Methods to improve the effectiveness and growth of immune cells

Abstract

To provide methods of making immune effector cells (e.g., T cells, NK cells) that can be engineered to express a chimeric antigen receptor (CAR), compositions comprising the same and reaction mixtures.SOLUTION: A method for expanding and / or activating a population of immune cells (e.g., immune effector cells) includes introducing a nucleic acid encoding a first chimeric antigen receptor (CAR) molecule into an immune cell population, under conditions suitable for transient expression of the CAR molecule, thereby producing a first CAR-expressing cell population, where the CAR molecule comprises an antigen binding domain of an antigen molecule; and contacting the first CAR-expressing cell population with a ligand of the CAR antigen binding domain selected from a cognate antigen molecule (e.g., a recombinant antigen) or an anti-idiotypic antibody molecule, under conditions such that immune cell expansion and / or activation occurs, thereby producing an expanded and / or activated immune cell population.SELECTED DRAWING: None

Description

[Technical Field] 【0001】 Related applications This application claims priority under U.S. Application No. 62 / 195,056 filed July 21, 2015, which is incorporated herein by reference in its entirety. 【0002】 Sequence List This application includes a sequence listing, which has been submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on 18 July 2016, is named N2067-7081WO_SL.txt and has a size of 2,053,055 bytes. [Background technology] 【0003】 Background of the Invention Until about 10 years ago, in vitro T cell activation was primarily achieved using mitotic lectins such as phytohemagglutinin (PHA) and concanavalin A (Con A). These mitotic molecules bind to glycoproteins on the cell surface. To achieve T cell receptor (TCR) complex-specific stimulation, surface molecule-specific antibodies, including CD2, CD3, CD28, and CD45, have been used. These antibodies provided the necessary co-stimulatory signals to induce full activation and proliferation of T cells in culture (Frauwirth and Thompson J Clin Invest (2002) Feb;109(3):295-9). This field has progressed towards immobilizing these antibodies on accessory cells, beads, or solid surfaces for strong T lymphocyte expansion (Trickett and Kwan J Immunol Methods (2003) Apr 1;275(1-2):251-5). 【0004】 However, existing protocols for T cell activation and proliferation still have limitations. These limitations include, for example, existing protocols rely on the presence of a functional TCR on the T cell surface. This limits T cell activation to such cells that possess a functional TCR. Primary T lymphocytes are a heterogeneous pool of cells that may include T cells without a functional TCR, thus limiting the T cell population that can be activated. The production, acquisition, and use of antibodies against cell surface molecules such as CD2, CD3, CD28, and CD45 are expensive and depend on the availability of such antibodies. Furthermore, the cost is even higher because complete T cell activation may require two different antibodies (a primary stimulant such as anti-CD3 and a secondary stimulant such as anti-CD28). In addition, since CD3 / CD28 stimulation generally persists in the culture medium for a long period, the TCR is exposed to repeated stimulation over a long period. Prolonged, high-level TCR stimulation leads to strong activation signals in naive T cells and simultaneous activation-induced cell death (AICD) of memory T cells (Collette Y, et al. Blood (1998) Aug 15;92(4):1350-63; Kerstan A and Huenig TJ Immunol (2004) Feb 1;172(3):1341-5; Noel, PJ et al. J Immunol. 1996 Jul 15;157(2):636-42). [Overview of the project] [Problems that the invention aims to solve] 【0005】 Therefore, there is a demand for improved in vitro enlargement and activation of immune cells, such as immune effector cells. [Means for solving the problem] 【0006】 Summary of the Invention The present invention relates, at least in part, to a method for improving the growth and / or activation (e.g., in vitro growth and / or activation) of immune cells (e.g., immune effector cells). One embodiment described herein provides the growth and / or activation of immune cells by transiently expressing a chimeric antigen receptor (CAR) molecule. The CAR-expressing immune cells can be activated via a ligand of the CAR molecule, for example, a ligand of the CAR antigen-binding domain (e.g., a homogeneous antigen molecule or an anti-idiotype antibody molecule). In one embodiment, the method disclosed herein enables the growth of immune cells without requiring the presence of a functional T cell receptor and / or without substantially altering the phenotype of the immune cells. For example, immune effector cells, including anergized T cells, hematopoietic stem cells, NK cells, and B cells, can be grown using the method described herein. Furthermore, immune cells can be grown without substantially altering their undifferentiated phenotype and / or without prolonged, repeated stimulation of the T cell receptor. In one embodiment, the method described herein enables superior proliferation and cell number yield compared to conventional TCR-stimulated growth. Therefore, the improved methods and compositions disclosed herein (e.g., modified immune cell populations, reaction mixtures) can offer significant benefits to cell therapies, such as immunotherapy. 【0007】 Accordingly, in one embodiment, the present invention relates to a method for increasing and / or activating a population of immune cells, for example, immune effector cells. The method comprises introducing a CAR molecule (e.g., a nucleic acid encoding the CAR molecule) into a population of immune cells under conditions suitable for the expression of the CAR molecule (e.g., transient expression) (e.g., thereby producing a “primary CAR-expressing cell population” or a “transient CAR-expressing cell population” as defined herein). In one embodiment, the CAR molecule includes an antigen-binding domain (e.g., the antigen-binding domain of an antibody molecule). The method comprises contacting the primary or transient CAR-expressing cell population with a ligand of the CAR molecule, for example, a ligand of the CAR antigen-binding domain (e.g., a homologous antigen molecule (e.g., a recombinant antigen) or an anti-idiotype antibody molecule) under conditions that result in immune cell increase and / or activation, thereby producing an “increased and / or activated immune cell population”. In one embodiment, the ligand of the CAR molecule is present in / on a substrate, for example, a substrate that does not exist naturally (e.g., immobilized or bound). The method may further comprise culturing a population of immune cells in the presence of the ligand of the CAR molecule. 【0008】 In a related embodiment, the present invention relates to a method for increasing and / or activating a population of immune cells, such as immune effector cells. The method includes providing a primary CAR-expressing cell population or a transient CAR-expressing cell population as described herein, and bringing the CAR-expressing cell population into contact with a ligand of a CAR molecule, such as a ligand of the CAR antigen-binding domain (e.g., a homologous antigen molecule (e.g., a recombinant antigen) or an anti-idiotype antibody molecule) under conditions that result in immune cell increase and / or activation, thereby producing an “increased and / or activated immune cell population”. In one embodiment, the ligand of the CAR molecule is present in / on a substrate, such as a substrate that does not exist in nature (e.g., immobilized or bound). The method may further include culturing a population of immune cells in the presence of the ligand of the CAR molecule. 【0009】 In one embodiment, transiently expressed CARs are produced by transiently introducing nucleic acids (e.g., RNA or DNA) encoding CARs into cells under conditions that enable CAR production. 【0010】 In one embodiment, the transiently expressed CAR is produced by the use of saltase. For example, saltase can be used to couple the extracellular domain (e.g., including an antigen-binding domain and a saltase-recognition motif) with a saltase-acceptor member (e.g., including a saltase-acceptor motif, a transmembrane domain, and optionally an intracellular signaling domain or switch domain). In one embodiment, the transiently expressed CAR includes, for example, a saltase-transduction signature resulting from the coupling of the saltase-recognition motif to the saltase-acceptor motif. In one embodiment, the saltase, CAR, or saltase-acceptor member is as described in PCT / CN2014 / 090503 filed November 6, 2014, or PCT / CN2014 / 082600 filed July 21, 2014, each of which is incorporated herein by reference in whole. 【0011】 The above method can be performed in vitro, ex vivo, or in vivo. 【0012】 In one embodiment, the population of immune cells used in the method described herein is obtained, for example, from a blood sample from a subject (e.g., a cancer patient). In another embodiment, the population of immune cells is obtained by apheresis. 【0013】 In one embodiment, the immune cell population includes, for example, immune effector cells as described herein. Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, bone marrow-derived phagocytic cells, or combinations thereof. 【0014】 In one embodiment, the immune cell population includes a subset of primary T cells or lymphocytes, such as anergized T cells, naive T cells, T regulatory cells, Th-17 cells, stem T cells, or a combination thereof. 【0015】 In one embodiment, the immune cell population includes peripheral blood mononuclear cells (PBMCs), umbilical cord blood cells, or a combination thereof. 【0016】 In one embodiment, the immune cell population includes cells that express or lack T cell receptors (e.g., functional T cell receptors) at low levels. In another embodiment, the immune cell population includes cells that have non-functional or substantially impaired T cell receptors. 【0017】 In one embodiment, the nucleic acid encoding the CAR molecule (e.g., the first CAR molecule) is an RNA molecule, such as in vitro transcription (IVT) RNA. In one embodiment, the CAR-coding RNA construct described herein is introduced into a population of immune cells by transfection or electroporation. In one embodiment, the CAR molecule is transiently expressed (e.g., the CAR molecule is not integrated into the cellular genome or is not substantially integrated). In one embodiment, the CAR molecule is expressed in a limited number of immune cells for a limited period of time for cell replication, for example, less than 50 days (e.g., 40, 30, 25, 20, 15, 10, 5 days or less). 【0018】 In one embodiment, the CAR molecule is transiently expressed on the surface of immune cells and internalized after stimulation with a single ligand (e.g., an antigen). In another embodiment, the immune cells are not subjected to repeated ligand (e.g., antigen) stimulation. 