hours after the start of the step of contacting the excitatory molecule with the agent that stimulates the excitatory molecule. In embodiments, the step of contacting the population of cells with a nucleic acid molecule encoding a CAR comprises The population of cells is then treated with an agent that stimulates the CD3 / TCR complex and / or a coactivator on the surface of the cells. The step of contacting the stimulatory molecule with the agent that stimulates the stimulatory molecule is performed within 19 hours after the start of the step. In an embodiment of the method, contacting the population of cells with a nucleic acid molecule encoding a CAR. The method comprises the steps of: treating a population of cells with an agent that stimulates the CD3 / TCR complex and / or with an agent that stimulates the CD3 / TCR complex on the surface of the cells; This is performed within 18 hours after the start of the step of contacting with the agent that stimulates the costimulatory molecule. In some embodiments, the step of contacting the population of cells with a nucleic acid molecule encoding a CAR. The group treats a population of cells with an agent that stimulates the CD3 / TCR complex and / or a receptor on the surface of the cells. The method is performed within 17 hours after the initiation of the step of contacting the cells with an agent that stimulates a costimulatory molecule of the present invention. In some embodiments, the population of cells is contacted with a nucleic acid molecule encoding a CAR. The triplicate is a method for treating a population of cells with an agent that stimulates the CD3 / TCR complex and / or the surface of the cells. The step of contacting the costimulatory molecule with an agent that stimulates the costimulatory molecule is carried out within 16 hours after the start of the step. In some embodiments, a population of cells is contacted with a nucleic acid molecule encoding a CAR. The step comprises treating a population of cells with an agent that stimulates the CD3 / TCR complex and / or the expression of said cells. The method is performed within 15 hours after the start of the step of contacting the surface with an agent that stimulates the costimulatory molecule. In some embodiments, the population of cells is contacted with a nucleic acid molecule encoding a CAR. The step comprises administering to the population of cells an agent that stimulates the CD3 / TCR complex and / or a therapeutic agent that stimulates the CD3 / TCR complex of the cells. Within 14 hours after the initiation of the step of contacting the costimulatory molecule on the surface with an agent that stimulates the costimulatory molecule. In some embodiments, the population of cells is contacted with a nucleic acid molecule encoding a CAR. The step of treating the population of cells with an agent that stimulates the CD3 / TCR complex and / or the cells The method is carried out within 14 hours after the start of the step of contacting the surface of the subject with an agent that stimulates the costimulatory molecule. In some embodiments, the population of cells is contacted with a nucleic acid molecule encoding a CAR. The step of inducing comprises treating the population of cells with an agent that stimulates the CD3 / TCR complex and / or the cells. Within 13 hours after the initiation of the step of contacting with an agent that stimulates a costimulatory molecule on the surface of the cell. In some embodiments, the population of cells is contacted with a nucleic acid molecule encoding a CAR. The step of allowing comprises treating the population of cells with an agent that stimulates the CD3 / TCR complex and / or the above-described Within 12 hours after initiation of the step of contacting the cells with an agent that stimulates costimulatory molecules on the surface of the cells. In some embodiments, the population of cells is contacted with a nucleic acid molecule encoding a CAR. The contacting step may involve contacting the population of cells with an agent and / or a surface active agent that stimulates the CD3 / TCR complex. 11 hours or less after initiation of the step of contacting the cells with an agent that stimulates the costimulatory molecules on the surface of the cells. In some embodiments, the population of cells is treated with a nucleic acid molecule encoding a CAR. The contacting step may comprise contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or 10 hours after the initiation of the step of contacting the cells with an agent that stimulates a costimulatory molecule on the surface of the cells. In some embodiments, the population of cells is subjected to a nucleic acid molecule encoding a CAR. The step of contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or is 9 hours after the start of the step of contacting the cells with an agent that stimulates the costimulatory molecules on the surface of the cells. In some embodiments, the population of cells is subjected to a nucleic acid molecule encoding a CAR. The step of contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or is 8 hours after the start of the step of contacting the cells with an agent that stimulates costimulatory molecules on the surface of the cells. In some embodiments, the population of cells is subjected to a nucleic acid molecule encoding a CAR. The step of contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or is 7 hours after the start of the step of contacting the cells with an agent that stimulates the costimulatory molecules on the surface of the cells. In some embodiments, the population of cells is subjected to a nucleic acid molecule encoding a CAR. The step of contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or is 6 hours after the start of the step of contacting the cells with an agent that stimulates costimulatory molecules on the surface of the cells. In some embodiments, the population of cells is subjected to a nucleic acid molecule encoding a CAR. The step of contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or is 5 hours after the start of the step of contacting the cells with an agent that stimulates the costimulatory molecules on the surface of the cells. In some embodiments, the population of cells is subjected to a nucleic acid molecule encoding a CAR. The step of contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or is 4 hours after the start of the step of contacting the cells with an agent that stimulates the costimulatory molecules on the surface of the cells. In some embodiments, the population of cells is subjected to a nucleic acid molecule encoding a CAR. The step of contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or is 3 hours after the start of the step of contacting the cells with an agent that stimulates the costimulatory molecules on the surface of the cells. In some embodiments, the population of cells is subjected to a nucleic acid molecule encoding a CAR. The step of contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or is 2 hours after the start of the step of contacting the cells with an agent that stimulates the costimulatory molecules on the surface of the cells. In some embodiments, the population of cells is subjected to a nucleic acid molecule encoding a CAR. The step of contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or is 1 hour after the start of the step of contacting the cells with an agent that stimulates the costimulatory molecules on the surface of the cells. In some embodiments, the population of cells is subjected to a nucleic acid molecule encoding a CAR. The step of contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or is 30 minutes after the start of the step of contacting the cells with an agent that stimulates the costimulatory molecules on the surface of the cells. This will be implemented within.

[0219] In some embodiments, the population of cells is harvested for storage or administration.

[0220] In some embodiments, the population of cells is treated with an agent that stimulates the CD3 / TCR complex and / or or 72 after the initiation of the step of contacting the cell with an agent that stimulates a costimulatory molecule on the surface of the cell. , 60, 48, 36, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, Harvesting a population of cells for storage or administration within 22, 21, 20, 19, or 18 hours In some embodiments, the population of cells is treated with an agent that stimulates the CD3 / TCR complex and / or or 26 after the initiation of the step of contacting the cell with an agent that stimulates a costimulatory molecule on the surface of the cell. Within hours, the population of cells is harvested for storage or administration. The population is treated with an agent that stimulates the CD3 / TCR complex and / or a costimulatory molecule on the surface of the cells. For storage or administration purposes within 25 hours after the start of the step of contacting the child with the stimulating agent. The population of cells is harvested. In some embodiments, the population of cells is subjected to a CD3 / TCR complex. and / or a co-stimulatory molecule on the surface of the cell. A population of cells is harvested for storage or administration within 24 hours of the start of the step. In one embodiment, the population of cells is treated with an agent that stimulates the CD3 / TCR complex and / or a cellular signaling pathway that stimulates the cells. Within 23 hours after the initiation of the step of contacting with an agent that stimulates costimulatory molecules on the surface of the cells. The population of cells is harvested for storage or administration purposes. In some embodiments, the population of cells is: Agents that stimulate the CD3 / TCR complex and / or costimulatory molecules on the surface of the cells The population of cells for storage or administration is prepared within 22 hours after the initiation of the step of contacting the population of cells with the agent. is collected.

[0221] In some embodiments, the population of cells is not expanded ex vivo.

[0222] In some embodiments, the population of cells is a population of cells that is amplified by, for example, a drug that stimulates the CD3 / TCR complex. A population of cells prior to contacting said population with an agent that stimulates co-stimulatory molecules on the surface of said cells. 5, 6, 7, 8, 9, 10, 11, 12, 1 3, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 In some embodiments, the CD3 / TCR complex is stimulated and expanded by no more than 55 or 60%. The cells prior to contact with an agent that stimulates co-stimulatory molecules on the surface of the cells and / or an agent that stimulates co-stimulatory molecules on the surface of the cells. Compared to the total cell population, the number of viable cells is estimated by less than 5%. In embodiments, the population of cells is treated with, for example, an agent that stimulates the CD3 / TCR complex and / or A population of cells that has survived for 10 years, compared to a population of cells that have not yet been contacted with an agent that stimulates co-stimulatory molecules on the surface of the cells. The cells are expanded by no more than 10% as assessed by cell number. The group may, for example, be an agent that stimulates the CD3 / TCR complex and / or a co-stimulatory agent on the surface of the cell. Assessed by the number of surviving cells compared to the population of cells prior to exposure to a stimulant agent. In some embodiments, the population of cells is expanded by no more than 15%. / An agent that stimulates the TCR complex and / or a costimulatory molecule on the surface of the cell The number of viable cells was assessed by 20% or less compared to the population of cells before contact with the In some embodiments, the population of cells is expanded, e.g., by stimulating the CD3 / TCR complex. and / or a costimulatory molecule on the surface of said cell prior to contact with said cell. Compared to the original population, the number of viable cells was estimated by expanding by 25% or less. In embodiments, the population of cells is treated with, for example, an agent that stimulates the CD3 / TCR complex and / or A population of cells that has survived for 10 years, compared to a population of cells that have not yet been contacted with an agent that stimulates co-stimulatory molecules on the surface of the cells. The cells are expanded by no more than 30% as assessed by cell number. The group may, for example, be an agent that stimulates the CD3 / TCR complex and / or a co-stimulatory agent on the surface of the cell. Assessed by the number of surviving cells compared to the population of cells prior to exposure to a stimulant agent. In some embodiments, the population of cells is expanded by no more than 35%. / An agent that stimulates the TCR complex and / or a costimulatory molecule on the surface of the cell The number of viable cells was assessed by 40% or less compared to the population of cells before contact with the They are multiplied.

[0223] In some embodiments, the population of cells is treated with, for example, one or more of the cytokines described above. The number of viable cells was assessed as 1, 1.5, 2, or 3, compared to the population of cells before exposure. 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20 , grown for no more than 24, 36 or 48 hours.

[0224] In some embodiments, the activation process is carried out in serum-free cell culture medium. In an embodiment, the activation process is mediated by IL-2, IL-15 (e.g., hetIL-1 5 (IL15 / sIL-15Ra)) or IL-6 (e.g., IL-6 / sIL-6Ra )) in a cell culture medium containing one or more cytokines selected from the following: In terms of morphology, hetIL-15 is [ka] In some embodiments, the hetIL-15 comprises the amino acid sequence of SEQ ID NO: 309. An amino acid sequence having at least about 70, 75, 80, 85, 90, 95, or 99% identity to In some embodiments, the activation process comprises a cell comprising an LSD1 inhibitor. In some embodiments, the activation process is carried out in a culture medium. In some embodiments, the serum-free cell culture medium is a blood substitute. In some embodiments, the serum replacement comprises CTS™ Immune Cell In some embodiments, The level of ICSR may be, for example, up to 5%, for example, about 1%, 2%, 3%, 4% or 5%. Without intending to be bound by theory, it is possible that the ICSR, e.g. Cell culture media such as Rapid Med (Rapid Med) shown in Table 21 or Table 25. ia) can be used to improve cell viability during the manufacturing processes described herein. can be done.

[0225] In some embodiments, the present disclosure provides a method for the production of a chimeric antigen receptor (CAR)-expressing cell (e.g., The present invention provides a method for producing a population of apoptotic cells (e.g., T cells) comprising: (a) administering to a subject an apoptotic cell population comprising: Leukapheresis samples (e.g., fresh or cryopreserved leukapheresis samples) (b) selecting T cells from the apheresis sample (e.g., by (c) isolating the cells using selective selection, positive selection or bead-free selection; For example, 1 x 10 T cells 6 ~1×10 7(d) T cells, inoculating at 100 cells / mL with an agent that stimulates T cells, e.g., an agent and / or cell that stimulates the CD3 / TCR complex. contacting the T cells with an agent that stimulates costimulatory molecules on the surface of the T cells (e.g., by contacting the T cells with anti-CD3 and / or contacting the T cells with an anti-CD28 antibody, e.g., contacting the T cells with TransAct) (e) administering to the patient a T cell that is a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding a CAR. For example, the T cells are contacted with the CAR for 6 to 48 hours, for example, for 20 to 28 hours. (f) contacting the virus with a virus containing a nucleic acid molecule to be encoded; and (f) storing (e.g., by freezing). (Formulating T cells in a freezing medium) or washing and harvesting T cells for administration In some embodiments, step (f) comprises step (d) or (e). Within 30 hours after the initiation of step (d) or (e), e.g., within 22, 23, 24 hours after the initiation of step (d) or (e), , 25, 26, 27, 28, 29 or 30 hours after the test.