【0019】 In other embodiments, the intensity of immune cell stimulation can be customized to a desired level by, for example, adjusting the CAR surface density or the ligand of the CAR antigen-binding domain, e.g., affinity for the antigen, or both. For example, increasing the CAR surface density of immune cells or increasing the affinity of the CAR-binding domain to the ligand (e.g., antigen) can increase the intensity of immune cell stimulation. 【0020】 In other embodiments, the nucleic acid encoding the CAR molecule (e.g., the first CAR molecule) is a DNA vector or an RNA vector. In one embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenovirus vector, or retroviral vector. In one embodiment, the vector is a lentivirus. In one embodiment, the nucleic acid is stably integrated into the cell genome. 【0021】 In one embodiment, the encoded CAR molecule is, for example, a tumor antigen-binding CAR (e.g., CD19 CAR) as described herein. 【0022】 In other embodiments, the ligand for the CAR molecule is a cancer-associated antigen, for example, a cancer-associated antigen recognized by the CAR molecule described herein, e.g., CD19 CAR. 【0023】 In one embodiment, the substrate is a noncellular substrate. The noncellular substrate may be a solid support selected from, for example, plates (e.g., microtiter plates), membranes (e.g., nitrocellulose membranes), matrices, chips, or beads. In one embodiment, the ligand of the CAR molecule is present on the substrate (e.g., on the substrate surface). The ligand may be immobilized, bound, or associated with the substrate covalently or noncovalently (e.g., by crosslinking). In one embodiment, the ligand is bound to beads (e.g., covalently). In the above embodiment, the immune cell population may be augmented in vitro or ex vivo. 【0024】 In other embodiments, the substrate is a cell, for example, a cell expressing a ligand, for example, a cell expressing a congenital antigen on its surface. In one embodiment, the congenital antigen is heterologous to the cell, for example, a recombinant antigen expressed on the cell surface. In another embodiment, the congenital antigen is endogenously expressed in a cell, for example, a tumor cell. In the above embodiments, the immune effector cell population can be increased in vitro, ex vivo, or in vivo. In one embodiment, T cells are increased in vivo, for example, by lymph node injection or injection of tumor-infiltrating lymphocytes (TILs) into a tumor. 【0025】 In one embodiment, CAR-expressing immune cells are cultured in the presence of a ligand for the CAR molecule for a predetermined time (e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 18 hours, 21 hours, 22 hours, 23 hours, or 24 hours) or (e.g., 1 day, 5 days, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, or 50 days). In one embodiment, CAR-expressing cells are cultured for a period of 4 to 9 days. In one embodiment, CAR-expressing cells are cultured for 8 days or less, for example, 7 days, 6 days, or 5 days. 【0026】 In one embodiment, the CAR-expressing immune cell population exhibits population doubling of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more populations. In another embodiment, the CAR-expressing immune cell population exhibits a total of 8 to 10 or approximately 9 population doublings. 【0027】 In one embodiment, the CAR-expressing immune cell population increases by a total of 200-, 300-, 400-, 450-, 500-, 550-, 600-, or 650-fold or more per cell. In one embodiment, the CAR-expressing immune cell population increases by approximately 500-fold. In one embodiment, on average, one cell proliferates to 400-600 cells or more than approximately 500 cells. In one embodiment, cell growth is measured by a method described herein, such as flow cytometry, after approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 days. In one embodiment, cell growth is measured 10-25 days after stimulation with a ligand. In one instance, the increase is measured 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days after stimulation with the ligand. 【0028】 In one embodiment, the increase and / or activation of an immune cell population using the method described herein does not substantially stimulate the TCR of the immune cells. In one embodiment, the method described herein results in less rapid differentiation of immune cells in culture and / or promotes a “younger” T cell phenotype. In one embodiment, the increased and / or activated immune cell population includes immune effector cells having a less differentiated phenotype, e.g., younger cells, e.g., immature T cells. In one embodiment, younger T cells are naive T cells (T N ), memory stem cells (T SCM ), central memory T cells (T CM ) or a combination of these. 【0029】 In one embodiment, the method disclosed herein further comprises contacting an augmented and / or activated immune cell population with a nucleic acid encoding a second CAR molecule, for example, a vector containing a nucleic acid encoding a second CAR, thereby producing a second CAR-expressing cell population. 【0030】 In one embodiment, the nucleic acid encoding the second CAR molecule is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenovirus vector, or retroviral vector. In one embodiment, the nucleic acid vector encoding the second CAR molecule is a lentivirus. 【0031】 In another embodiment, the nucleic acid encoding the second CAR molecule is IVT RNA. 【0032】 In one embodiment, the first CAR and the second CAR molecule target the same antigen, e.g., the same tumor cell antigen. In another embodiment, the first CAR and the second CAR molecule are the same CAR molecule. In such embodiments, a population of immune cells expressing the first CAR (e.g., transiently) is increased and / or activated in vitro or ex vivo by contacting the immune cell population with, for example, a tumor cell antigen or anti-idiotype antibody against the CAR-binding antibody molecule (e.g., CD19 antigen or anti-CD19 idiotype antibody immobilized on a non-cellular or cellular substrate as described herein). Separately or in combination therewith, a population of immune cells expressing the second CAR (e.g., stably) is increased and / or activated in vivo by contacting, for example, an endogenous tumor cell antigen (e.g., CD19). In another embodiment, the second CAR-expressing immune cells are administered to a subject, for example, as part of a therapeutic protocol. 【0033】 In other embodiments, the first CAR and second CAR molecules target different antigens, e.g., different tumor cell antigens. In some embodiments, the first CAR and second CAR molecules are different CAR molecules (e.g., the first CAR and the second CAR molecule). In such embodiments, a population of immune cells expressing the first CAR (e.g., transiently) is increased and / or activated in vitro or ex vivo by contacting the immune cell population with the first tumor cell antigen or a first anti-idiotype antibody against the antigen-binding domain of the CAR (e.g., an anti-idiotype antibody against the mesoserine-binding domain of the mesoserine antigen or CAR molecule immobilized on a non-cellular or cellular substrate as described herein). Separately or in combination therewith, a population of immune cells expressing the second CAR (e.g., stably) is increased and / or activated in vivo by contacting, for example, an endogenous second tumor cell antigen (e.g., CD19). In some embodiments, the second CAR-expressing immune cells are administered to a subject, for example, as part of a therapeutic protocol. 【0034】 In one embodiment, the first CAR and the second CAR are selected from ROR1 CAR, CD19 CAR, CD20 CAR, CD22 CAR, CD123 CAR, CD10 CAR, CD34 CAR, FLT-3 CAR, CD79b CAR, CD179b CAR, Mesoserine CAR, or CD79a CAR, for example, the CARs described herein. In one embodiment, the first CAR and the second CAR are the same. In another embodiment, the first CAR and the second CAR are different. Any combination of the first CAR and the second CAR can be used in the method disclosed herein. 【0035】 In one embodiment, the method further includes storing the augmented and / or activated immune cell population after a suitable growth period. In one embodiment, the augmented and / or activated immune cell population is cryopreserved according to the method described herein. In one embodiment, the augmented and / or activated immune cell population is cryopreserved in a suitable medium, for example, an injectable medium. 【0036】 In other embodiments, the present invention relates to a method for treating a disorder or condition (e.g., the disorders or conditions described herein) in a subject. The method comprises administering to the subject an enlarged and / or activated immune cell population prepared by one or more of the methods described herein. In some embodiments, the method comprises obtaining (e.g., acquiring) the enlarged and / or activated immune cell population. The enlarged and / or activated immune cell population can be obtained from suitable storage conditions, for example, cryopreservation. 【0037】 In one embodiment, the immune cell population includes, for example, immune effector cells as described herein. Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, hematopoietic stem cells (HSCs), bone marrow-derived phagocytic cells, or combinations thereof. 【0038】 In one embodiment, the immune cell population includes, for example, a subset of primary T cells or lymphocytes, including anergic T cells, naive T cells, T regulatory cells, Th-17 cells, stem T cells, or combinations thereof. 【0039】 In one embodiment, the immune cell population includes peripheral blood mononuclear cells (PBMCs), umbilical cord blood cells, or a combination thereof. 【0040】 In yet another embodiment, the present invention relates to a method for treating a subject having cancer or providing antitumor immunity. The method comprises administering to the subject an effective amount of an immune effector cell population expressing a CAR molecule (e.g., the first and / or second CAR molecule described herein) (e.g., an enlarged and / or activated immune cell population described herein), either alone or in combination with further treatment, e.g., the second treatment described herein. 【0041】 In one embodiment, the treatment method includes obtaining (e.g., acquiring) an enlarged and / or activated immune cell population using one or more of the methods described herein. For example, the enlarged and / or activated immune cell population may have been obtained beforehand by introducing a first CAR molecule (e.g., a nucleic acid molecule encoding the first CAR molecule as described herein, e.g., IVT RNA encoding the first CAR) under conditions suitable for the expression (e.g., transient expression) of the CAR molecule, and then contacting the CAR-expressing cell population with a ligand of the CAR molecule, e.g., a ligand for the CAR antigen-binding domain (e.g., a homologous antigen molecule (e.g., a recombinant antigen) or an anti-idiotype antibody molecule) under conditions that result in immune cell enlargement and / or activation. In one embodiment, the ligand of the CAR molecule is present in / on a substrate, e.g., a non-naturally occurring substrate as described herein (e.g., immobilized or bound). The enlarged and / or activated immune cell population can be stored under suitable conditions, e.g., by cryopreservation as described herein. 【0042】 In one embodiment, the treatment method disclosed herein further includes acquiring (e.g., obtaining) a secondary CAR-expressing cell population, for example, a secondary CAR-expressing cell population. For example, the augmented and / or activated immune cell population may have been previously contacted with a nucleic acid encoding the secondary CAR molecule, for example, a vector containing the nucleic acid encoding the secondary CAR. In one embodiment, the nucleic acid encoding the secondary CAR molecule is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenovirus vector, or retroviral vector. In one embodiment, the nucleic acid vector encoding the secondary CAR molecule is a lentivirus. 【0043】 In one embodiment, the first CAR and the second CAR molecule target the same antigen molecule, e.g., the same cancer-associated antigen. In another embodiment, the first CAR and the second CAR molecule are the same CAR molecule. In such embodiments, a population of immune cells expressing the first CAR (e.g., transiently) is previously enlarged and / or activated in vitro or ex vivo by, for example, contact between the immune cell population and an anti-idiotype antibody against the cancer-associated antigen or CAR-binding antibody molecule (e.g., CD19 antigen or anti-CD19 idiotype antibody immobilized on a non-cellular or cellular substrate as described herein). In another embodiment, the second CAR-expressing immune cells are administered to a subject, for example, as part of a therapeutic protocol. 【0044】 In other embodiments, the first CAR and second CAR molecules are directed toward different antigens, e.g., different cancer-associated antigens. In some embodiments, the first CAR and second CAR molecules are different CAR molecules (e.g., the first CAR and the second CAR molecule). In such embodiments, the immune cell population expressing the first CAR (e.g., transiently expressing it) has been previously enlarged and / or activated in vitro or ex vivo by, for example, contact between the immune cell population and the first cancer-associated antigen or a first anti-idiotype antibody molecule against the antigen-binding domain of the CAR (e.g., an antigen or anti-idiotype antibody against the binding domain of a CAR molecule immobilized on a non-cellular or cellular substrate as described herein). In some embodiments, the second CAR-expressing immune cells are administered to a subject, for example, as part of a therapeutic protocol. 【0045】 In one embodiment, the first CAR and the second CAR molecules are each independently selected from ROR1 CAR, CD19 CAR, CD20 CAR, CD22 CAR, CD123 CAR, CD10 CAR, CD34 CAR, FLT-3 CAR, CD79b CAR, CD179b CAR, mesoserine CAR, or CD79a CAR, for example, the CARs described herein. In one embodiment, the first CAR and the second CAR are the same. In another embodiment, the first CAR and the second CAR are different. Any combination of the first CAR and the second CAR can be used in the method disclosed herein. 【0046】 In one exemplary embodiment, the first CAR is directed towards mesoserine, and mesoserine CAR-expressing cells are contacted with a mesoserine antigen or an anti-idiotype antibody against the mesoserine antigen-binding domain of the CAR, while the second CAR is directed towards CD19 (e.g., the CD19 CAR disclosed herein). In another exemplary embodiment, the first CAR is directed towards CD19, and CD19 CAR-expressing cells are contacted with a CD19 antigen or an anti-idiotype antibody against the CD19 antigen-binding domain of the CAR, while the second CAR is directed towards mesoserine (e.g., the mesoserine CAR disclosed herein). 【0047】 In one embodiment, the immune cell population used in the therapeutic method includes, for example, immune effector cells as described herein. Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, hematopoietic stem cells (HSCs), bone marrow-derived phagocytic cells, or combinations thereof. 【0048】 In yet another embodiment, the present invention relates, for example, to a population of immune effector cells (e.g., comprising a first and / or second CAR molecule or nucleic acids encoding a first and / or second CAR molecule), an immunocell preparation, or a reaction mixture prepared according to the method described herein. In one embodiment, the first and second CAR molecules are expressed simultaneously (e.g., fully or partially overlapping) or sequentially. 【0049】 Further characteristics or embodiments of any of the above methods, preparations, and reaction mixtures include one or more of the following: 【0050】 Immune cell enlargement and / or activation In one embodiment, the method disclosed herein involves increasing and / or activating a population of immune cells, e.g., immune effector cells. The method includes acquiring a population of immune cells, exposing the cells and nucleic acids encoding CAR molecules to CAR molecule expression (e.g., transient expression) under appropriate conditions (where the CAR molecule binds to a ligand, e.g., a congener antigen molecule (e.g., recombinant antigen) or an anti-idiotype antibody to the antigen-binding domain of the CAR molecule), and culturing the population of immune cells in the presence of the congener antigen molecule or the anti-idiotype antibody. 【0051】 In one embodiment, the population of immune effector cells is self-referential to the subject to which the cells are administered for treatment. In another embodiment, the population of immune effector cells is homogeneous to the subject to which the cells are administered for treatment. 【0052】 In one embodiment, the population of immune effector cells is T cells isolated from peripheral blood lymphocytes. In one embodiment, the population of T cells is obtained by erythrolysis and / or monocyte depletion. In one embodiment, the population of T cells is isolated from peripheral lymphocytes, for example, using the method described herein. In one embodiment, the T cells are CD4 + Includes T cells. In another embodiment, T cells are CD8 +In one embodiment, the immune effector cells include T cells. In another embodiment, the T cells include regulatory T cells. In yet another embodiment, the T cells include naive T cells. In one embodiment, the immune effector cells include hematopoietic stem cells (e.g., umbilical cord blood cells). In another embodiment, the immune effector cells include B cells. In yet another embodiment, the immune effector cells include NK cells. In another embodiment, the immune effector cells include NKT cells. In yet another embodiment, the immune effector cells include Th-17 cells. 【0053】 In one embodiment, immune effector cells have reduced T cell receptor levels or lack T cell receptors. In another embodiment, immune effector cells have non-functional or substantially impaired T cell receptors. 【0054】 In one embodiment, a population of immunoeffector cells can be obtained, for example, from a blood sample from a subject obtained by apheresis. In one embodiment, the immunoeffector cells obtained by apheresis are washed to remove the plasma fraction, and optionally, the cells are provided in a suitable buffer or culture medium for processing. In one embodiment, the cells are washed with a buffer such as phosphate-buffered saline (PBS). In one embodiment, the cells are washed with a washing solution lacking one or more divalent cations, such as calcium and magnesium. In one embodiment, the immunoeffector cells are washed with a buffer that is substantially free of divalent cations. 【0055】 In one embodiment, the method comprises producing a population of RNA-modified cells that transiently express exogenous RNA from a population of immune effector cells. The method comprises introducing into cells from a population of in vitro transcription RNA or synthetic RNA, wherein the RNA comprises nucleic acids encoding CARs, for example, the CAR described herein, for example, the CD19 CAR described herein. 【0056】 In one embodiment, RNA is introduced into immunoeffector cells by the method described herein (e.g., electroporation). In one embodiment, at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the immunoeffector cells express CAR mRNA. 【0057】 In other embodiments, at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the immune effector cells express CAR on their cell surface. 【0058】 In one embodiment, immune effector cells are enlarged and / or activated by culturing them in the presence of a ligand, such as a congenital antigen molecule or an anti-idiotype antibody. In one embodiment, immune effector cells are contacted with the congenital antigen molecule or anti-idiotype antibody for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 28, 32, 36, or 48 hours after RNA has been introduced into the immune effector cells. In one embodiment, immune effector cells are contacted with the congenital antigen molecule or anti-idiotype antibody for a shorter period than 24, 15, 12, 10, or 8 hours after RNA has been introduced into the immune effector cells. 【0059】 In one embodiment, the ligand is a molecule that binds to and / or activates a CAR (e.g., the CAR described herein, e.g., the CD19 CAR described herein) on the cell surface of a population of immunoeffector cells expressing the CAR (e.g., transiently). In one embodiment, the congenital antigen molecule is a congenital antigen of the CAR. In one embodiment, the congenital antigen molecule is a recombinant antigen recognized by the antigen-binding moiety of the CAR. In one embodiment, the congenital antigen molecule is a cancer-associated antigen, e.g., the cancer-associated antigen described herein, e.g., CD19. In one embodiment, the ligand is an anti-idiotype antibody (e.g., an antibody molecule that binds to the antigen-binding domain of the CAR), e.g., an anti-CD19 idiotype antibody. 【0060】 In one embodiment, the ligand is bound to the substrate. In one embodiment, the substrate is a solid support. In one embodiment, the substrate is selected from a microtiter plate (e.g., an ELISA plate), a membrane (e.g., a nitrocellulose membrane, a PVDF membrane, a nylon membrane, an acetic acid derivative, and combinations thereof), a fiber matrix, a Sepharose matrix, a sugar matrix, a plastic chip, a glass chip, or any type of bead (e.g., Luminex beads, DynaBeads, magnetic beads, flow cytometry beads, and combinations thereof). In one embodiment, the substrate is an ELISA plate. In another embodiment, the substrate is a bead, for example, DynaBeads. 