[0226] Populations of CAR-expressing cells produced by the processes disclosed herein In some embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising the steps of, for example, any of the methods of manufacture described herein. and immune effector cells (e.g., T cells) engineered to express a CAR. In one embodiment, the engineered immune effector cells are characterized as tumor-specific inflammatory cells (e.g., NK cells or NK cells), where the engineered immune effector cells have anti-tumor properties. In some embodiments, the CAR comprises an antigen binding domain, a transmembrane domain and and an intracellular signaling domain. Exemplary antigens are the cancer associated antigens described herein. In some embodiments, the cell (e.g., a T cell or an NK cell) is transfected with a CAR. In some embodiments, the cell (e.g., T cells or NK cells) are transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the cells can stably express the CAR. The cell (e.g., T cell or NK cell) is administered a nucleic acid, e.g., an mRNA, cDNA, In some such embodiments, the cells are transfected with NA or DNA. The CAR can be transiently expressed.

[0227] In some embodiments, the methods of manufacture described herein (e.g., the methods of manufacture described herein) are These are generated by either an inhibitory or activation process and are adapted to express CAR. A collection of cells (e.g., immune effector cells, such as T cells or NK cells) that have been engineered to A team is provided.

[0228] In some embodiments, at the end of the manufacturing process (e.g., the cytokines described herein) are Naive cells in a population of cells (either in-process or at the end of an activation process), e.g. The percentage of isolated T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, is At the start of the manufacturing process (e.g., at the start of the cytokine process or activation process described herein), Naive cells, e.g., naive T cells, e.g., CD4 (1) is the same as the percentage of 5RA+CD45RO-CCR7+ T cells or (2) for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 % or less, or (3) at least 5, 6, 7, 8, 9, 10, 11, 12, 1 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 In some embodiments, at the end of the manufacturing process (e.g., At the end of the cytokine process or activation process described in Super-continuous (e.g., lasting for more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or or more than 3 days (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or Similar methods are used, but include expanding the population of cells in vitro for approximately 15 days. As compared to cells produced by the method, naive cells, e.g., naive T cells, e.g., C A higher percentage of CD45RA+CD45RO-CCR7+ T cells (e.g., At least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50% higher).

[0229] In some embodiments, at the end of the manufacturing process (e.g., the cytokines described herein) are Naive cells in a population of cells (either in-process or at the end of an activation process), e.g. The percentage of isolated T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, is , 20, 25, 30, 35, 40, 45, 50, 55 or 60% or more.

[0230] In some embodiments, at the end of the manufacturing process (e.g., the cytokines described herein) are central memory cells in a population of cells (either in-process or at the end of an activation process), For example, the percentage of central memory T cells, e.g., CD95+ central memory T cells The input is at the beginning of the manufacturing process (e.g., the cytokine process or Central memory cells in a population of cells (at the beginning of the activation process), e.g. compared with the percentage of CD95+ central memory T cells. (1) are the same, or (2) are, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1 (3) differ by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2 In some embodiments, the end of the manufacturing process is reduced by 3, 24, or 25%. At the end of the cytokine or activation process described herein, The cell population persists for, e.g., more than 26 hours (e.g., 5, 6, 7, 8, 9, 10, 11, or or persisting for more than 12 days), or by expanding a population of cells in vitro for, e.g., more than 3 days. (For example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 1 A similar method, but including the step of expanding the population of cells in vitro for 5 days. Compared to cells generated by For example, lower potency of central memory T cells, e.g., CD95+ central memory T cells -Percentage (e.g. at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50% lower ) is shown.

[0231] In some embodiments, at the end of the manufacturing process (e.g., the cytokines described herein) are central memory cells in a population of cells (either in-process or at the end of an activation process), For example, the percentage of central memory T cells, e.g., CD95+ central memory T cells The percentage is less than or equal to 40, 45, 50, 55, 60, 65, 70, 75 or 80%.

[0232] In some embodiments, at the end of the manufacturing process (e.g., the cytokines described herein) are The population of cells (either in-process or at the end of the activation process) is administered in vivo, For example, lasting more than 26 hours (for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or by expanding a population of cells in vitro for, e.g., more than 3 days (e.g., , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days (b) expanding the population of cells in vitro. The cell culture medium is then incubated for a period of time that is longer lasting or higher (e.g., at least 20, 25, 30 , 35, 40, 45, 50, 55, 60, 70, 75, 80, 85 or 90% higher) Proliferate at levels (e.g., as assessed using the methods described in Example 1 with reference to FIG. 4C) as it should be).

[0233] In some embodiments, the population of cells may be cultured prior to the start of the manufacturing process (e.g., as described herein). Prior to the initiation of the described cytokine or activation process), IL6R-expressing cells (e.g. , IL6Rα and / or IL6Rβ). The population of cells may be, for example, at the beginning of a manufacturing process (e.g., a cytogenetic analysis of the cells described herein). At the beginning of the activation process or activation process, for example, 30, 35, 40, 45, 50, 5 5, 60, 65, 70, 75, or 80% or less of IL6R-expressing cells (e.g., IL6Rα and and / or IL6Rβ positive cells).

[0234] Pharmaceutical Compositions Further, the present disclosure provides CAR-expressing cell compositions and methods for treating cancer or other diseases, among others, as described herein. Any malignant or autoimmune disease containing cells or tissues expressing the tumor antigens described in the document. In some embodiments, the present invention provides a method for treating a disease comprising administering to a patient a therapeutically effective amount of a medicament for the treatment of a disease. The present invention is directed to a method for treating or preventing atopic dermatitis in combination with one or more pharma- ceutically or physiologically acceptable carriers, diluents, or excipients. The manufacturing processes described herein (e.g., cytokine processes or activation processes described herein) A pharmaceutical comprising a CAR-expressing cell, e.g., a plurality of CAR-expressing cells, produced by the process. A composition is provided.

[0235] Chimeric antigen receptors (CARs) The present invention provides a method for targeting immune effector cells to cancer, comprising administering one or more CARs to the cancer. The present invention provides immune effector cells (e.g., T cells or NK cells) that have been engineered to This is achieved through an antigen-binding domain on the CAR that is specific for a cancer-associated antigen. There are two classes of cancer-associated antigens (tumor antigens) that can be targeted by the CARs described herein: (1) a cancer-associated antigen that is expressed on the surface of a cancer cell; and (2) itself within the cell. However, fragments (peptides) of such antigens are not bound to the MHC (major histocompatibility complex). A cancer-associated antigen that is displayed on the surface of cancer cells.

[0236] Thus, immune effector cells (e.g., those obtained by the methods described herein) may be used to ) is engineered to contain a CAR that targets one of the following cancer-associated antigens (tumor antigens): Can be used to detect: CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, P SMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PS CA, VEGFR2, Lewis Y, CD24, PDGFR-β, PRSS21, SSEA- 4. CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosin GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor body β, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CX ORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, Globo H, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, Reg Mine, HPV E6, E7, MAGE-A1, MAGE A1, ETV6-AML, Child protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, F os-related antigen 1, p53, p53 mutants, prostein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutants, hTERT, Sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA1 7, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2 , CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKA P-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal cancer carboxyl esterase and mut hsp70-2.

[0237] Non-limiting example sequences of various components that can be part of the CAR molecules described herein are shown in Table 1. The sequences are listed in Table 1, where "aa" represents an amino acid and "na" represents the corresponding peptide. It represents the nucleic acid that encodes it.

[0238] [Table 1]

[0239] [Table 2]

[0240] [Table 3]

[0241] [Table 4]

[0242] [Table 5]

[0243] Bispecific CAR In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody molecule has specificity for two or less antigens. A first immunoglobulin variable domain sequence having a combined specificity and a second immunoglobulin variable domain sequence for a second epitope. It is characterized by a second immunoglobulin variable domain sequence that has binding specificity. In one embodiment, the first and second epitopes are located on the same antigen, e.g., the same protein ( or subunits of a multimeric protein. In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes are distinct antigens, e.g. In some embodiments, the nucleotide sequence is in a multimeric protein (or in different subunits of a multimeric protein). In the present invention, a bispecific antibody molecule comprises a heavy chain variable domain having binding specificity for a first epitope. A light chain variable domain sequence and a heavy chain variable domain sequence having binding specificity for a second epitope. In some embodiments, the bispecific antibody comprises a light chain variable domain sequence and a light chain variable domain sequence. The molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In some embodiments, the bispecific antibody molecule comprises a half antibody having a first epitope. A half antibody or fragment thereof having binding specificity for a second epitope and In some embodiments, bispecific antibodies include half antibodies or fragments thereof having the same functional groups as the antibody. The molecule comprises an scFv or fragment thereof having binding specificity for a first epitope and a second epitope. The present invention includes an scFv or fragment thereof having binding specificity for an epitope of the present invention.

[0244] In certain embodiments, the antibody molecule is a multispecific (e.g., bispecific or trispecific) antibody. Protocols and methods for producing bispecific or heterodimeric antibody molecules are provided. Various configurations of bispecific antibody molecules are described, for example, in International Publication No. 2015 / 03 / 134, filed on March 13, 2015. The same is described in paragraphs 455 to 458 of the brochure No. 2015 / 142675, is incorporated by reference in its entirety.

[0245] In some embodiments, the bispecific antibody molecule comprises a first immunoglobulin variable domain sequence The antibody is characterized by a sequence, e.g., scFv, which has binding specificity for CD19, e.g., For example, the antibody may comprise an scFv as described herein or may be derived from an scFv as described herein. a light chain CDR and / or a heavy chain CDR and a second epitope having binding specificity for said ... The two immunoglobulin variable domain sequences are associated with different antigens.

[0246] Chimeric TCR In some embodiments, the antigens and antigen fragments of the invention (e.g., CD19 antibodies) and fragments) of one of the T cell receptor ("TCR") chains to create a chimeric TCR. One or more constant domains can be grafted onto, for example, the TCR α or TCR β chain. However, the chimeric TCR is thought to transmit signals through the TCR complex upon antigen binding. For example, an scFv as described herein can be used to bind to a TCR chain, e.g., a TCR α chain and and / or a constant domain of the TCR β chain, e.g., at least an extracellular constant domain, a transmembrane domain As another example, antibody fragments, e.g., A VL domain as described herein can be grafted onto the constant domain of the TCR α chain to produce an antibody against The VH domain as described herein may be coupled to the constant domain of the TCR β chain. domain (or alternatively, the VL domain can be grafted onto the constant domain of the TCR β chain). (The VH domain can be grafted onto the TCR α chain.) As another example, an antibody or antibody fragment can be The CDRs of the target gene can be grafted onto the TCR α and / or β chains to create a chimeric TCR. For example, the LCDRs described herein may be grafted into the variable domain of a TCR α chain, It is possible to graft the HCDRs described in the above onto the variable domain of a TCR β chain or vice versa. Such chimeric TCRs can be produced, for example, by methods known in the art (e.g. , Willemsen RA et al,Gene Therapy 2000;7: 1369-1377;Zhang T et al,Cancer Gene Ther 2004;11:487-496;Aggen et al,Gene Ther.2 012 Apr;19(4):365-74).

[0247] Non-antibody scaffolds In embodiments, the antigen binding domain is a non-antibody scaffold, e.g., fibronectin, Antibodies, domain antibodies, lipocalins, small modular immunopharmaceuticals, maxibodies, proteins The non-antibody scaffold contains affinins or affilins. The non-antibody scaffold has the ability to bind to target antigens on cells. In embodiments, the antigen binding domain is a naturally occurring protein expressed on a cell. In some embodiments, the antigen-binding domain is a polypeptide of a desired quality or a fragment thereof. The inclusion of such an antibody scaffold allows the resulting polypeptide to specifically bind to a target antigen on a target cell. A variety of non-antibody scaffolds can be used, so long as they contain at least one binding region that binds to the antibody. .

[0248] Non-antibody scaffolds include fibronectin (Novartis, MA), ankyrin (Molecular cular Partners AG, Zurich, Switzerland), domain antibodies (Dom antis, Ltd., Cambridge, MA and Ablynx nv, Zwijn aarde, Belgium), lipocalin (Pieris Proteolab AG, Fr eising, Germany), small modular immunopharmaceuticals (Trubion Pharma uticals Inc., Seattle, WA), Maxibody (Avidia, nc., Mountain View, CA), Protein A (Affibody AG , Sweden) and affilin (γ-crystallin or ubiquitin) (Scil Pro teins GmbH, Halle, Germany).