【0061】 In one embodiment, CAR-expressing immunoeffector cells are brought into contact with ligand-coated beads, such as antigen-coated beads, in a ratio of 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or 15:1 beads per immunoeffector cell. In another embodiment, CAR-expressing immunoeffector cells are brought into contact with antigen-coated beads in a ratio of 3:1 beads per immunoeffector cell. 【0062】 In one embodiment, immune effector cells are grown in a suitable medium (e.g., the medium described herein) which may optionally contain one or more factors for proliferation and / or viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-21, TGFβ and TNF-α or any other additives for cell growth. In one embodiment, cells are grown in the presence of IL-15 and / or IL-7 (e.g., IL-15 and IL-7). In one embodiment, immune effector cells are grown in the presence of IL-2. 【0063】 In one embodiment, immunoeffector cells transduced with nucleic acids encoding a CAR, for example, the CAR described herein, for example, the CD19 CAR described herein, are cultured for several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 40 days (e.g., day 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, or 40) to increase in size. In one embodiment, cells are grown for a period of 4 to 9 days. In another embodiment, cells are grown for a period of 8 days or less, for example, 7, 6, 5, 4, or 3 days. 【0064】 The efficacy of immune effector cells can be defined, for example, by various T cell functions, such as proliferation, target cell death, cytokine production, activation, migration, or a combination thereof. In one embodiment, immune effector cells grown for 5 days, such as the CD19 CAR cells described herein, show at least a 1-fold, 2-fold, 3-fold, or 4-fold increase in cell doubling upon antigen stimulation compared to the same cells grown for 9 days under the same culture conditions. In one embodiment, immune effector cells, such as cells expressing the CD19 CAR described herein, are grown for 5 days, and the resulting cells show higher pro-inflammatory cytokine production, such as IFN-γ and / or GM-CSF levels, compared to the same cells grown for 9 days under the same culture conditions. In one embodiment, immune effector cells enlarged for 5 days, such as the CD19 CAR cells described herein, show at least a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or greater increase in pro-inflammatory cytokine production at pg / ml levels, such as IFN-γ and / or GM-CSF levels, compared to the same cells enlarged by 9 days of culture under the same culture conditions. 【0065】 In one embodiment, immune effector cells are enlarged by at least 200 times (e.g., 200, 250, 300, 350, 400, 450, 500, 550, or 650 times) by the methods described herein, such as flow cytometry. In one embodiment, the cells are enlarged by approximately 500 times. 【0066】 In one embodiment, cell growth is measured approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 days after stimulation with a ligand, e.g., a congenital antigen molecule. In one embodiment, cell growth is measured 10 to 25 days after stimulation with a ligand, e.g., a congenital antigen molecule. In one embodiment, growth is measured 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days after stimulation with a ligand, e.g., a congenital antigen molecule. 【0067】 In one embodiment, immunoeffector cells are cryopreserved after an appropriate growth period. In another embodiment, cells are cryopreserved according to the method described herein. In another embodiment, the grown cells are cryopreserved in an appropriate culture medium, for example, an insoluble medium as described herein. 【0068】 In one embodiment, the method involves contacting immune effector cells with nucleic acid encoding a first CAR (e.g., in vitro transcription RNA) under conditions suitable for transient expression of the first CAR (where the first CAR targets a congeneral antigen molecule), increasing the population of immune effector cells by culturing the first CAR-expressing immune effector cells in the presence of the congeneral antigen molecule, and further contacting the cells with a vector containing nucleic acid encoding a second CAR. In one embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, or retroviral vectors. In one embodiment, cells from the population of immune effector cells are transduced with the vector within, for example, one day after the population of immune effector cells was obtained, for example, from a blood sample from a subject obtained by apheresis. 【0069】 In one embodiment, the first CAR targets a congeneral antigen molecule, and the second CAR targets the same congeneral antigen molecule. In another embodiment, the first CAR targets a congeneral antigen molecule, and the second CAR targets a different congeneral antigen molecule. In yet another embodiment, the first CAR targets the cancer-associated antigen described herein, and the second CAR targets the same cancer-associated antigen described herein. In yet another embodiment, the first CAR targets the cancer-associated antigen described herein, and the second CAR targets a different cancer-associated antigen described herein. In one embodiment, the first CAR is one of the ROR1 CAR, CD19 CAR, CD20 CAR, CD22 CAR, CD123 CAR, CD10 CAR, CD34 CAR, FLT-3 CAR, CD79b CAR, CD179b CAR, mesoserine CAR, or CD79a CAR listed herein, and the second nucleic acid codes one of the ROR1 CAR, CD19 CAR, CD20 CAR, CD22 CAR, CD123 CAR, CD10 CAR, CD34 CAR, FLT-3 CAR, CD79b CAR, CD179b CAR, mesoserine CAR, or CD79a CAR listed herein. 【0070】 In another embodiment, the present invention relates to a reaction mixture comprising a population of immunoeffector cells, wherein a plurality of cells in the population in the reaction mixture comprises nucleic acid molecules comprising a CAR-coding sequence, for example, the CD19 CAR-coding sequence described herein, for example, in vitro transcription RNA or synthetic RNA. 【0071】 In one embodiment, at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the immune effector cells express CAR mRNA. 【0072】 In other embodiments, at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the immune effector cells express CAR on their cell surface. 【0073】 In one embodiment, the reaction mixture may further include a ligand as described herein (e.g., a congener antigen molecule or an anti-idiotype antibody). In one embodiment, the ligand is a molecule that expresses a CAR, e.g., the CAR described herein, e.g., the CD19 CAR described herein, e.g., transiently, and binds to and / or activates a CAR on the cell surface of a population of immune effector cells. In one embodiment, the ligand is a congener antigen of the CAR. In one embodiment, the congener antigen is a cancer-associated antigen, e.g., the cancer-associated antigen described herein, e.g., CD19. In another embodiment, the ligand is an anti-idiotype antibody, e.g., an anti-CD19 idiotype antibody. 【0074】 In one embodiment, a ligand, such as a congener antigen molecule or an anti-idiotype antibody, is bound to the substrate. In one embodiment, the substrate is a solid support. In one embodiment, the substrate is selected from microtiter plates (e.g., ELISA plates), membranes (e.g., nitrocellulose membranes, PVDF membranes, nylon membranes, acetic acid derivatives, and combinations thereof), fiber matrices, Sepharose matrices, sugar matrices, plastic chips, glass chips, or any type of beads (e.g., Luminex beads, magnetic beads (e.g., DynaBeads), flow cytometry beads, and combinations thereof). In one embodiment, the substrate is an ELISA plate. In another embodiment, the substrate is magnetic beads, such as DynaBeads. 【0075】 In one embodiment, CAR-expressing immunoeffector cells and ligand (e.g., antigen) coated beads are present in a ratio of 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or 15:1 beads per immunoeffector cell. In another embodiment, CAR-expressing immunoeffector cells and ligand (e.g., antigen) coated beads are present in a ratio of 3:1 beads per immunoeffector cell. 【0076】 In one embodiment, the reaction mixture further comprises one or more factors for growth and / or viability enhancement, including serum (e.g., fetal bovine or human serum), e.g., interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-21, TGFβ and TNF-α, or any other additives for cell growth. In one embodiment, the reaction mixture further comprises IL-15 and / or IL-7. In one embodiment, cells are enlarged in the presence of IL-2. 【0077】 In one embodiment, a population of cells in the reaction mixture contains one or both of a nucleic acid molecule encoding a first CAR and a nucleic acid molecule encoding a second CAR molecule, for example, the CARs described herein. 【0078】 In one embodiment, the nucleic acid encoding the first CAR is the in vitro transcription RNA described herein. 【0079】 In one embodiment, the nucleic acid encoding the second CAR is a vector selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenovirus vector, or retroviral vector. 【0080】 In one embodiment, the first CAR targets a congener antigen molecule, and the second CAR targets the same congener antigen molecule. 【0081】 In one embodiment, the first CAR targets a congeneral antigen molecule, and the second CAR targets a different congeneral antigen molecule. 【0082】 In one embodiment, the first CAR targets the cancer-associated antigen described herein, and the second CAR targets the same cancer-associated antigen described herein. 【0083】 In one embodiment, the first CAR targets a cancer-associated antigen described herein, and the second CAR targets a different cancer-associated antigen described herein. 【0084】 In one embodiment, the first CAR is selected from the ROR1 CAR, CD19 CAR, CD20 CAR, CD22 CAR, CD123 CAR, CD10 CAR, CD34 CAR, FLT-3 CAR, CD79b CAR, CD179b CAR, mesoserine CAR, or CD79a CAR listed herein, and the second nucleic acid codes for the ROR1 CAR, CD19 CAR, CD20 CAR, CD22 CAR, CD123 CAR, CD10 CAR, CD34 CAR, FLT-3 CAR, CD79b CAR, CD179b CAR, mesoserine CAR, or CD79a CAR listed herein. 【0085】 In one embodiment, the reaction mixture further comprises, for example, saccharides, oligosaccharides, polysaccharides and polyols (e.g., trehalose, mannitol, sorbitol, lactose, sucrose, glucose and dextran), salts and cryoprotectants or stabilizers such as crown ethers. In one embodiment, the cryoprotectant is dextran. 【0086】 Further characteristics and embodiments of the method are described in a chapter titled “Further Embodiments of the Method, Preparations, and Reaction Mixtures.” 【0087】 CAR molecule The methods, preparations, and reaction mixtures described herein can be used to modify, for example, immune effector cells obtained by the methods described herein to include CAR molecules (hereinafter also referred to as "CARs") that target one or more cancer-associated antigens. In one embodiment, the tumor antigen is the tumor antigen described in International Application WO2015 / 142675, filed March 13, 2015, which is incorporated herein by reference in its entirety. 【0088】 In one embodiment, cancer-associated antigens (tumor antigens) include CD19, CD123, CD22, CD30, CD171, CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), type C lectin-like molecule-1 (CLL-1 or CLECL1), CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), TNF receptor family member B cell maturation (BCMA), and Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), mesoserine, interleukin-11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), proteaseserine 21 (testicin or PRSS21), vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-beta), Oncogene fusion protein (bcr-abl) consisting of stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor alpha, receptor tyrosine-protein kinase ERBB2 (Her2 / neu), mucin 1, cell surface-related (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutation (ELF2M), ephrin B2, fibroblast-activating protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (Macropain) subunit, beta type, 9 (LMP2), glycoprotein 100 (gp100), cleavage region (BCR), and Abelson mouse leukemia virus oncogene homolog 1 (Abl),Tyrosinase, ephrin type A receptor 2 (EphA2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), protein G-coupled receptor Condition class C group 5, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), hexasaccharide portion of globoH glycoceramide (GloboH), mammary gland differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cytogenic receptor 1 (HAVCR1), adrenergic receptor beta 3 (ADRB3), panexin 3 (PANX3), protein G-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K 9 (LY6K), olfactory receptor 51E2 (OR51E2), TCR gamma alternating reading frame protein (TARP), Wilms tumor protein (WT1), cancer / testicular antigen 1 (NY-ESO-1), cancer / testicular antigen 2 (LAGE-1a), melanoma-associated antigen 1 (MAGE-A1), ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family, member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2) Melanoma carcinoma testicular antigen-1 (MAD-CT-1), Melanoma carcinoma testicular antigen-2 (MAD-CT-2), Fos-related antigen 1, tumor protein p53 (p53), p53 variants, prostain, survivor, telomerase, prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MART1), rat sarcoma (Ras) variant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane protease,Serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyl-transferase V (NA17), paired-box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytosis virus oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), CCCTC binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen 3 (SART3) recognized by T cells, paired box protein Pax-5 (PAX5), proacrosin-binding protein p32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchor protein 4 (AKAP-4), synovial sarcoma, X-cut point 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), regmine, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), enteric carboxylesterase, heat shock protein 70-2 mutation (mut It is one or more selected from hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), type C lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), and immunoglobulin lambda-like polypeptide 1 (IGLL1). 【0089】 In one embodiment, the cancer-associated antigen targeted by the CAR molecule is CD19, for example, the CD19 CAR described herein (e.g., CTL019). In one embodiment, the CD19 CAR contains the amino acids or has the nucleotide sequence shown in Table 4. 【0090】 In one embodiment, the antigen-binding domain molecule of the CAR includes an antibody, an antibody fragment, scFv, Fv, Fab, (Fab')2, a single-domain antibody (SDAB), a VH or VL domain, or a camel VHH domain. 【0091】 In one embodiment, the transmembrane domain of the CAR molecule is the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD1 8) ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R Beta, IL2R Gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, I TGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7 , TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55) comprises a transmembrane domain selected from the transmembrane domains of PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C. 【0092】 In one embodiment, the transmembrane domain of the CAR molecule contains an amino acid sequence of a CD8 transmembrane domain having at least 1, 2, or 3 modifications to SEQ ID NO: 6, but with no more than 20, 10, or 5 modifications, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 6. In one embodiment, the transmembrane domain contains the sequence of SEQ ID NO: 6. 【0093】 In another embodiment, the nucleic acid sequence encoding the CD8 transmembrane domain includes the sequence of SEQ ID NO: 17 or a sequence having 95-99% identity thereto. 【0094】 In one embodiment, the antigen-binding domain is connected to the transmembrane domain by a hinge region. In one embodiment, the hinge region includes a CD8 hinge, e.g., the amino acid sequence of SEQ ID NO: 2, or an IgG4 hinge, e.g., the amino acid sequence of SEQ ID NO: 36, or a sequence having 95-99% identity with SEQ ID NO: 2 or 36. In another embodiment, the nucleic acid sequence encoding the hinge region includes the sequence of SEQ ID NO: 13 or SEQ ID NO: 37, or a sequence having 95-99% identity with SEQ ID NO: 13 or 37, respectively, corresponding to the CD8 hinge or the IgG4 hinge. 【0095】 In other embodiments, the CAR includes an intracellular signaling domain, for example, a primary signaling domain and / or a co-stimulatory signaling domain. In one embodiment, the intracellular signaling domain includes a primary signaling domain. In one embodiment, the intracellular signaling domain includes a co-stimulatory signaling domain. In one embodiment, the intracellular signaling domain includes a primary signaling domain and a co-stimulatory signaling domain. 【0096】 In one embodiment, the primary signaling domain includes a functional signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc epsilon R1b), CD79a, CD79b, Fc gamma RIIa, DAP10, and DAP12. 【0097】 In one embodiment, the primary signaling domain of the CAR molecule includes a functional signaling domain of CD3 zeta. The CD3 zeta primary signaling domain may include an amino acid sequence having at least one, two, or three modifications to SEQ ID NO: 9 or SEQ ID NO: 10, but with no more than 20, 10, or 5 modifications, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment, the primary signaling domain includes the sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In another embodiment, the nucleic acid sequence encoding the primary signaling domain includes the sequence of SEQ ID NO: 20 or SEQ ID NO: 21 or a sequence having 95-99% identity with it. 【0098】 In one embodiment, the intracellular signaling domain of a CAR molecule includes a co-stimulatory signaling domain. For example, the intracellular signaling domain may include a primary signaling domain and a co-stimulatory signaling domain. In one embodiment, the co-stimulatory signaling domain is a ligand that specifically binds to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, CDDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGA It contains one or more functional signaling domains of proteins selected from M, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, or NKG2D. 【0099】 In one embodiment, a population of immune effector cells, such as T cells, comprises a mixture of cells containing CAR molecules having two or more intracellular signaling domains. In another embodiment, a population of immune effector cells contains one or more CARs containing a CD28 signaling domain and a 4-1BB signaling domain. For example, the first immune effector cell contains a CAR molecule containing a CD28 signaling domain, and the second immune effector cell contains a CAR molecule containing a 4-1BB signaling domain. The expression of the CAR molecules containing the CD28 signaling domain and / or the 4-1BB signaling domain may be transient or stable. 【0100】 In one embodiment, the co-stimulatory signaling domain of the CAR molecule includes an amino acid sequence having at least one, two, or three modifications to SEQ ID NO: 7 or SEQ ID NO: 16, but with no more than 20, 10, or 5 modifications, or a sequence that is 95-99% identical to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 16. In another embodiment, the co-stimulatory signaling domain includes the sequence of SEQ ID NO: 7 or SEQ ID NO: 16. In yet another embodiment, the nucleic acid sequence encoding the co-stimulatory signaling domain includes the sequence of SEQ ID NO: 18 or SEQ ID NO: 15, or a sequence that is 95-99% identical thereto. 【0101】 In another embodiment, the intracellular domain of the CAR molecule comprises the sequence of SEQ ID NO: 9 or SEQ ID NO: 10 and the sequence of SEQ ID NO: 7 or SEQ ID NO: 16, wherein the amino acid sequence containing the intracellular signaling domain is expressed in the same frame and as a single polypeptide chain. 【0102】 In one embodiment, the nucleic acid sequence encoding the intracellular signaling domain includes the sequence of SEQ ID NO: 18 or SEQ ID NO: 15 or a sequence that is 95-99% identical thereto, and the sequence of SEQ ID NO: 20 or SEQ ID NO: 21 or a sequence that is 95-99% identical thereto. 【0103】 In one embodiment, CAR further includes a leader array. In one embodiment, the leader array includes the array of sequence number 1. 【0104】 In one embodiment, the antigen-binding domain molecule of CAR is 10 -4 M~10 -8 It has binding affinity KD for M. 【0105】 In one embodiment, the antigen-binding domain molecule of the CAR is the antigen-binding domain described herein, for example, the antigen-binding domain described herein for the above target. 【0106】 In one embodiment, CAR includes ROR1 CAR, CD19 CAR, CD20 CAR, CD22 CAR, CD123 CAR, CD10 CAR, CD34 CAR, FLT-3 CAR, CD79b CAR, CD179b CAR, mesoserine CAR, or CD79a CAR as described herein. 【0107】 In one embodiment, the CAR includes a CD19 CAR, for example, the CD19 CAR described herein. In one embodiment, the CD19 CAR includes, for example, the antigen-binding domain described herein in Table 1 or 4. 【0108】 In other embodiments, the antigen-binding portion of the CAR recognizes and binds to the extracellular domain of a mesoserine protein. An example of a mesoserine CAR sequence can be found, for example, in International Publication WO2013 / 040557A2, which is incorporated herein by reference in its entirety. 