[0249] In some embodiments, the antigen binding domain binds to a counter ligand on the surface of a target cell. The present invention includes the extracellular domain of a molecule that binds to the ligand or a corresponding ligand-binding fragment thereof.

[0250] The immune effector cells contain a recombinant DNA construct that includes a sequence encoding a CAR. and the CAR can be a tumor antigen, e.g., a tumor antigen described herein, and an intracellular signaling an antigen-binding domain (e.g., an antibody or antibody fragment, The intracellular signaling domain contains a co-stimulatory signal. It may include a transduction domain and / or a primary signaling domain, e.g., the ζ chain. As described in, the methods described herein can be used to culture cells (e.g., T regulatory depleted cells). and (from the population) with a nucleic acid encoding a CAR, e.g., a CAR described herein. The method may include transducing the

[0251] In some embodiments, the CAR comprises an scFv domain, and the scFv is preceded by the sequence There may be an optional leader sequence as provided in SEQ ID NO:1, followed by SEQ ID NO:2 or Optional hinge sequences as provided in SEQ ID NO: 36 or SEQ ID NO: 38, An intracellular signaling domain comprising a transmembrane domain as provided, SEQ ID NO: 7 or SEQ ID NO: 16. The CD3ζ sequence may include the CD3ζ domain and SEQ ID NO:9 or SEQ ID NO:10, for example. The domains are contiguous and in the same reading frame to form a single fusion protein. Form.

[0252] In some embodiments, the exemplary CAR constructs include an optional leader sequence (e.g., a leader sequence as described herein), an extracellular antigen binding domain (e.g., a an antigen binding domain mounted on the membrane; a hinge (e.g., a hinge region described herein); A transmembrane domain (e.g., a transmembrane domain as described herein) and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain described herein). In particular, the exemplary CAR constructs can include an optional leader sequence (e.g., a leader sequence described herein). an extracellular antigen-binding domain (e.g., an antigen-binding domain described herein); ), a hinge (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain as described herein), an intracellular costimulatory signaling domain (e.g., The co-stimulatory signaling domains described herein and / or the intracellular primary signaling domains The polypeptides include a primary signaling domain (e.g., a primary signaling domain as described herein).

[0253] An exemplary leader sequence is provided as SEQ ID NO: 1. Examples of hinge / spacer sequences Exemplary transmembrane domains are provided as SEQ ID NO: 2, SEQ ID NO: 36, or SEQ ID NO: 38. The main sequence is provided as SEQ ID NO: 6. The sequence of the signal transduction domain is provided as SEQ ID NO: 7. An example of a signaling domain sequence is provided as SEQ ID NO: 16. The domain sequence is provided as SEQ ID NO:9 or SEQ ID NO:10.

[0254] In some embodiments, the immune effector cell comprises a nucleic acid molecule encoding a CAR. The nucleic acid molecule includes a nucleic acid sequence encoding an antigen-binding domain. This sequence is contiguous with the nucleic acid sequence encoding the intracellular signaling domain, Exemplary intracellular signaling domains that can be used in CARs include The main antibodies include, but are not limited to, one or more of CD3-zeta, CD28, 4-1BB, etc. In some examples, the CAR comprises an intracellular signaling domain of CD3-ζ, CD 28, CD27, 4-1BB, and any combination thereof.

[0255] The nucleic acid sequence encoding the desired molecule can be prepared using standard techniques, e.g., to express the nucleic acid molecule. by screening libraries from cells known to contain it or by deriving the nucleic acid molecule from a vector that contains it, or directly from cells and tissues that contain it. It can be obtained using recombinant methods known in the art, such as by isolating Alternatively, the nucleic acid of interest can be produced synthetically rather than cloned. .

[0256] The nucleic acid encoding the CAR can be, for example, a retroviral or lentiviral vector construct. can be used to introduce into immune effector cells.

[0257] The nucleic acid encoding the CAR can be, for example, an RNA construct that can be directly transfected into a cell. The immune effector cells can also be transfected using Methods for producing mRNA for this purpose include in vivo amplification of the template with specially designed primers. Intracellular transcription (IVT) followed by poly(A) addition, resulting in 3' and 5' untranslated sequences ("UTRs") (e.g., 3' and / or 5' UTRs as described herein), 5' capsid a 5′ cap as described herein and / or an internal ribosome entry site ( IRES) (e.g., an IRES described herein), the nucleic acid to be expressed and poly( A) A construct including a tail, typically 50 to 2000 bases long (e.g., as described in the Examples). The RNA thus produced is a different In some embodiments, the template can be efficiently transfected into cells of the same species. The rate comprises the sequence of the CAR. In some embodiments, the RNA CAR vector comprises: Cells, such as T cells, are transduced by electroporation.

[0258] Antigen-binding domain In some embodiments, a collection of multiple immune effector cells, such as T regulatory depleted cells, The panel includes a nucleic acid encoding a CAR that includes a target-specific binding element, also referred to as an antigen-binding domain. The choice of binding member will depend on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain may bind to a cell surface marker on a target cell that is associated with a particular disease state. Thus, the CAs described herein can be selected to recognize ligands that act as ligands. Examples of cell surface markers that can act as ligands for the antigen-binding domain in R include These include those associated with viral, bacterial and parasitic infections, autoimmune diseases and cancer cells.

[0259] In some embodiments, the portion of the CAR that comprises the antigen binding domain is a tumor antigen, e.g. For example, the antigen-binding domains include those that target a tumor antigen as described herein.

[0260] The antigen-binding domain may be, but is not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, or a recombinant protein. Recombinant antibodies, human antibodies, humanized antibodies and functional fragments thereof, including but not limited to , a heavy chain variable domain (VH), a light chain variable domain (VL) and a camelid-derived nanobody Single domain antibodies, such as the variable domain of the antibody (VHH) and recombinant fibronectin domain Main, T cell receptor (TCR) or a fragment thereof, such as a single chain TCR, antigen Any alternative scaffold known in the art to function as a binding domain may be used to bind to the antigen. In some examples, the antigen-binding domain is a domain that binds to the CAR. It is beneficial that it is derived from the same species in which it will ultimately be used; e.g. For use in this study, the antigen-binding domain of the CAR is linked to the antigen-binding domain of an antibody or antibody fragment. It may be beneficial to include humanized or humanized residues.

[0261] CD19CAR In some embodiments, the CAR-expressing cells described herein express CD19CAR. The present cell is a cell (e.g., a cell expressing a CAR that binds to human CD19).

[0262] In some embodiments, the antigen binding domain of the CD19CAR is et al.Mol.Immun.34(16-17):1157-1165(199 7) The same or similar binding specificity as the FMC63scFv fragment described in In some embodiments, the antigen binding domain of the CD19CAR has the structure lson et al.Mol.Immun.34(16-17):1157-1165 (1997) and the scFv fragments described therein.

[0263] In some embodiments, the CD19CAR is a compound according to the invention as disclosed in WO 2014 / 153270. Antigen-binding domains (e.g., For example, a humanized antigen-binding domain. The present invention also describes methods for assaying the binding and efficacy of various CAR constructs.

[0264] In some embodiments, the parent mouse scFv sequence is CAR19 constructs described in Brochure No. 0 (incorporated herein by reference). In some embodiments, the anti-CD19 binding domain is This is the scFv described in brochure no. 79000.

[0265] In some embodiments, the CAR molecule is The fusion polypeptide sequence is set forth in SEQ ID NO: 12 of the Rett protocol, which is a human CD19 The present invention provides a mouse-derived scFv fragment that specifically binds to

[0266] In some embodiments, the CD19CAR is a compound according to the invention as disclosed in WO 2012 / 079000. The amino acid sequence is set forth in SEQ ID NO: 12 of the accompanying specification.

[0267] In some embodiments, the amino acid sequence is [ka] or a substantially compatible sequence thereto.

[0268] In some embodiments, CD19CAR is designated by the USAN name TISAGENLECLEU In an embodiment, CTL019 is a T cell-specific T cell that has been genetically modified to This modification contains the CTL019 transgene under the control of the EF-1α promoter. Stable transduction with self-inactivating, replication-deficient lentiviral (LV) vectors CTL019 is a marker for the expression of IL-16, mediated by targeted insertion of IL-16. The T cells may be a mixture of transgene-positive and transgene-negative T cells delivered to the subject via a transgene-positive T cell line.

[0269] In other embodiments, the CD19CAR is Antigen-binding domains listed in Table 3 of Patent Publication No. 2014 / 153270 (e.g., humanized antigen-binding domain).

[0270] Humanization of the mouse CD19 antibody involves the substitution of mouse-specific residues for the CART19 treatment, i.e., Human-anti-mouse antigen in patients treated with T cells transduced with the AR19 construct In clinical settings where it may be possible to induce a humanized CD19CAR sequence, Production, characterization and efficacy, WO 2014 / 153270 (the entire contents of which are incorporated herein by reference) 159) and others.

[0271] In some embodiments, the CAR molecule is [ka] It is a humanized CD19CAR comprising the amino acid sequence:

[0272] In some embodiments, the CAR molecule is [ka] It is a humanized CD19CAR comprising the amino acid sequence:

[0273] Any known CD19 CAR, e.g., the CD19 antigen of any known CD19 CAR. Binding domains are known in the art and can be used in accordance with the present disclosure. For example, LG -740; U.S. Patent No. 8,399,645; U.S. Patent No. 7,446,190 Details;Xu et al.,Leuk Lymphoma.2013 54(2):25 5-260(2012);Cruz et al.,Blood 122(17):29 65-2973(2013);Brentjens et al.,Blood,118 (18):4817-4828(2011);Kochenderfer et al. ,Blood 116(20):4099-102(2010);Kochenderf er et al., Blood 122(25):4129-39(2013); and 16th Annu Meet Am Soc Gen Cell Ther(ASGC T)(May 15-18,Salt Lake City)2013,Abst 10 The CD19CAR is described in

[0274] Exemplary CD19CARs include those described herein or those described by Xu et al. l.Blood 123.24(2014):3750-9;Kochenderfer et al.Blood 122.25(2013):4129-39,Cruz e t al.Blood 122.17(2013):2965-73、NCT00586 391、NCT01087294、NCT02456350、NCT00840853、 NCT02659943、NCT02650999、NCT02640209、NCT0 1747486、NCT02546739、NCT02656147、NCT02772 198、NCT00709033、NCT02081937、NCT00924326、 NCT02735083、NCT02794246、NCT02746952、NCT0 1593696、NCT02134262、NCT01853631、NCT02443 831、NCT02277522、NCT02348216、NCT02614066、 NCT02030834、NCT02624258、NCT02625480、NCT0 2030847、NCT02644655、NCT02349698、NCT02813 837、NCT02050347、NCT01683279、NCT02529813、 NCT02537977、NCT02799550、NCT02672501、NCT0 2819583、NCT02028455、NCT01840566、NCT01318 317、NCT01864889、NCT02706405、NCT01475058、 NCT01430390、NCT02146924、NCT02051257、NCT0 2431988、NCT01815749、NCT02153580、NCT01865 617、NCT02208362、NCT02685670、NCT02535364、 NCT02631044, NCT02728882, NCT02735291, NCT0 1860937, NCT02822326, NCT02737085, NCT02465 983, NCT02132624, NCT02782351, NCT01493453, NCT02652910, NCT02247609, NCT01029366, NCT0 1626495, NCT02721407, NCT01044069, NCT00422 383, NCT01680991, NCT02794961 or NCT02456207 and the anti-CD19 CARs described in Can be enjoyed.

[0275] In some embodiments, the CD19CAR comprises the CDRs, VHs, , VL, scFv or the complete CAR sequence or at least 80%, 85%, 90% thereof , including sequences having 95% or 99% identity.

[0276] [Table 6]

[0277] [Table 7]

[0278] [Table 8]

[0279] [Table 9]

[0280] BCMA CAR In some embodiments, the CAR-expressing cells described herein are BCMA CAR An exemplary B cell is a cell expressing a CAR that binds to human BCMA. CMA CAR is incorporated herein by reference in its entirety. The BCMA CAR structure may include a sequence disclosed in Table 1 or 16 of the International Publication No. 2006 / 023991, which is incorporated herein by reference. The construct may include an optional leader sequence; an optional hinge domain, e.g., a CD8 hinge domain; transmembrane domain, e.g., CD8 transmembrane domain; intracellular domain, e.g., 4-1BB an intracellular domain; and a functional signaling domain, such as a CD3 zeta domain. In certain embodiments, the domains are contiguous and in the same reading frame. In other embodiments, the domains are combined to form a single fusion protein, e.g., Like the RCAR molecules described herein, they are present within separate polypeptides.