【0109】 Treatment / combination therapy methods In other embodiments, the present invention relates to a method for treating a subject having cancer or providing antitumor immunity. The method comprises administering an effective amount of an immune effector cell population to a subject, wherein the immune effector cell population is brought into contact with a nucleic acid encoding a CAR (where the CAR targets a congener antigen molecule) under conditions suitable for transient expression of a CAR, and the immune effector cell population is augmented or pre-augmented by culturing it in the presence of a ligand, e.g., a congener antigen molecule or an anti-idiotype antibody molecule. In one embodiment, the nucleic acid is RNA, e.g., in vitro transcription RNA. In another embodiment, the congener antigen molecule is a cancer-associated antigen molecule. In one embodiment, the congener antigen molecule or anti-idiotype antibody molecule is conjugated to a substrate, e.g., beads. 【0110】 In one embodiment, the method further comprises administering a secondary CAR (e.g., a vector containing nucleic acid encoding the secondary CAR) to a population of immune effector cells, where the immune effector cell population is augmented or pre-augmented as described herein. In one embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, or retroviral vectors. 【0111】 In one embodiment, a population of immune effector cells is transduced with a vector within, for example, one day after the population of immune effector cells is obtained from a blood sample from a subject, for example, by apheresis. In one embodiment, the first CAR targets a congeneral antigen molecule, and the second CAR targets the same congeneral antigen molecule. In one embodiment, the first CAR targets a congeneral antigen molecule, and the second CAR targets a different congeneral antigen molecule. In one embodiment, the first CAR targets the cancer-associated antigen described herein, and the second CAR targets the same cancer-associated antigen described herein. In one embodiment, the first CAR targets the cancer-associated antigen described herein, and the second CAR targets a different cancer-associated antigen described herein. 【0112】 In one embodiment, the first CAR is one of the ROR1 CAR, CD19 CAR, CD20 CAR, CD22 CAR, CD123 CAR, CD10 CAR, CD34 CAR, FLT-3 CAR, CD79b CAR, CD179b CAR, mesoserine CAR, or CD79a CAR listed herein, and the second nucleic acid codes one of the ROR1 CAR, CD19 CAR, CD20 CAR, CD22 CAR, CD123 CAR, CD10 CAR, CD34 CAR, FLT-3 CAR, CD79b CAR, CD179b CAR, mesoserine CAR, or CD79a CAR listed herein. 【0113】 In one embodiment, a method for treating a disorder (e.g., cancer) and providing the antitumor immunity described herein includes administering to a subject a population of CAR molecules or immune effector cells produced by the method described herein. In one embodiment, a population of immune effector cells is modified to express a CAR molecule, for example, the CAR described herein, for example, the CD19 CAR described herein. 【0114】 Also provided herein are compositions comprising immune effector cells (e.g., a population of immune effector cells prepared as described herein) containing a CAR molecule (e.g., the CAR molecule described herein) for use in treating subjects having a disease associated with the expression of a tumor antigen, for example, the disorder described herein. 【0115】 In one embodiment, cancer is a blood cancer such as ALL or CLL. In another embodiment, cancer is a blood cancer or lymphoma such as those described herein, such as leukemia (e.g., ALL or CLL). 【0116】 In some embodiments, the disease associated with the tumor antigen, e.g., the tumor antigen described herein, e.g., CD19, is selected from proliferative disorders such as cancer or malignant tumors or precancerous conditions such as spinal dysplasia, myelodysplastic syndromes or preleukemic states, or is a non-cancer-related sign associated with the expression of the tumor antigen described herein. In some embodiments, the disease is the cancer described herein, e.g., the cancer described herein as being associated with the target described herein. In some embodiments, the hematological cancer is leukemia. In one embodiment, cancer includes, but is not limited to, one or more acute leukemias, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), one or more chronic leukemias, B-cell prelymphocytic leukemia, blastocyte plasmacytoid dendritic cell neoplasms, Burkitt lymphoma, generalized large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative state, MALT lymphocyte The group consists of further hematological malignancies or hematological conditions, including, but not limited to, parma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, spinal dysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, Waldenström hypergammaglobulinemia and / or “preleukemic conditions” (e.g., a diverse collection of hematological conditions grouped by inability (or malformation) of bone marrow blood cells). In some embodiments, the diseases associated with the expression of the tumor antigens described herein include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions or proliferative disorders expressing the tumor antigens described herein, and any combination thereof. 【0117】 In one embodiment, the disease associated with the expression of a tumor antigen is selected from the group consisting of proliferative disorders, precancerous conditions, cancers, and non-cancer-related signs associated with the expression of a tumor antigen. 【0118】 In other embodiments, the diseases associated with the tumor antigens described herein are solid tumors. In some embodiments, cancers include colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian duct cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma, endocrine cancer, thyroid cancer, Selection is made from parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of these cancers, and their metastatic lesions. 【0119】 In one embodiment of the method or use described above, the tumor antigens associated with the disease are CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1 (CLECL1), CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesoserine, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, FAP, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variant, prostain, survivor and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras variant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, regmine, HPV E6, E7, intestinal carboxylesterase, mutIt is one or more selected from hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5 and IGLL1. 【0120】 In certain embodiments, the population of cells is autologous to the subject to whom the population is administered. In certain embodiments, the population of cells is allogeneic to the subject to whom the population is administered. In certain embodiments, the subject is human. 【0121】 In certain embodiments, a population of immune effector cells transduced with a nucleic acid encoding a CAR, such as a CAR described herein, such as a CD19 CAR described herein, is expanded, for example, by the methods described herein. In certain embodiments, the cells are expanded for a period of 8 days or less, such as 7 days, 6 days, 5 days, 4 days or 3 days. In certain embodiments, the cells, such as the CD19 CAR cells described herein, are expanded in 5 days of culture and the resulting cells are more potent than the same cells expanded in 9 days of culture under the same culture conditions, as described herein. 【0122】 In certain embodiments, 10 4 ~10 6 immune effector cells are administered per kg of the subject's body weight. In certain embodiments, the subject is initially administered a population of immune effector cells (e.g., 10 4 ~10 6 immune effector cells per kg of the subject's body weight), such as a population of immune effector cells that includes a nucleic acid encoding a CAR, such as a CAR described herein, such as a CD19 CAR described herein, and one or more subsequent administrations of the population of immune effector cells (e.g., 10 4 ~10 5 immune effector cells per kg of the subject's body weight), such as a population of immune effector cells that includes a nucleic acid encoding a CAR, such as a CAR described herein, such as a CD19 CAR described herein. 4 ~10 6 immune effector cells per kg of the subject's body weight), such as a population of immune effector cells that includes a nucleic acid encoding a CAR, such as a CAR described herein, such as a CD19 CAR described herein. 4 ~10 5The subject receives one or more consecutive doses of immune effector cells (some of which contain nucleic acids encoding CARs, e.g., the CARs described herein, e.g., the CD19 CAR described herein). In one embodiment, the one or more consecutive doses are administered within 15 days after the previous dose, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days, e.g., within 4, 3, or 2 days after the previous dose. In one embodiment, the subject receives a total of approximately 10 doses per kg of body weight over at least three doses of the immune effector cell population. 6 After receiving administration of immune effector cells, for example, the subject is 1 × 10 5 Initial administration of immune effector cells, 3 x 10 5 Second dose of immune effector cells and 6 × 10 5 After receiving the third dose of immune effector cells, each dose is administered, for example, within 4, 3, and 2 days following the previous dose. 【0123】 In one embodiment, the method or use is performed in combination with a drug that enhances the effectiveness of immune effector cells, such as the drugs described herein. 【0124】 For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGF beta. In one embodiment, the agent that inhibits an inhibitory molecule includes a primary polypeptide, e.g., the inhibitory molecule, which is conjugated to a secondary polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the drug comprises a first polypeptide which is an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGF beta or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., a co-stimulatory domain (e.g., 41BB, CD27, or CD28 as described herein) and / or a primary signaling domain (e.g., the CD3 zeta signaling domain as described herein)). In one embodiment, the drug comprises a first polypeptide of PD1 or a fragment of PD1 (e.g., at least a portion of the extracellular domain of PD1) and a second polypeptide which is an intracellular signaling domain (e.g., the CD28 signaling domain and / or the CD3 zeta signaling domain as described herein). 【0125】 In one embodiment, a CAR molecule, for example, cells expressing the CAR molecule described herein, is administered in combination with a drug that reduces one or more side effects associated with the administration of cells expressing the CAR molecule, for example, a drug described herein. 【0126】 In one embodiment, a CAR molecule, for example, one of the CAR molecules described herein, is administered in combination with a B cell inhibitor. For example, CD19 CAR-expressing cells are administered in combination with one or more additional B cell inhibitors. In one embodiment, the B cell inhibitor is a second CD19 inhibitor. In one embodiment, the B cell inhibitor is one or more inhibitors of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a. 