[0281] In some embodiments, a BCMA CAR molecule comprises one or more CDRs, a VH, a VL, scFv or BCMA- 1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6, BCM A-7, BCMA-8, BCMA-9, BCMA-10, BCMA-11, BCMA-1 2, BCMA-13, BCMA-14, BCMA-15, 149362, 149363, 149364, 149365, 149366, 149367, 149368, 14936 9, BCMA_EBB-C1978-A4, BCMA_EBB-C1978-G1, BC MA_EBB-C1979-C1, BCMA_EBB-C1978-C7, BCMA_E BB-C1978-D10, BCMA_EBB-C1979-C12, BCMA_EBB -C1980-G4, BCMA_EBB-C1980-D2, BCMA_EBB-C19 78-A10, BCMA_EBB-C1978-D4, BCMA_EBB-C1980- A2, BCMA_EBB-C1981-C3, BCMA_EBB-C1978-G4, A The full length sequence of 7D12.2, C11D5.3, C12A3.2 or C13F12.1 or a sequence substantially (e.g., 95 to 99%) identical thereto.

[0282] Another exemplary BCMA targeting sequence that can be used in an anti-BCMA CAR construct is , International Publication No. 2017 / 021450, International Publication No. 2017 / 011804, International Publication No. 2017 / 025038, 2016 / 090327, 2016 / 130598, 2016 / 090327, 2016 / 130598, No. 2016 / 210293, No. 2016 / 090320, No. 2016 / 0147 No. 89, No. 2016 / 094304, No. 2016 / 154055, No. 2015 / 166073, 2015 / 188119, 2015 / 158671 Frett, U.S. Patent Nos. 9,243,058, 8,920,776, and 9,27 Nos. 3,141, 7,083,785, and 9,034,324, U.S. Pat. Application No. 2007 / 0049735, No. 2015 / 0284467, No. 2015 / No. 0051266, No. 2015 / 0344844, No. 2016 / 0131655 , Same No. 2016 / 0297884, Same No. 2016 / 0297885, Same No. 2017 / No. 0051308, No. 2017 / 0051252, No. 2017 / 0051252 , International Publication No. 2016 / 020332, International Publication No. 2016 / 087531, International Publication No. No. 016 / 079177, No. 2015 / 172800, No. 2017 / 008169 No. 9,340,621, U.S. Patent Application No. 2013 / 0273 No. 055, No. 2016 / 0176973, No. 2015 / 0368351, No. 2 017 / 0051068, 2016 / 0368988 and 2015 / 023 No. 2557, which is incorporated herein by reference in its entirety. In some embodiments, another exemplary BCMA CAR construct is disclosed in International Publication No. No. 2012 / 0163805, the entirety of which is incorporated herein by reference. The VH and VL sequences are generated from

[0283] In some embodiments, the BCMA CAR comprises a sequence, for example, a sequence disclosed in Tables 3-15. CDR, VH, VL, scFv or complete CAR sequence or at least 80 %, 85%, 90%, 95% or 99% identity to the sequence. In some embodiments, the antigen-binding domain comprises a human antibody or a human antibody fragment. In embodiments, human anti-BCMA binding domains are described herein (e.g., in Tables 3-10 and 12-15) of one or more (e.g., all three) of the human anti-BCMA binding domains described herein. LC CDR1, LC, CDR2 and LC CDR3 and / or the amino acids described herein (e.g., One or more of the human anti-BCMA binding domains (e.g., For example, the HC CDR1, HC CDR2 and HC CDR3 are all three. In one embodiment, the human anti-BCMA binding domains are described herein (e.g., in Tables 3, 7 and 12). ) and / or the human VLs described herein (e.g., in Tables 3, 7 and 12). In some embodiments, the anti-BCMA binding domain comprises a VH. In some embodiments, the anti-BCM antibody is an scFv comprising the amino acid sequences VL and VH. The A binding domain (e.g., scFv) comprises the amino acids listed in Tables 3, 7 and 12. At least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence. , but having no more than 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions). or a sequence having 95 to 99% identity with the amino acid sequences in Tables 3, 7 and 12. and / or at least one of the amino acid sequences set forth in Tables 3, 7 and 12, Two or three modifications (e.g., substitutions, e.g., conservative substitutions), but not more than 30, 20 or or an amino acid sequence having 10 or fewer modifications (e.g., substitutions, e.g., conservative substitutions) or VH comprising a sequence having 95-99% identity with amino acid sequences 7 and 12.

[0284] [Table 10]

[0285] [Table 11]

[0286] [Table 12]

[0287] [Table 13]

[0288]

Table 14

[0289]

Table 15

[0290]

Table 16

[0291]

Table 17

[0292]

Table 18

[0293]

Table 19

[0294]

Table 20

[0295]

Table 21

[0296]

Table 22

[0297]

Table 23

[0298]

Table 24

[0299]

Table 25

[0300]

Table 26

[0301]

Table 27

[0302]

Table 28

[0303]

Table 29

[0304]

Table 30

[0305]

Table 31

[0306]

Table 32

[0307]

Table 33

[0308]

Table 34

[0309] [Table 35]

[0310] [Table 36]

[0311] [Table 37]

[0312] [Table 38]

[0313] [Table 39]

[0314] [Table 40]

[0315] [Table 41]

[0316] [Table 42]

[0317] [Table 43]

[0318] In some embodiments, the human anti-BCMA binding domain comprises HC CDR1, HC CDR2, HC CDR3, HC CDR4, HC CDR5, HC CDR6, HC CDR7, HC CDR8, HC CDR9, HC CDR10, HC CDR11, HC CDR12, HC CDR13, HC CDR14, HC CDR15, HC CDR16, HC CDR17, HC CDR LC CDR1, LC CDR2 and LC CDR3.

[0319] In certain embodiments, the CAR molecule described herein or the anti-BCM molecule described herein The A binding domain comprises: (1) one, two, or three light chain (LC) CDRs selected from: (i) LC CDR1 of SEQ ID NO: 54, LC CDR2 of SEQ ID NO: 55 and SEQ ID NO: 5 LC CDR3 of 6; and / or (2) one, two, or three heavy chain (HC) CDRs from one of the following: (i) HC CDR1 of SEQ ID NO: 44, HC CDR2 of SEQ ID NO: 45 and SEQ ID NO: 8 (ii) HC CDR1 of SEQ ID NO: 44, HC CDR2 of SEQ ID NO: 45; (iii) HC CDR1 of SEQ ID NO: 44, (iv) a HC CDR2 of SEQ ID NO: 45 and a HC CDR3 of SEQ ID NO: 68; or HC CDR1 of SEQ ID NO: 44, HC CDR2 of SEQ ID NO: 45 and HC CDR of SEQ ID NO: 76 R3.

[0320] In certain embodiments, the CAR molecule described herein or the anti-BCM molecule described herein The A binding domain comprises: (1) one, two, or three light chain (LC) CDRs from one of the following: (i) an LC CDR1 of SEQ ID NO: 95, an LC CDR2 of SEQ ID NO: 131, and an LC CDR3 of SEQ ID NO: (ii) LC CDR1 of SEQ ID NO: 95, LC CDR2 of SEQ ID NO: 96 (iii) LC CDR2 and CDR3 of SEQ ID NO: 97; 1, LC CDR2 of SEQ ID NO: 114 and LC CDR3 of SEQ ID NO: 115; or (iv LC CDR1 of SEQ ID NO: 95, LC CDR2 of SEQ ID NO: 114 and LC CDR3 of SEQ ID NO: 97 LC CDR3; and / or (2) one, two, or three heavy chain (HC) CDRs from one of the following: (i) HC CDR1 of SEQ ID NO: 86, HC CDR2 of SEQ ID NO: 130 and SEQ ID NO: (ii) HC CDR1 of SEQ ID NO: 86, HC CDR3 of SEQ ID NO: 87 CDR2 and HC CDR3 of SEQ ID NO: 88; or (iii) HC CDR of SEQ ID NO: 86 R1, HC CDR2 of SEQ ID NO:109 and HC CDR3 of SEQ ID NO:88.

[0321] In certain embodiments, the CAR molecule described herein or the anti-BCM molecule described herein The A binding domain comprises: (1) one, two, or three light chain (LC) CDRs from one of the following: (i) LC CDR1 of SEQ ID NO: 147, LC CDR2 of SEQ ID NO: 182 and SEQ ID NO: (ii) LC CDR3 of SEQ ID NO: 183; (ii) LC CDR1 of SEQ ID NO: 147, SEQ ID NO: 148 and an LC CDR3 of SEQ ID NO: 149; or (iii) an LC CDR2 of SEQ ID NO: 147 LC CDR1 of SEQ ID NO: 170, LC CDR2 of SEQ ID NO: 171 and LC CDR 3; and / or (2) one, two, or three heavy chain (HC) CDRs from one of the following: (i) HC CDR1 of SEQ ID NO: 179, HC CDR2 of SEQ ID NO: 180 and SEQ ID NO: (ii) HC CDR3 of SEQ ID NO: 181; (ii) HC CDR1 of SEQ ID NO: 137, SEQ ID NO: 138 and a HC CDR2 of SEQ ID NO: 139; or (iii) a HC CDR3 of SEQ ID NO: 160 HC CDR1 of SEQ ID NO: 161, HC CDR2 of SEQ ID NO: 162 and HC CDR 3.

[0322] In some embodiments, the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 are SEQ ID NOs: 44, 45 and 84, respectively. In some embodiments, the HC CDR1 comprises the amino acid sequence of , HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR 3 contain the amino acid sequences of SEQ ID NOs: 44, 45, 46, 54, 55 and 56, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 are SEQ ID NOs: 44, 45 and 68, respectively. In some embodiments, the HC CDR1 comprises the amino acid sequence of , HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR 3 contain the amino acid sequences of SEQ ID NOs: 44, 45, 76, 54, 55 and 56, respectively. .

[0323] In some embodiments, the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 are SEQ ID NOs: 47, 48 and 84, respectively. In some embodiments, the HC CDR1 comprises the amino acid sequence of , HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR 3 contain the amino acid sequences of SEQ ID NOs: 47, 48, 46, 57, 58 and 59, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 are SEQ ID NOs: 47, 48 and 68, respectively. In some embodiments, the HC CDR1 comprises the amino acid sequence of , HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR 3 contain the amino acid sequences of SEQ ID NOs: 47, 48, 76, 57, 58 and 59, respectively. .

[0324] In some embodiments, the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 are SEQ ID NOs: 49, 50, 85, respectively. In some embodiments, the HC CDR1 , HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR 3 contain the amino acid sequences of SEQ ID NOs: 49, 50, 51, 60, 58 and 56, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 are SEQ ID NOs: 49, 50 and 69, respectively. In some embodiments, the HC CDR1 , HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR 3 contain the amino acid sequences of SEQ ID NOs: 49, 50, 77, 60, 58 and 56, respectively. .

[0325] In some embodiments, a human anti-BCMA binding domain comprises a VH (e.g., Some scFvs include those comprising a VH (e.g., a VL as described herein) and a VL (e.g., a VL as described herein). In embodiments, the VH is linked to a linker, e.g., a linker described herein, e.g., a linker in Table 1. In some embodiments, the human anti-BCMA binding site is linked to the VL via a linker as described herein. The binding domain is (Gly 4 -Ser)n linker, where n is 1, 2, 3, 4 , 5 or 6, preferably 3 or 4 (SEQ ID NO: 26). and the heavy chain variable region, for example, in the following orientation: light chain variable region-linker-heavy chain variable region or It may be either heavy chain variable region-linker-light chain variable region.

[0326] In some embodiments, the anti-BCMA binding domain is a fragment, such as a single chain variable fragment ( In some embodiments, the anti-BCMA binding domain is an Fv, Fab, (scFv). Fab')2 or a bifunctional (e.g., bispecific) hybrid (e.g., Lan zavecchia et al.,Eur.J.Immunol.17,105(19 87). In some embodiments, the antibodies and fragments thereof of the present invention are either wild-type or have increased affinity. It binds to the BCMA protein in a potent manner.

[0327] In some embodiments, scFvs can be prepared according to methods known in the art. (e.g., Bird et al., (1988) Science 242:423 -426 and Huston et al., (1988) Proc. Natl. Acad. .Sci.USA 85:5879-5883). scFv molecules are flexible polypeptides. The VH and VL domains can be linked together using a peptide linker. The scFv molecules can be modified with a linker having an optimized length and / or amino acid composition. (e.g., a Ser-Gly linker). The linker length determines how the scFv molecule In fact, the variable regions of short polypeptides can greatly affect how they fold and interact with one another. The use of a linker (e.g., 5–10 amino acids) prevents intrachain folding. Interchain folding also brings the two variable regions together to form functional epitopes. necessary to form a binding site. For example, the linker orientation and size may be Hollinger et al.1993 Proc Natl Acad.Sci .USA90:6444-6448, U.S. Patent Application No. 2005 / 0100543 , 2005 / 0175606, 2007 / 0014794 and International Publication No. 2006 / 020258 Brochure and No. 2007 / 024715 Brochure See Lett, incorporated herein by reference.

[0328] An scFv may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 The linker may be 35, 40, 45, 50 or more amino acid residues. The anchor sequence may comprise any naturally occurring amino acid. The linker sequence comprises the amino acids glycine and serine. In some embodiments, the linker sequence is , a series of glycine and serine repeats, e.g. (Gly 4 Ser)n (where n is 1 or more In some embodiments, the linker comprises (Gl y 4 Ser) 4 (SEQ ID NO: 27) or (Gly 4 Ser) 3 (SEQ ID NO:28) Varying the linker length either retains or increases activity, resulting in superior efficacy in activity tests. obtain.

[0329] CD20 CAR In some embodiments, the CAR-expressing cells described herein are CD20 CAR-expressing In some embodiments, the cell is a human CD20-binding CAR. In this regard, the CD20 CAR expressing cells are described in International Publication No. 2013 / 013363, which is incorporated herein by reference. Anti-corrosion measures in accordance with Brochure No. 16164731 and Brochure No. 2018067992 Exemplary CD20 binding sequences or CD20 CAR sequences include, for example, These are disclosed in Tables 1 to 5 of International Publication No. 2018067992. In embodiments, the CD20 CAR is a polypeptide as described in WO2018067992 or The CDR, variable region, scFv or contains the full length sequence.

[0330] CD22 CAR In some embodimen...

Claims

1. Methods for generating populations of cells (e.g., T cells) expressing chimeric antigen receptors (CARs) And, (i) cells (e.g., T cells, e.g., frozen or fresh leukapheresis products) The population of T cells isolated from the subject is treated with an agent that stimulates the CD3 / TCR complex and / or contacting (e.g., binding) an agent that stimulates a costimulatory molecule on the surface of the cell; (ii) administering to the population of cells (e.g., T cells) a nucleic acid molecule encoding the CAR (e.g., For example, a DNA or RNA molecule, thereby producing a cell (e.g., providing a population of T cells; (iii) preservation (e.g., reformulating the population of cells in a cryopreservation medium); or Harvesting the population of cells (e.g., T cells) for administration. Including, (a) Step (ii) is performed together with step (i) or 2 min after the start of step (i). Within 0 hours, for example, 12, 13, 14, 15, 16, 17 or 20 hours after the start of step (i). at or within 18 hours, e.g., within 18 hours after the initiation of step (i), and Step (iii) may be carried out within 30 (e.g., 26) hours after the initiation of step (i), e.g. For example, 22, 23, 24, 25, 26, 27, 28, 29 or 3 after the start of step (i). or within 0 hours, e.g. within 24 hours after the initiation of step (i); (b) Step (ii) may be performed together with step (i) or 2 hours after the start of step (i). Within 0 hours, for example, 12, 13, 14, 15, 16, 17 or 20 hours after the start of step (i). at or within 18 hours, e.g., within 18 hours after the initiation of step (i), and Step (iii) may be carried out within 30 hours after the initiation of step (ii), for example, step ( ii) within 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the onset of be carried out or (c) the population of cells from step (iii) is, for example, or not proliferated as assessed by the number of viable cells compared to a population of said cells of Proliferate by 0, 15, 20, 25, 30, 35, or 40% or less, for example by 10% or less. 、 Optionally, the nucleic acid molecule in step (ii) is on a viral vector; Optionally, the nucleic acid molecule in step (ii) is an RNA molecule on a viral vector. and optionally, step (ii) comprises subjecting said population of cells (e.g., T cells) to a transducing a viral vector comprising a nucleic acid molecule encoding the CAR. 。

2. The agent that stimulates the CD3 / TCR complex may be an agent that stimulates CD3 (e.g., an anti-CD 3 antibody), and the agent that stimulates a costimulatory molecule is CD28, ICOS, CD27, H VEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, An agent that stimulates TIM1, CD2, CD226, or any combination thereof, Optionally, the agent stimulates the CD3 / TCR complex or the agent stimulates a costimulatory molecule. can be used to generate antibodies (e.g., single domain antibodies (e.g., heavy chain variable domain antibodies), peptibodies, , Fab fragment or scFv), a small molecule or a ligand (e.g., a naturally occurring a ligand that is capable of binding to a target ligand, a recombinant ligand or a chimeric ligand, and optionally The agent that stimulates the .DELTA..TCR complex or the agent that stimulates the costimulatory molecule comprises beads. First, optionally, the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody; and the agent that stimulates costimulatory molecules comprises an anti-CD28 antibody, and optionally a CD3 / T The agent that stimulates the CR complex is covalently attached to the colloidal polymer nanomatrix. The agent comprising an anti-CD3 antibody and stimulating a costimulatory molecule is a colloidal polymer nanoparticle. and optionally, a CD3 / TCR complex. The agent that stimulates and the agent that stimulates costimulatory molecules are T Cell TransAc 10. The method of claim 1 , comprising:

3. Step (i) comprises cross-reacting with CAR-expressing cells in the population of cells from step (iii). For example, the population of cells from step (iii) is The CAR-expressing cells were compared to cells generated by a similar method but without step (i). A higher percentage of cells (e.g., at least 10, 20, 30, 40, 50, or 6 3. The method of claim 1 or 2, wherein the .alpha.-amino acid concentration is 0.0% higher.

4. (a) selecting a naive cell in the population of cells from step (iii), such as a naive T The percentage of cells, e.g., CD45RA+CD45RO-CCR7+ T cells, is A naive cell, e.g., a naive T cell, in the population of cells at the start of step (i), e.g. The percentage of CD45RA+CD45RO-CCR7+ cells was equal to or 5 times younger than the percentage of CD45RA+CD45RO-CCR7+ cells. or differ by less than 10%; (b) selecting a naive cell in the population of cells from step (iii), such as a naive T The percentage of cells, e.g., CD45RA+CD45RO-CCR7+ T cells, e.g., For example, naive cells, e.g., naive T cells, in the population of cells at the start of step (i). , for example, is low compared to the percentage of CD45RA+CD45RO-CCR7+ cells. All are 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8 or 3 Is it increased by a factor of 1? (c) a CAR-expressing naive T cell, e.g., a CAR-expressing CD45RA T cell, in the population of cells. The percentage of CD45RO-CCR7+ T cells increases during step (ii). For example, at least 30, 35, 40 , increased by 45, 50, 55 or 60%; or (d) selecting a naive cell in the population of cells from step (iii), such as a naive T The percentage of cells, e.g., CD45RA+CD45RO-CCR7+ T cells, is A naive cell, e.g., a naive T cell, in the population of cells at the start of step (i), e.g. The percentage of CD45RA+CD45RO-CCR7+ cells is not decreased or The method according to any one of claims 1 to 3, wherein the is reduced by no more than 5 or 10%.

5. (a) the population of cells from step (iii), wherein step (iii) comprises the steps of: More than 26 hours after the initiation of step (i), e.g., 5, 6, 7, 8, 9, 10, 11 or 12 days after the first injection. As compared to cells that have been subjected to the A higher percentage of RO-CCR7+ T cells (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 0 or 40% higher); (b) selecting a naive cell in the population of cells from step (iii), such as a naive T The percentage of cells, e.g., CD45RA+CD45RO-CCR7+ T cells, is Step (iii) is continued for more than 26 hours after the initiation of step (i), e.g. Unless it is carried out more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of the Naive cells, such as naive T cells, e.g., C cells, among cells produced by similar methods. A higher percentage of CD45RA+CD45RO-CCR7+ T cells (e.g., fewer At most 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8 or 3 times higher) or; (c) detecting a CAR-expressing naive T cell in the population of cells from step (iii), such as For example, the percentage of CAR-expressing CD45RA+CD45RO-CCR7+ T cells is Step (iii) is continued for more than 26 hours after the initiation of step (i), e.g. Unless it is carried out more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of the CAR-expressing naive T cells, e.g., CAR-expressing CTCs, among cells produced by similar methods. A higher percentage of CD45RA+CD45RO-CCR7+ T cells (e.g., fewer at least 4, 6, 8, 10 or 12 times higher; (d) the population of cells from step (iii) is subjected to step (ii) and step (iii) the population of cells (e.g., T cells) has been incubated in vitro for more than 3 days, e.g., 5, 6 The cells were cultured in a similar manner, but further comprising the step of growing the cells for 7, 8 or 9 days. naive cells, e.g., naive T cells, e.g., CD45RA T cells, as compared to cells generated by A higher percentage of CD45RO-CCR7+ T cells (e.g., at least 10 , 20, 30 or 40% higher); (e) selecting a naive cell in the population of cells from step (iii), such as a naive T The percentage of cells, e.g., CD45RA+CD45RO-CCR7+ T cells, is After step (ii) and before step (iii), the population of cells (e.g., T cells) is in vitro. The method further comprises growing the culture in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. However, naive cells, such as naive T cells, may be produced by the same method. , for example, a higher percentage of CD45RA+CD45RO-CCR7+ T cells (e.g. 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 higher); or (f) detecting a CAR-expressing naive T cell in the population of cells from step (iii), such as For example, the percentage of CAR-expressing CD45RA+CD45RO-CCR7+ T cells is After step (ii) and before step (iii), the population of cells (e.g., T cells) is in vitro. The method further comprises growing the culture in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. CAR-expressing naive T cells, e.g., C A higher percentage of AR-expressing CD45RA+CD45RO-CCR7+ T cells (e.g. For example, at least 4, 6, 8, 10 or 12 times higher), Method of posting.

6. (a) a central memory cell in the population of cells from step (iii), such as Percentage of central memory T cells, e.g., CD95+ central memory T cells The cell population at the start of step (i) is a central memory cell, e.g., a cell Percentage of central memory T cells, e.g., CD95+ central memory T cells or differs by no more than 5 or 10%; (b) a central memory cell in the population of cells from step (iii), e.g. The percentage of central memory T cells, e.g., CCR7+CD45RO+ T cells, In the population of cells at the start of step (i), a central memory cell, e.g., compared to the percentage of primary memory T cells, e.g., CCR7+CD45RO+ T cells. is reduced by at least 20, 25, 30, 35, 40, 45, or 50%; (c) a CAR-expressing central memory T cell, e.g., a CAR-expressing CCR7+CD45R The percentage of O+ cells is decreased during step (ii), e.g., during step (i). i) by at least 8, 10, 12, 14, 16, or 20% at 18 to 24 hours after the start of Decrease; or (d) a central memory cell in the population of cells from step (iii), e.g. The percentage of central memory T cells, e.g., CCR7+CD45RO+ T cells, In the population of cells at the start of step (i), a central memory cell, e.g., compared to the percentage of primary memory T cells, e.g., CCR7+CD45RO+ T cells. The method according to any one of claims 1 to 5, wherein the amount of the stoichiometric ratio is not increased or increased by no more than 5 or 10%. Method of posting.