【0127】 In one embodiment, the B cell inhibitor is a small molecule inhibitor, a polypeptide that binds to a B cell antigen (e.g., one or more of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a), such as a soluble ligand, an antibody or antigen-binding fragment thereof, or an inhibitory nucleic acid (e.g., double-stranded RNA (dsRNA), small interfering RNA (siRNA), or small hairpin RNA (shRNA)). In another embodiment, the B cell inhibitor is a cell expressing a CAR that binds to a B cell antigen (e.g., one or more of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a) (e.g., a CAR-expressing immunoeffector cell). 【0128】 In one embodiment, a CAR (e.g., CD19 CAR, mesoserine CAR, ROR1 CAR, CD20 CAR, CD22 CAR, CD123 CAR, CD10 CAR, CD34 CAR, FLT-3 CAR, CD79b CAR, CD179b CAR, or CD79a CAR) comprises an arbitrary leader sequence (e.g., any leader sequence described herein), an extracellular antigen-binding domain, a hinge (e.g., a hinge described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain described herein). In one embodiment, an example of a CAR construct comprises an arbitrary leader sequence (e.g., a leader sequence described herein), an extracellular antigen-binding domain, a hinge, a transmembrane domain, an intracellular co-stimulatory domain (e.g., an intracellular co-stimulatory domain described herein), and an intracellular stimulatory domain. 【0129】 subject In one embodiment, the subject, for example, a subject that has acquired immune cells and / or is treated, is a human, for example, a cancer patient. 【0130】 In one embodiment, the subject has a disease associated with the expression of tumor or cancer-related antigens, for example, the disease described herein. In another embodiment, the subject has cancer, for example, the cancer described herein. 【0131】 In one embodiment, the subject has a cancer selected from blood cancers, solid tumors, or metastatic lesions thereof. Examples of cancer include, but are not limited to, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell promyelocytic leukemia, blastocyte plasmacytoid dendritic cell neoplasms, Burkitt lymphoma, generalized large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative states, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, spinal dysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, and Waldenström hypergammaglobulinemia. In one embodiment, cancer is ALL. In another embodiment, cancer is CLL. 【0132】 In one embodiment, the subjects do not have recurrent cancer. In another embodiment, the subjects have recurrent cancer. 【0133】 In one embodiment, immune cells (e.g., a population of immune effector cells) are obtained, for example, from subjects having hematological cancers, such as leukemia, such as CLL, ALL, or lymphoma, such as MCL, NHL, or HL. 【0134】 Further embodiments of the method, preparations, and reaction mixtures In some embodiments, by means of treatment and / or the preparation and reaction mixture described herein (e.g., amplification and / or activation), the method further includes removing T regulatory cells, e.g., CD25+ T cells, from the immune cell population, for example, thereby providing a population of T regulatory depleted cells, e.g., CD25+ depleted cells, suitable for CAR expression. 【0135】 In one embodiment, the T regulatory depletion cell population contains 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and less than 1% CD25+ cells. 【0136】 In one embodiment, the immune cell population includes cells of a subject with cancer, such as chronic lymphocytic leukemia (CLL), or, for example, a subject with CD25-expressing cancer. In one embodiment, the T regulatory depletion cell population includes less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% CD25+ cells and less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% tumor cells. 【0137】 In one embodiment, the immune cell population is self to the subject to which the cells are administered for treatment. In another embodiment, the immune effector cell population is homogeneous to the subject to which the cells are administered for treatment. 【0138】 In one embodiment, T regulatory cells, such as CD25+ T cells, are removed from the population using an anti-CD25 antibody or its fragment or a CD25-binding ligand, such as IL-2. In one embodiment, the anti-CD25 antibody or its fragment or a CD25-binding ligand is conjugated to a substrate, such as beads, or coated to a substrate, such as beads, in another manner. In one embodiment, the anti-CD25 antibody or its fragment is conjugated to the substrate described herein. 【0139】 In one embodiment, T regulatory cells, for example, CD25+ T cells, are used in Milteny TM Remove from the population using a CD25 depletor. In one embodiment, the cell-to-CD25 depletor ratio is 1e 7 Cells per 20 μL or 1 e 7 Cell pair 15 μL or 1 e 7 Cells per 10 μL or 1 e 7 Cells per 5 μL or 1 e 7 Cell pair 2.5 μL or 1e 7 Each cell contains 1.25 μL. 【0140】 In one embodiment, a population of T regulatory depletion cells, for example, CD25+ depletion cells, is suitable for the expression of the CAR described herein, for example, the CD19 CAR described herein. In one embodiment, the population of T regulatory depletion cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% of leukemia cells, for example, CLL cells, ALL cells, or lymphoma cells, for example, MCL cells, NHL cells, or HL cells. In one embodiment, a population of immune effector cells is obtained from a subject having CLL, and the population of T regulatory depletion cells, for example, CD25+ depletion cells, contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% of leukemia cells, for example, CLL cells, and is suitable for the expression of the CD19 CAR described herein. In one embodiment, the T regulatory depletion cell population comprises 15%, 10%, 5%, 4%, 3%, 2%, and less than 1% CD25+ cells and tumor cells, e.g., CD25-expressing tumor cells, e.g., CLL cells, comprising 15%, 10%, 5%, 4%, 3%, 2%, and less than 1%. 【0141】 In one embodiment, the production method further includes removing cells from a population expressing tumor antigens, e.g., tumor antigens that do not contain CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14, or CD11b, thereby providing a population of T-regulatory depleted cells, e.g., CD25+ depleted and tumor antigen depleted cells, suitable for the expression of CARs, e.g., the CARs described herein. In one embodiment, tumor antigen-expressing cells are removed simultaneously with T-regulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody or its fragment and an anti-tumor antigen antibody or its fragment may be conjugated to the same substrate, e.g., beads, that can be used for cell removal, or the anti-CD25 antibody or its fragment or the anti-tumor antigen antibody or its fragment may be conjugated to separate beads, and a mixture thereof can be used for cell removal. In another embodiment, the removal of T-regulatory cells, e.g., CD25+ cells and tumor antigen-expressing cells is sequential and can be performed in any order, e.g. In one embodiment, the production method further includes removing one or more (e.g., 1, 2, or 3) cells expressing checkpoint inhibitors, e.g., the checkpoint inhibitors described herein, e.g., PD1+ cells, LAG3+ cells, and TIM3+ cells, from the population, thereby providing a population of T-regulatory depletion, e.g., CD25+ depleted cells and checkpoint inhibitor depleted cells, e.g., PD1+, LAG3+, and / or TIM3+ depleted cells. In one embodiment, checkpoint inhibitor-expressing cells are removed simultaneously with T-regulatory cells, e.g., CD25+ cells. For example, an anti-CD25 antibody or a fragment thereof and an anti-checkpoint inhibitor antibody or a fragment thereof may be conjugated to the same beads that can be used for cell removal, or the anti-CD25 antibody or a fragment thereof and an anti-checkpoint inhibitor antibody or a fragment thereof may be conjugated to separate beads, and the mixture thereof can be used for cell removal. In another embodiment, the removal of T-regulatory cells, e.g., CD25+ cells, and the removal of checkpoint inhibitor-expressing cells are sequential and can be performed in any order, e.g. 【0142】 In one embodiment, the population of cells to be removed is neither regulatory T cells nor tumor cells, but cells that otherwise negatively affect the growth and / or function of CART cells, such as cells expressing CD14, CD11b, CD33, CD15, or other markers that may be expressed by immunosuppressive cells. In one embodiment, such cells are intended to be removed simultaneously with or after the depletion of regulatory T cells and / or tumor cells, or in any other order. 【0143】 In one embodiment, the method further includes removing CD14-expressing cells from the population, thereby providing a population of T-regulatory depletion, e.g., CD25+ depleted cells and CD14+ depleted cells. In one embodiment, CD14+ cells are removed simultaneously with T-regulatory cells, e.g., CD25+ cells. For example, an anti-CD25 antibody or a fragment thereof and an anti-CD14 antibody or a fragment thereof may be conjugated to the same beads that can be used for cell removal; or the anti-CD25 antibody or a fragment thereof and the anti-CD14 antibody or a fragment thereof may be conjugated to separate beads, and the mixture thereof can be used for cell removal. In another embodiment, the removal of T-regulatory cells, e.g., CD25+ cells and CD14+ cells is sequential and can be performed in any order, e.g. 【0144】 In one embodiment, the population of immune effector cells provided is selected based on the expression of one, two, three, four, five, six, seven or more markers, such as CD3, CD28, CD4, CD8, CD27, CD127, CD45RA, and CD45RO, for example, the population of immune effector cells provided (e.g., T cells) is CD3+ and / or CD28+. 【0145】 In one embodiment, the method further includes obtaining a population of immunoeffector cells, e.g., T cells, enriched with the expression of one or more markers, e.g., CD3, CD28, CD4, CD8, CD27, CD127, CD45RA, and CD45RO, e.g., 1, 2, 3, 4, 5, 6, 7 or more. In one embodiment, the population of immunoeffector cells is enriched with CD3+ and / or CD28+ cells. For example, isolated T cells are obtained by incubation with anti-CD3 / anti-CD28 conjugate beads. In one embodiment, the method further includes selecting cells from a population of T regulatory depletion cells, e.g., CD25+ depletion cells, that express one or more markers, e.g., CD3, CD28, CD4, CD8, CD45RA, and CD45RO, e.g., 1, 2, 3, 4, 5, 6, 7 or more. 【0146】 In one embodiment, the method further includes, for example, activation of a population of T regulatory depleted cells, such as CD25+ depleted cells, by the method described herein. 