7. (a) the population of cells from step (iii), wherein step (iii) comprises the steps of: More than 26 hours after the initiation of step (i), e.g., 5, 6, 7, 8, 9, 10, 11 or 12 days after the first injection. Compared to cells that have been treated with IFN-γ, central memory cells, e.g., central memory T cells, e.g., C A lower percentage of D95+ central memory T cells (e.g., at least 10 , 20, 30 or 40% lower); (b) a central memory cell in the population of cells from step (iii), e.g. The percentage of central memory T cells, e.g., CCR7+CD45RO+ T cells, Step (iii) is performed more than 26 hours after the start of step (i), e.g., step (i) that is carried out more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of the Others have reported that central memory cells, such as central The percentage of primary memory T cells, e.g., CCR7+CD45RO+ T cells ( For example, at least 20, 30, 40 or 50% lower; (c) detecting a CAR-expressing central memory T cell in the population of cells from step (iii). The percentage of cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, may be determined by steps (iii) is performed more than 26 hours after the start of step (i), e.g., The same applies except that the implementation is carried out more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of the program. CAR-expressing central memory T cells, e.g., CAR A lower percentage of CCR7+CD45RO+ T cells expressing CCR7+CD45RO+ T cells (e.g., at least 1 0, 20, 30 or 40% lower); (d) the population of cells from step (iii) is subjected to step (ii) and step (iii) the population of cells (e.g., T cells) has been incubated in vitro for more than 3 days, e.g., 5, 6 The cells were cultured in a similar manner, but further comprising the step of growing the cells for 7, 8 or 9 days. Compared to cells generated by For example, a lower percentage of CD95+ central memory T cells (e.g., at least 10, 20, 30 or 40% lower; (e) a central memory cell in the population of cells from step (iii), e.g. The percentage of central memory T cells, e.g., CCR7+CD45RO+ T cells, after step (ii) and before step (iii), Further, the step of growing in vitro for more than 3 days, e.g., 5, 6, 7, 8 or 9 days. Central memory cells, e.g., cells produced by a similar method except that they include For example, the percentage of central memory T cells, e.g., CCR7+CD45RO+ T cells is lower (e.g., at least 20, 30, 40, or 50% lower); or (f) detecting a CAR-expressing central memory T cell in the population of cells from step (iii). The percentage of cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, may be determined by steps After step (ii) and prior to step (iii), subjecting the population of cells (e.g., T cells) to in vitro The method further comprises the step of growing the culture at 4° C. for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. Others have reported CAR-expressing central memory T cells produced by similar methods, e.g. For example, a lower percentage of CAR-expressing CCR7+CD45RO+ T cells (e.g., The method according to any one of claims 1 to 6, wherein the 。

8. (a) selecting stem memory T cells in the population of cells from step (iii), such as C Percentage of D45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells The page includes a stem memory T cell, e.g., a C cell, in the population of cells at the start of step (i). Percentage of D45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells is increased compared to the page; (b) a CAR-expressing stem memory T cell in the population of cells from step (iii). , for example, CAR expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L + T cells in the population of cells at the start of step (i). Stem memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+ is increased relative to the percentage of CCR7+CD62L+ T cells; (c) stem memory T cells in the population of cells from step (iii), such as C Percentage of D45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells The process may further comprise the step (iii) being carried out for more than 26 hours after the initiation of step (i), e.g. (i) is performed more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). Stem memory T cells, e.g., CD4+ T cells, were generated by a similar method, but without the need for a stem cell line. Percentage of 45RA+CD95+IL-2 receptor beta+CCR7+CD62L+T cells higher than (d) a CAR-expressing stem memory T cell in the population of cells from step (iii). , for example, CAR expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L The percentage of + T cells is determined by the step (iii) at 26 hours after the start of the step (i). For example, 5, 6, 7, 8, 9, 10, 11 or 12 hours after the start of step (i). A similar method was used to generate CAR expression sequences in cells, but the procedure was performed after 1 day. stem memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+C a higher percentage of CR7+CD62L+ T cells; (e) stem memory T cells in the population of cells from step (iii), such as C Percentage of D45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells The page may further comprise: The population is expanded in vitro for more than 3 days, e.g., 5, 6, 7, 8 or 9 days. stem memory T cells in cells produced by a similar method, but further comprising For example, CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cell population -percentage higher; or (f) a CAR-expressing stem memory T cell in the population of cells from step (iii). , for example, CAR expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L The percentage of + T cells after step (ii) and before step (iii) The population (e.g., T cells) is cultured in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. C in cells produced by a similar method, but further comprising the step of propagating the cells by AR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2-receptor The present invention relates to a method for treating a patient having a pulmonary edema, comprising administering to said patient a therapeutically effective amount of at least one of said at least one T cells, said at least one T cell being at least one CCR7+CD62L+T cell level. The method according to any one of claims 1 to 5.

9. (a) the median GeneSetScore ( Up TEM vs. Down TSCM refers to the concentration of the cells at the start of step (i). The GeneSetScore median of the group (Up TEM vs. Down TSCM) are approximately the same or differ by no more than about 25, 50, 75, 100, or 125% (e.g., if so, it will be increased by less; (b) the median GeneSetScore ( Up TEM vs. Down TSCM) Step (iii) is continued for more than 26 hours after the initiation of step (i), e.g. (i) that is carried out more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of the Other examples include cells produced by similar methods, or After step (ii) and before step (iii), subjecting the population of cells (e.g., T cells) to Further, the step of growing in vitro for more than 3 days, e.g., 5, 6, 7, 8 or 9 days. Cells produced by a similar method except that Lower than the median GeneSetScore (Up TEM vs. Down TSCM) (e.g., at least about 100, 150, 200, 250, or 300% lower); (c) the median GeneSetScore ( Up Treg vs. Down Teff of the cells at the start of step (i). Median GeneSetScore of the population (Up Treg vs. Down Teff) about the same as or differing by no more than about 25, 50, 100, 150, or 200% (e.g., increased by less than that); (d) the median GeneSetScore ( Up Treg vs. Down Teff Step (iii) is continued for more than 26 hours after the initiation of step (i), e.g. (i) that is carried out more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of the Other examples include cells produced by similar methods, or After step (ii) and before step (iii), subjecting the population of cells (e.g., T cells) to Further, the step of growing in vitro for more than 3 days, e.g., 5, 6, 7, 8 or 9 days. Cells produced by a similar method except that From the GeneSetScore median (Up Treg vs. Down Teff) low (e.g., at least about 50, 100, 125, 150, or 175% lower); (e) the median GeneSetScore ( Down stemness refers to the gene down-stemness of the population of cells at the start of step (i). SetScore median (Down stemness) or about 25 , differ by less than 50, 100, 150, 200, or 250% (e.g., (increased by 10%); (f) the median GeneSetScore ( Down stemness Step (iii) is continued for more than 26 hours after the initiation of step (i), e.g. (i) that is carried out more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of the Other examples include cells produced by similar methods, or After step (ii) and before step (iii), subjecting the population of cells (e.g., T cells) to Further, the step of growing in vitro for more than 3 days, e.g., 5, 6, 7, 8 or 9 days. Cells produced by a similar method except that Lower than the GeneSetScore median (Down stemness) of at least about 50, 100, or 125% lower; (g) the median GeneSetScore ( Up hypoxia) refers to the GeneSet of the population of cells at the start of step (i). Score median (Up hypoxia) is approximately equal to or about 125, 150, differs (e.g., increased by less than) 175 or 200%; (h) the median GeneSetScore ( Up hypoxia is Step (iii) is continued for more than 26 hours after the initiation of step (i), e.g. (i) that is carried out more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of the Other examples include cells produced by similar methods, or After step (ii) and before step (iii), subjecting the population of cells (e.g., T cells) to Further, the step of growing in vitro for more than 3 days, e.g., 5, 6, 7, 8 or 9 days. Cells produced by a similar method except that Lower than the GeneSetScore median (Up hypoxia) (e.g., about 40, 50, 60, 70 or 80% lower); (j) the median GeneSetScore ( Up autophagy refers to the GeneS of the population of cells at the start of step (i). approximately equal to or about 180 median etScore (Up autophagy); differs by (e.g., is increased by) no more than 190, 200, or 210% or (k) the median GeneSetScore ( Up autophagy) is Step (iii) is continued for more than 26 hours after the initiation of step (i), e.g. (i) that is carried out more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of the Other examples include cells produced by similar methods, or After step (ii) and before step (iii), subjecting the population of cells (e.g., T cells) to Further, the step of growing in vitro for more than 3 days, e.g., 5, 6, 7, 8 or 9 days. Cells produced by a similar method except that Lower than the GeneSetScore median (Upautophagy) of The method of any one of claims 1 to 8, wherein the concentration of the ion exchange resin is at least about 20, 30 or 40% lower.

10. The population of cells from step (iii) expresses an antigen recognized by the CAR. After incubation with cells expressing Step (iii) is a step of determining whether the More than 26 hours after initiation, for example 5, 6, 7, 8, 9, 10 hours after initiation of step (i). Cells produced by the same method, but performed after 11 or 12 days. or collection of said cells (e.g., T cells) after step (ii) and before step (iii). The cells are grown in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. The cells produced have higher levels (e.g., , or at least 2, 4, 6, 8, 10, 12, or 14 times higher) of IL-2. Item 10. The method according to any one of items 1 to 9.

11. The population of cells from step (iii) is administered in vivo and then subjected to step (i ii) is for more than 26 hours after the initiation of step (i), e.g. , 5, 6, 7, 8, 9, 10, 11, or 12 days after the date of the or after step (ii) and step (iii), ) the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8 or 9 days. The cells persist longer or proliferate at higher levels compared to cells produced by As assessed using the method described in Example 1 for C), any of claims 1 to 10 The method according to any one of claims 1 to 5.

12. The population of cells from step (iii) is administered in vivo and then subjected to step (i ii) is for more than 26 hours after the initiation of step (i), e.g. , 5, 6, 7, 8, 9, 10, 11, or 12 days after the date of the or a cell produced by the method of any one of claims 1 to 5 after step (ii) and before step (iii). The population of cells (e.g., T cells) is incubated in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. Cells made by a similar method, but with the additional step of growing the cells for 1 day. More potent antitumor activity than the cells (e.g., low doses, e.g., 0.15×10 ６ , 0.2×10 ６ , 0.25×10 ６ or 0.3 x 10 ６ Stronger antitumor activity in viable CAR-expressing cells The method according to any one of claims 1 to 11, wherein

13. The population of cells from step (iii) is the same as the population of cells at the start of step (i). compared to the population, e.g., no proliferation or 5, 10, 15, or 20% proliferation as assessed by the number of viable cells. , 20, 25, 30, 35, or 40% or less, and optionally, The number of viable cells in the population of cells from step (ii) is The method of any one of claims 1 to 12, wherein the number of viable cells in the population is reduced.

14. The population of cells from step (iii) is the same as the population of cells at the start of step (i). The cells are not multiplied or are multiplied for less than 2 hours, e.g., less than 1 or 1.5 hours, relative to the population. The method according to any one of claims 1 to 13,

15. Steps (i) and / or (ii) may include administering to the mammalian subject an IL-2, IL-15 (e.g., hetIL- 15 (IL15 / sIL-15Ra)), IL-7, IL-21, IL-6 (e.g., I LSD1 inhibitor, MALT1 inhibitor, or a combination thereof The method according to any one of claims 1 to 14, wherein the method is carried out in a cell culture medium (e.g., a serum-free medium) comprising: The method described above.

16. Steps (i) and / or (ii) are carried out in a serum-free cell culture medium containing a serum replacement. The method according to any one of claims 1 to 15,

17. The serum replacement is CTS™ Immune Cell Serum Replacement 17. The method of claim 16, wherein the sol is a sol-gel cement (ICSR).

18. Before step (i), (iv) (Optionally) obtain fresh leukocyte samples from an entity, e.g., a laboratory, hospital, or healthcare provider; Pheresis products (or fresh whole blood products, fresh bone marrow products, or fresh tumor or organ products) Alternative sources of hematopoietic tissue, such as organ biopsy or resection (e.g., fresh product from a thymus removal) ), and (v) fresh leukapheresis product (or fresh whole blood product, fresh bone marrow product or may be a fresh tumor or organ biopsy or resection (e.g., fresh product from a thymus gland ablation), etc. from an alternative source of hematopoietic tissue (e.g., T cells) contacted in step (i). and / or CD4+ T cells) from the target cell. and optionally further comprising: Step (iii) may be performed within 35 hours after the initiation of step (v), for example step (v Within 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the onset of For example, within 30 hours after the initiation of step (v), or The population of cells from step (iii) is the population of cells at the end of step (v). Compared to the group, no proliferation was observed or 5, 10, 15, 20, or 30% proliferation was observed as assessed by the number of viable cells. , 25, 30, 35 or 40%, for example 10% or less.

18. The method according to any one of claims 17 to 17.

19. Prior to step (i), leukocyte apheresis may be obtained from an entity, e.g., a laboratory, hospital, or healthcare provider. lysis products (or whole blood, bone marrow, or tumor or organ biopsy or resection (e.g., thymus Alternative sources of hematopoietic tissue, such as cryopreserved T cells isolated from The method according to any one of claims 1 to 17, further comprising the step of receiving stored T cells. Law.

20. Before step (i), (iv) (Optionally) cryopreserved white blood cells from an entity, e.g., a laboratory, hospital, or healthcare provider. Apheresis products (or cryopreserved whole blood products, cryopreserved bone marrow products, or cryopreserved tumor tissue products) or from organ biopsies or resections (e.g., cryopreserved products from thymus removal) of hematopoietic tissue. receiving an alternative supply source; and (v) Cryopreserved leukapheresis products (or cryopreserved whole blood products, cryopreserved bone marrow) Products or cryopreserved tumor or organ biopsies or resections (e.g., cryopreserved tissue from thymus removal) and (b) contacting the cells (e.g., from an alternative source of hematopoietic tissue, such as a hematopoietic product) with the cells (e.g., For example, isolating a population of T cells, e.g., CD8+ and / or CD4+ T cells. and optionally further comprising: Step (iii) may be performed within 35 hours after the initiation of step (v), for example step (v Within 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the onset of For example, within 30 hours after the initiation of step (v), or The population of cells from step (iii) is the population of cells at the end of step (v). Compared to the group, no proliferation was observed or 5, 10, 15, 20, or 30% proliferation was observed as assessed by the number of viable cells. , 25, 30, 35 or 40%, for example 10% or less.

18. The method according to any one of claims 17 to 17.

21. Step (vi): separating a portion of the population of cells from step (iii) into at least two portions; 2.5, 3, 3.5, 4, 4.5, 5.5, 6, 6.5 or 7 days, for example at least 2 days and culturing the cells for 7 days or less, and measuring the level of CAR expression in the portion (e.g., (b) measuring the percentage of viable CAR-expressing cells in said portion. and optionally further comprising: Step (iii) comprises harvesting and freezing the population of cells (e.g., T cells). and step (vi) comprises thawing a portion of the population of cells from step (iii). and dividing said portion by at least 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 6, 6.5 or is cultured for 7 days, for example, for at least 2 days and not more than 7 days, and The expression level is measured (e.g., the percentage of viable CAR-expressing cells in the portion is measured). The method of any one of claims 1 to 20, comprising:

22. Methods for generating populations of cells (e.g., T cells) expressing chimeric antigen receptors (CARs) And, (1) Cells (e.g., T cells, e.g., T cells isolated from frozen leukapheresis products) The population of cells is treated with IL-2, IL-7, IL-15, IL-21, IL-6 or any of their respective a cytokine selected from the combination of (2) Transfecting a population of cells (e.g., T cells) with a nucleic acid molecule encoding the CAR (e.g., For example, a DNA or RNA molecule, thereby producing a cell that contains the nucleic acid molecule (e.g., providing a population of T cells; (3) Preservation (e.g., reformulating the population of cells in cryopreservation medium) or administration harvesting the population of cells (e.g., T cells) for Including, (a) Step (2) is performed simultaneously with step (1) or 5 hours after the start of step (1). for example, within 1, 2, 3, 4, or 5 hours after the initiation of step (1); Beauty Step (3) is carried out within 26 hours after the initiation of step (1), for example, Within 22, 23 or 24 hours after initiation, for example within 24 hours after initiation of step (1). be carried out or (b) the population of cells from step (3) is, for example, Compared to the population of cells described above, the cells either did not proliferate or showed no significant changes in cell viability, as assessed by the number of viable cells. proliferate by no more than 15, 20, 25, 30, 35, or 40%, e.g., no more than 10%, Optionally, the nucleic acid molecule in step (2) is on a viral vector, optionally Optionally, the nucleic acid molecule in step (ii) is an RNA molecule on a viral vector. and optionally, step (ii) comprises subjecting the population of cells (e.g., T cells) to transducing with a viral vector comprising a nucleic acid molecule encoding a CAR.

23. Step (1) comprises contacting the population of cells (e.g., T cells) with IL-2.

23. The method of claim 22, comprising:

24. Step (1) comprises contacting the population of cells (e.g., T cells) with IL-7.

23. The method of claim 22, comprising:

25. Step (1) comprises inducing a population of cells (e.g., T cells) to IL-15 (e.g., het The method according to claim 22, comprising contacting the antibody with IL-15 (IL15 / sIL-15Ra). Method of posting.

26. Step (1) includes contacting the population of cells (e.g., T cells) with IL-21.

23. The method of claim 22, comprising:

27. Step (1) comprises inducing a population of cells (e.g., T cells) with IL-6 (e.g., IL-6 23. The method of claim 22, comprising contacting the antibody with IL-6. / sIL-6Ra.

28. Step (1) comprises inducing a population of cells (e.g., T cells) to IL-7 and IL-15 (e.g., For example, the method includes contacting the antibody with hetIL-15 (IL15 / sIL-15Ra).

23. The method according to claim 22.

29. Step (1) comprises contacting a population of cells (e.g., T cells) with IL-7 and IL-21.

23. The method of claim 22, comprising contacting.

30. Step (1) comprises inducing a population of cells (e.g., T cells) to IL-15 (e.g., het IL-15 (IL15 / sIL-15Ra) and IL-21, 23. The method of claim 22.

31. Step (1) comprises inducing a population of cells (e.g., T cells) to IL-7, IL-15 (e.g., For example, contacting with hetIL-15 (IL15 / sIL-15Ra) and IL-21 The method of claim 22, comprising:

32. Step (1) comprises inducing a 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-1 23. The method of claim 22, comprising contacting said compound with 5Ra).

33. Step (1) comprises treating a population of cells (e.g., T cells) with IL-2 and IL-6 (e.g., 23. The method of claim 22, comprising contacting said IL-6 / sIL-6Ra with said IL-6 / sIL-6Ra.

34. The population of cells from step (3) may be contacted with, for example, an anti-CD3 antibody. The method further comprises the step of: A higher percentage of naive cells among R-expressing cells (e.g., at least 10, 1 5, 20, 25, 30, 35 or 40% higher The method described above.

35. Naive cells, e.g., naive T cells, in the population of cells from step (3), e.g., For example, the percentage of CD45RA+CD45RO-CCR7+ T cells is (a) a naive cell, such as a naive T cell, in the population of cells at the start of step (1); cells, e.g., CD45RA+CD45RO-CCR7+ cells. or differ by no more than 5 or 10%; (b) a naive cell, such as a naive T cell, in the population of cells at the start of step (1). cells, e.g., CD45RA+CD45RO-CCR7+ cells. Any of claims 22 to 34, wherein the .alpha.-methyl-2-propanol is added, for example increased by at least 10 or 20%.

3. The method according to claim 1 .

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

37. The population of cells from step (3) is pretreated after step (2) and before step (3). The population of cells (e.g., T cells) is cultured in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. Cells produced by a similar method, but further comprising the step of growing the cells for compared to naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO- A higher percentage of CCR7+ T cells (e.g., at least 10, 20, 30 or The method according to any one of claims 22 to 36, wherein the IL-10 concentration is 40% higher than the IL-10 concentration.

38. The population of cells from step (3) is administered in vivo, after which step (3) , More than 26 hours after the start of step (1), for example, 5, 6 hours after the start of step (1) , 7, 8, 9, 10, 11, or 12 days after the cells that persist longer or proliferate to a higher degree (e.g., 4C), as assessed using the method described in Example 1 with respect to FIG.

37. The method according to any one of claims 37 to 37.

39. The population of cells from step (3) is administered in vivo after step (2). and prior to step (3), the population of cells (e.g., T cells) is cultured in vitro for more than 3 days, e.g., The same method is used, but further includes the step of growing the cells for, for example, 5, 6, 7, 8, or 9 days. persist longer or proliferate to higher levels compared to cells produced by the method (e.g., as assessed using the method described in Example 1 with respect to FIG. 4C ) Item 39. The method according to any one of items 22 to 38.

40. The population of cells from step (3) may be different from the population of cells at the start of step (1). In comparison, no proliferation or 5, 10, 15, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 0, 25, 30, 35 or 40% or less, e.g., 10% or less, optionally The number of viable cells in the population of cells from step (3) is The method according to any one of claims 22 to 39, wherein the number of viable cells in the population of cells is reduced. How to.

41. The population of cells from step (3) may be different from the population of cells at the start of step (1). is not expanded or expanded for less than 2 hours, e.g., less than 1 or 1.5 hours, compared to The method according to any one of claims 22 to 40.

42. The population of cells may be treated with an agent that stimulates the CD3 / TCR complex and / or ... When not contacted in vitro with an agent that stimulates the costimulatory molecule of said 2. The method of claim 1, wherein the contacting step is for less than 2 hours, e.g., 1 or 1.5 hours or less.

42. The method according to any one of claims 2 to 41.

43. The agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3 (e.g., an anti-CD3 antibody). The agent that stimulates costimulatory molecules is selected from the group consisting of CD28, ICOS, CD27, H VEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, An agent that stimulates TIM1, CD2, CD226, or any combination thereof, Optionally, the agent stimulates the CD3 / TCR complex or the agent stimulates a costimulatory molecule. can be used to generate antibodies (e.g., single domain antibodies (e.g., heavy chain variable domain antibodies), peptibodies, , Fab fragment or scFv), a small molecule or a ligand (e.g., a naturally occurring 43. The method according to claim 42, wherein the ligand is selected from the group consisting of a recombinant ligand and a chimeric ligand. Method of posting.

44. Steps (1) and / or (2) 5, 4, 3, 2, 1 or 0% or less serum, optionally comprising step (1) and / or or (2) is carried out in a cell culture medium containing about 2% serum, or LSD1 inhibitor or MALT1 inhibitor The method according to any one of claims 22 to 43, wherein the method is carried out in a cell culture medium comprising:

45. An entity, such as a laboratory, hospital, or health care provider, may purchase cryopreserved leukapheresis products (or or cryopreserved whole blood products, cryopreserved bone marrow products, or cryopreserved tumor or organ biopsies or Receive an alternative source of hematopoietic tissue, such as a cryopreserved product from a thymus removal The method of any one of claims 22 to 44, further comprising the steps of:

46. The population of cells at the start of step (i) or step (1) is an IL6R-expressing cell ( For example, cells that are positive for IL6Rα and / or IL6Rβ The method according to any one of claims 1 to 45.

47. The population of cells at the start of step (i) or step (1) may be 50, 60 or 70 % or more of IL6R expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ) 47. The method of any one of claims 1 to 46, comprising administering to said subject a therapeutically effective amount of ... medicament for which said therapeutically effective amount is less than or equal to 100 mg / mL.

48. Steps (i) and (ii) or steps (1) and (2) may be performed using IL-15 (e.g. The method is carried out in a cell culture medium containing hetIL-15 (IL15 / sIL-15Ra).

48. The method according to any one of claims 1 to 47.

49. IL-15 may be involved in the ability of said cell population to expand after, for example, 10, 15, 20 or 25 days.

49. The method of claim 48, wherein the

50. IL-15 increases the percentage of IL6Rβ-expressing cells in the cell population 49. The method of claim 48.

51. The CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The method of any one of claims 1 to 50, comprising:

52. The antigen-binding domain may be CD19, CD20, CD22, BCMA, mesothelin, E GFRvIII, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptide, sTn- O-glycopeptide, PSMA, CD97, TAG72, CD44v6, CEA, EPC AM, KIT, IL-13Ra2, Legman, GD3, CD171, IL-11Ra, P SCA, MAD-CT-1, MAD-CT-2, VEGFR2, Lewis Y, CD24, P DGFR-β, SSEA-4, folate receptor α, ERBB (e.g., ERBB2), Her 2 / neu, MUC1, EGFR, NCAM, ephrinB2, CAIX, LMP2, sL e, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TE M7R, FAP, legumain, HPV 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, AF P, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, human thyroglobulin melase reverse transcriptase, intestinal carboxylesterase, mut hsp 70-2, NA-1 7, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO -1, GPR20, Ly6k, OR51E2, TARP, GFRα4 or MHC-presented 52. The method of claim 51, wherein the peptide binds to an antigen selected from any of the peptides of the antigens. method.

53. The antigen binding domain may be any of the CDR, VH, VL, scFv or CDR domains disclosed herein. AR sequence, and optionally (a) the antigen-binding domain binds to BCMA and is a CD4 antigen-binding domain disclosed in Tables 3-15 R, VH, VL, scFv or CAR sequence or at least 80%, 85%, 90%, or contains a sequence with 0%, 95% or 99% identity; (b) the antigen-binding domain binds to CD19 and has the CDRs, V and VV disclosed in Table 2. H, VL, scFv or CAR sequence or at least 80%, 85%, 90% thereof, contains a sequence with 95% or 99% identity; (c) the antigen-binding domain binds to CD20 and has a CDR as disclosed herein. , VH, VL, scFv or CAR sequence or at least 80%, 85%, 90%, or %, 95% or 99% identity; or (d) the antigen-binding domain binds to CD22 and has a CDR as disclosed herein. , VH, VL, scFv or CAR sequence or at least 80%, 85%, 90%, or 53. The method of claim 51 or 52, comprising a sequence having 95% or 99% identity to said Law.

54. The antigen-binding domain comprises a VH and a VL, and 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. The method according to any one of claims 51 to 53.

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

56. The antigen binding domain is linked to the transmembrane domain by a hinge region, and optionally In the choice, (a) the hinge region comprises an amino acid sequence of SEQ ID NO: 2, 3, or 4, or at least one amino acid sequence selected from the group consisting of and each of the sequences has about 85%, 90%, 95% or 99% sequence identity. or (b) the nucleic acid molecule comprises a nucleic acid sequence encoding the hinge region, the nucleic acid sequence comprising 13, 14 or 15, or at least about 85%, 90% thereof; Any of claims 51 to 55, comprising a nucleic acid sequence having 95% or 99% sequence identity.

3. The method according to claim 1 .

57. The intracellular signaling domain comprises a primary signaling domain, and optionally The primary signaling domains are CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ , CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (IC OS), functional signals derived from FcεRI, DAP10, DAP12 or CD66d a transmission domain, and optionally (a) the primary signaling domain is a functional signaling domain derived from CD3ζ; Includes in (b) the primary signaling domain has the amino acid sequence of SEQ ID NO: 9 or 10, or having at least about 85%, 90%, 95% or 99% sequence identity thereto or (c) the nucleic acid molecule comprises a nucleic acid sequence encoding the primary signaling domain; The nucleic acid sequence is a nucleic acid sequence of SEQ ID NO: 20 or 21, or at least about 85% thereof; Claims 51 to 56, comprising a nucleic acid sequence having 90%, 95% or 99% sequence identity 2. The method according to claim 1 .

58. The intracellular signaling domain comprises a costimulatory signaling domain, and optionally , the costimulatory signaling domain is selected from the group consisting of MHC class I molecules, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytes Activation of lymphocytes by activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll liga androgenic 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, SL AMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19 , CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, V LA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD 49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29 , ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRAN CE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD2 29), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69 , SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, I PO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​L AT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, C and optionally a ligand that specifically binds to D28-4-1BB or CD83. (a) the costimulatory signaling domain is a functional signaling domain derived from 4-1BB Include the domain, or (b) the costimulatory signaling domain has the amino acid sequence of SEQ ID NO: 7 or at least one amino acid sequence thereof. and an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity with the Muka, or (c) the nucleic acid molecule comprises a nucleic acid sequence encoding the costimulatory signaling domain. The nucleic acid sequence is a nucleic acid sequence of SEQ ID NO: 18 or at least about 85%, 90%, 90%, 100%, or 150% identical thereto. Any of claims 51 to 57, comprising a nucleic acid sequence having 5% or 99% sequence identity.

13. The method according to claim 1.

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

60. 5. The CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO:

1.

60. The method of any one of 1 to 59.

61. A CAR-expressing cell (e.g., For example, a population of autologous or allogeneic CAR-expressing T cells or NK cells.

62. A population of cells engineered to express a CAR (a "population of CAR-expressing cells") 、 (a) a naive cell in the same population of cells prior to being engineered to express said CAR; For example, compared to the percentage of naive T cells, e.g., CD45RO-CCR7+ cells. , about the same percentage of naive cells, e.g., naive T cells, e.g., CD45RO -CCR7+ cells; (b) naive cells in the same population of cells prior to being engineered to express said CAR; For example, compared to the percentage of naive T cells, e.g., CD45RO-CCR7+ cells. , about 5% of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ cells ~ within about 10% change; (c) naive cells in the same population of cells prior to being engineered to express said CAR; For example, compared to the percentage of naive T cells, e.g., CD45RO-CCR7+ cells. , increased, for example, by at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2 .4, 2.6, 2.8 or 3 fold increased percentage of naive cells, e.g. Breast cancer cells, e.g., CD45RO-CCR7+ cells; (d) a central memory in the same population of cells before they were engineered to express the CAR; Lee cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells. Compared to the percentage of central memory cells, which is roughly the same percentage as central memory T cells, e.g., CCR7+CD45RO+ T cells; (e) a central memory in the same population of cells before they were engineered to express the CAR; Lee 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, For example, a change within about 5% to about 10% of CCR7+CD45RO+ T cells; (f) a central memory in the same population of cells before they were engineered to express the CAR; Lee cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells. Reduced, e.g., by at least 20, 25, 30, 35, 40, as compared to percentage , 45 or 50% reduced percentage of central memory cells, e.g., CENTRA full memory T cells, e.g., CCR7+CD45RO+ T cells; (g) stem memory in the same population of cells before they were engineered to express the CAR. T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T Compared to the percentage of cells, there is a roughly similar percentage of stem memory T cells, e.g. CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells; (h) stem memory in the same population of cells before they were engineered to express the CAR. T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T Compared to the percentage of cells that are stem memory T cells, e.g., CD45RA+CD95 + A change of about 5% to about 10% of IL-2 receptor beta CCR7 CD62L T cells; or teeth (i) stem memory in the same population of cells before they were engineered to express the CAR; T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T An increased percentage of stem memory T cells compared to a percentage of cells, e.g. For example, CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells A population of cells containing

63. A population of cells engineered to express a CAR (a "population of CAR-expressing cells") 、 (a) Median GeneSetScore of the cell population (Up TEM vs. Do tscm) is a genomic DNA fragment of the same population of cells before they were engineered to express the CAR. Almost the same as the neSetScore median (Up TEM vs. Down TSCM) or differs by no more than about 25, 50, 75, 100, or 125% (e.g., (increased by: (b) Median GeneSetScore of the cell population (Up Treg vs. D own Teff) is the G of the population of cells before they are engineered to express the CAR. Almost the same as eneSetScore median (Up Treg vs. Down Teff) are the same or differ by no more than about 25, 50, 100, 150, or 200% (e.g. , or less); (c) The median GeneSetScore of the population of cells (Down stemnes s) is a GeneSetS of the population of cells before they are engineered to express the CAR. Approximately the same as the down stemness or about 25, 50, Different by no more than 100, 150, 200, or 250% (e.g., increased by no more than that) (Can be); (d) The median GeneSetScore (Up hypoxia) of the cell population is GeneSetScore of the population of cells before they are engineered to express the CAR e Approximately the same as the median (Up hypoxia) or approximately 125, 150, 175 or differs by (e.g., is increased by) no more than 200%; or (e) the median GeneSetScore of the cell population (Up autophagy ) is a GeneSetSc of the population of cells before they are engineered to express the CAR. or approximately 180, 190, A population of cells that differs (e.g., is increased by) no more than 200 or 210% Group.

64. A population of CAR-expressing cells according to any one of claims 61 to 63, and a pharma- ceutically acceptable and a carrier comprising the compound.

65. A method for increasing an immune response in a subject, comprising administering to a subject a compound according to any one of claims 61 to 63. Administering to the subject a population of CAR-expressing cells or the pharmaceutical composition of claim 64, thereby increasing the immune response in said subject.

66. A method for treating cancer in a subject, comprising administering to a subject a CAR-expressing antibody according to any one of claims 61 to 63.

65. The method of claim 64, further comprising administering to said subject a population of present cells or a pharmaceutical composition of claim 64, thereby treating said cancer in an elephant.

67. 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, colon cancer, prostate cancer , cervical cancer, skin cancer, melanoma, kidney cancer, liver cancer, brain tumor, thymoma, sarcoma, carcinoma, uterine cancer, kidney Cancer of the digestive tract, urothelial cancer, pharyngeal cancer, head and neck cancer, rectal cancer, esophageal cancer or bladder cancer 67. The method of claim 66, wherein the cancer is a solid cancer or a metastasis thereof selected from one or more of the following:

68. The cancer may be, for example, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), , multiple myeloma, acute lymphoblastic leukemia (ALL), Hodgkin's lymphoma, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphocytic leukemia (TALL), small lymphocytic leukemia ( SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt Lymphoma lymphoma, diffuse large B-cell lymphoma (DLBCL), DLBCL with chronic inflammation, Chronic myeloid leukemia, myeloproliferative neoplasms, follicular lymphoma, childhood follicular lymphoma, hairy leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MA LT lymphoma (mucosa-associated lymphoid tissue type extranodal marginal zone lymphoma), marginal zone lymphoma, bone Myelodysplastic syndromes, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid Dendritic cell neoplasm, Waldenstrom's macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma Lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia - variant Atypical, lymphoplasmacytic lymphoma, heavy chain disease, plasma cell myeloma, solitary bone plasmacytoma, Extraskeletal plasmacytoma, nodal marginal zone lymphoma, childhood nodal marginal zone lymphoma, primary cutaneous follicular Cardiac lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, endovascular Large B-cell lymphoma, ALK+ large B-cell lymphoma, HHV8-associated multicentric lymphoma Large B-cell lymphoma, primary effusion lymphoma, and B-cell lymphoma occurring in Muscleman's disease a liquid cancer selected from acute myeloid leukemia (AML) or unclassifiable lymphoma; 67. The method of claim 66.

69. 69. The method of claim 65, further comprising administering a second therapeutic agent to the subject.

3. The method according to claim 1 .

70. The second therapeutic agent is IL-15 (e.g., hetIL-15 (IL15 / sIL-1 5Ra)).

71. 70. The method of claim 69, wherein the second therapeutic agent is ibrutinib.

72. Ibrutinib is about 600 mg, about 550 mg, about 500 mg, about 480 mg, about 46 0 mg, about 440 mg, about 420 mg, about 400 mg, about 350 mg, about 300 mg, about 280mg, about 250mg, about 200mg, about 190mg, about 180mg, about 170mg , about 160 mg, about 150 mg, about 140 mg, about 130 mg, about 120 mg or about 100 72. The method of claim 71, wherein the compound is administered once daily at a dose of about 100 mg.

73. Ibrutinib is administered once daily at doses of 420 mg, 280 mg, or 140 mg.

72. The method of claim 71 .

74. Ibrutinib is administered prior to, concurrently with, or after said administration of the population of CAR-expressing cells.

74. The method according to any one of claims 71 to 73, wherein

75. The population of CAR-expressing cells is the percentage of CAR-expressing cells measured in claim 21. The method according to any one of claims 65 to 74, wherein the dose is determined based on the Method of posting.

76. The population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is about 0.5 x 10 ６ ~50 x 10 ６ 10 viable CAR-expressing cells, e.g., about 5 x 10 ６ of viable CAR-expressing cells and optionally, said CAR-expressing cells (e.g., CD19 CAR-expressing cells) The population is 5 x 10 ６ 76. The method of claim 65, wherein the medicament is administered in a dose of 100 viable CAR-expressing cells.

2. The method according to any one of claims 1 to 11.

77. The population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is about 2.5 x 10 ６ ~2.5 x 10 ８ 10 viable CAR-expressing cells, e.g., about 2.5 x 10 ７ Viable CAR expression and optionally, the CAR-expressing cells (e.g., CD19 CAR-expressing The population of cells was 2.5 × 10 ７ 6. The method of claim 5, wherein the subject is administered a dose of viable CAR-expressing cells. 5-75. The method according to any one of claims 5 to 75.

78. The population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is about 1.25 x 10 ７ ~1.25 x 10 ９ 10 viable CAR-expressing cells, e.g., about 1.25 x 10 ８ Individual Survival CA and optionally, the CAR-expressing cells (e.g., CD19CA The population of R-expressing cells was 1.25 × 10 ８ 100 mg of viable CAR-expressing cells, 76. The method according to any one of claims 65 to 75.

79. The population of CAR-expressing cells (e.g., BCMA CAR-expressing cells) is about 2.5 x 10 ６ ~2.5 x 10 ８ 10 viable CAR-expressing cells, e.g., about 1 x 10 ７ Or 5 x 10 ７ Individual life The method of any one of claims 65 to 75, wherein the tumor necrosis factor is administered in a dose of resident CAR-expressing cells.

80. The method according to any one of claims 66 to 79, wherein the subject has CLL or SLL. Law.

81. The subject has DLBCL, e.g., relapsed and / or refractory DLBCL.

80. The method of any one of claims 66 to 79.

82. The subject may be, for example, at least 2, 2.5, 3, 3.5, or 4 days, for example, about 3 days. The method according to any one of claims 65 to 81, wherein the patient is monitored for signs of cytokine release syndrome. The method according to any one of claims 1 to 5.

83. 2. For use in a method of increasing an immune response in a subject, said method comprising administering to a subject an effective amount of administering to the subject the population of CAR-expressing cells or an effective amount of the pharmaceutical composition. A population of CAR-expressing cells according to any one of claims 61 to 63 or a population of CAR-expressing cells according to claim 64. The pharmaceutical composition described.

84. The method comprises administering to a subject a therapeutically effective amount of said CAI. administering to said subject a population of R-expressing cells or an effective amount of said pharmaceutical composition. The population of CAR-expressing cells according to any one of claims 61 to 63 or the pharmaceutical composition according to claim 64. Pharmaceutical composition.

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