【0147】 In one embodiment, the manufacturing method further includes transducing cells from a population of T regulatory depletion cells, for example, a population of CD25+ depletion cells, with a vector containing a nucleic acid encoding a CAR, for example, the CAR described herein, for example, the CD19 CAR described herein. In one embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, or retroviral vectors. In one embodiment, cells from a population of T regulatory depletion cells, for example, a population of CD25+ depletion cells, are transduced with the vector within, for example, one day after being obtained from a blood sample from a subject, for example, by apheresis, in a population of immune effector cells. 【0148】 In one embodiment, the method further comprises producing a population of RNA-modified cells that transiently express exogenous RNA from a population of T regulatory depletion cells, for example, a population of CD25+ depletion cells. The method comprises introducing in vitro transcription RNA or synthetic RNA into cells from the population, wherein the RNA comprises nucleic acids encoding CARs, for example, the CAR described herein, for example, the CD19 CAR described herein. 【0149】 In one embodiment, cells are grown in a suitable medium (e.g., the medium described herein) which may optionally contain one or more factors for proliferation and / or viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-21, TGFβ and TNF-α or any other additives for cell growth. 【0150】 In one embodiment, cells are grown in a suitable medium containing one or more interleukins (e.g., the medium described herein), which results in at least a 200-fold increase in the cells over a 14-day growth period, as measured by the method described herein, such as flow cytometry. In one embodiment, cells are grown in the presence of IL-15 and / or IL-7 (e.g., IL-15 and IL-7). 【0151】 In one embodiment, cells are cryopreserved after a suitable growth period. In another embodiment, cells are cryopreserved according to the method described herein. In another embodiment, the grown cells are cryopreserved in a suitable culture medium, for example, an insoluble medium as described herein. 【0152】 In one embodiment, the manufacturing method further includes contacting a population of immune effector cells with a nucleic acid encoding a telomerase subunit, such as hTERT. In one embodiment, the nucleic acid is DNA or RNA. 【0153】 In one embodiment, the method further includes removing T regulatory cells, e.g., CD25+ T cells, from the population before growth, thereby providing a population of T regulatory depleted cells, e.g., CD25+ depleted cells, which grows. In one embodiment, T regulatory cells, e.g., CD25+ cells, are removed by the method described herein. 【0154】 In one embodiment, the method further includes removing T regulatory cells, e.g., CD14+ cells, from the population before growth, thereby providing a population of CD14+ depleted cells that grows. In one embodiment, T regulatory cells, e.g., CD14+ cells, are removed by the method described herein. 【0155】 In one embodiment, the method further comprises contacting a population of immune effector cells with a nucleic acid encoding a telomerase subunit, such as hTERT. In one embodiment, the nucleic acid is DNA or RNA. 【0156】 In one embodiment, the method involves contacting a population of immunoeffector cells with a nucleic acid encoding CAR and a telomerase subunit, such as the nucleic acid encoding hTERT, under conditions that enable CAR and telomerase expression. 【0157】 In one embodiment, the nucleic acid encoding the telomerase subunit is RNA. In another embodiment, the nucleic acid encoding the telomerase subunit is DNA. In one embodiment, the nucleic acid encoding the telomerase subunit includes a promoter that can drive the expression of the telomerase subunit. 【0158】 In one embodiment, the production method includes contacting a population of immunoeffector cells with a nucleic acid encoding CAR and a telomerase subunit, such as RNA encoding hTERT, under conditions that enable CAR and telomerase expression. 【0159】 In one embodiment, the nucleic acid encoding the CAR and the RNA encoding the telomerase subunit are part of the same nucleic acid molecule. In another embodiment, the nucleic acid encoding the CAR and the RNA encoding the telomerase subunit are part of separate nucleic acid molecules. 【0160】 In one embodiment, the method includes contacting a population of immune effector cells with a nucleic acid encoding CAR and RNA encoding a telomerase subunit substantially simultaneously. In another embodiment, the production method includes contacting a population of immune effector cells with the nucleic acid encoding CAR before contacting a population of immune effector cells with RNA encoding a telomerase subunit. In yet another embodiment, the method includes contacting a population of immune effector cells with the nucleic acid encoding CAR after contacting a population of immune effector cells with RNA encoding a telomerase subunit. 【0161】 In one embodiment, the RNA encoding the telomerase subunit is mRNA. In one embodiment, the RNA encoding the telomerase subunit contains a poly(A) tail. In one embodiment, the RNA encoding the telomerase subunit contains a 5' cap structure. 【0162】 In one embodiment, the method comprises transfecting immune effector cells with RNA encoding a telomerase subunit. In another embodiment, the production method comprises transducing immune effector cells with RNA encoding a telomerase subunit. In yet another embodiment, the production method comprises electroporating immune effector cells with RNA encoding a telomerase subunit under conditions that enable CAR and telomerase expression. 【0163】 In one embodiment, the method provides a population of immunoeffector cells (e.g., T cells or NK cells) containing nucleic acids that express and / or encode CAR, and brings the population of immunoeffector cells into contact with a nucleic acid encoding a telomerase subunit, such as hTERT, under conditions that enable hTERT expression. 【0164】 In one embodiment, the method includes providing a population of immune effector cells (e.g., T cells or NK cells) expressing a telomerase subunit, for example, a nucleic acid encoding hTERT, and bringing the population of immune effector cells into contact with the nucleic acid encoding CAR under conditions that enable CAR expression. 【0165】 Immunoeffector cell preparations In one embodiment, the immunoeffector cell preparation described herein (e.g., a reaction mixture or a population of immunoeffector cells) is prepared by the method described herein. 【0166】 In one embodiment, a population of immune effector cells is selected based on the expression of one or more markers, such as CCR7, CD62L, CD45RO, and CD95, for example, the population of immune effector cells (e.g., T cells) is CCR7+ and CD62L+. 【0167】 In one embodiment, naive T cells are identified based on the expression patterns of CCR7+, CD62L+, CD45RO-, and CD95-, while stem central memory T cells are identified based on the expression patterns of CCR7+, CD62L+, CD45RO-, and CD95+, and central memory T cells are identified based on the expression patterns of CCR7+, CD62L+, CD45RO+, and CD95+. 【0168】 In one embodiment, the immunoeffector cell preparation described herein comprises a nucleic acid encoding a CAR, for example, the CAR described herein. 【0169】 In one embodiment, the immunoeffector cell preparation described herein comprises a nucleic acid encoding an exogenous telomerase subunit, such as hTERT. In one embodiment, the nucleic acid encoding the exogenous telomerase subunit is RNA, such as mRNA. 【0170】 In one embodiment, the immunoeffector cell preparation described herein comprises a CAR, e.g., the CAR described herein, and an exogenous telomerase subunit, e.g., hTERT. In one embodiment, the cells do not contain DNA encoding the exogenous telomerase subunit. For example, the cells may be in contact with mRNA encoding the exogenous telomerase subunit. 【0171】 In one embodiment, the immunoeffector cell preparation is a population of T regulatory depleted cells containing less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% CD25+ cells. In another embodiment, the immunoeffector cell preparation is a population of T regulatory depleted cells containing less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% CD25+ cells and less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% CD25-expressing tumor cells, such as CLL cells. In one embodiment, the immunoeffector cell preparation contains 15%, 10%, 5%, 4%, 3%, 2%, and less than 1% of CD25+ cells and tumor cells, e.g., CD25-expressing tumor cells, e.g., CLL cells, in proportion to 15%, 10%, 5%, 4%, 3%, 2%, and less than 1%. In one embodiment, the immunoeffector cell preparation contains 10%, 5%, 4%, 3%, 2%, and less than 1% of CD25+ cells and tumor cells, e.g., CD25-expressing tumor cells, e.g., CLL cells, in proportion to 10%, 5%, 4%, 3%, 2%, and less than 1%. 【0172】 In one embodiment, the immunoeffector cell preparation is a population of T regulatory depleted cells containing less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% CD25+ cells and less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% checkpoint inhibitor-expressing cells, such as PD1+ cells, LAG3+ cells, or TIM3+ cells. 【0173】 In one embodiment, the immunoeffector cell preparation is a population of T regulatory depleted cells containing 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and less than 1% CD25+ cells and 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and less than 1% CD14+ cells. 【0174】 In one embodiment, the immunoeffector cell preparation described herein comprises a population of autoimmune effector cells, for example, a portion thereof, transfected or transduced with a vector comprising a nucleic acid molecule encoding a CAR, for example, the CAR described herein, for example, the CD19 CAR described herein, wherein the immunoeffector cell preparation comprises less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% CD25+ cells and less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% tumor cells, for example, CLL cells. In one embodiment, the immunoeffector cell preparation contains 15%, 10%, 5%, 4%, 3%, 2%, and less than 1% of CD25+ cells and tumor cells, e.g., CD25-expressing tumor cells, e.g., CLL cells, in proportion to 15%, 10%, 5%, 4%, 3%, 2%, and less than 1%. In one embodiment, the immunoeffector cell preparation contains 10%, 5%, 4%, 3%, 2%, and less than 1% of CD25+ cells and tumor cells, e.g., CD25-expressing tumor cells, e.g., CLL cells, in proportion to 10%, 5%, 4%, 3%, 2%, and less than 1%. 【0175】 In one embodiment, the reaction mixture may further include a drug that activates and / or enlarges the population of cells, for example, a drug that stimulates CD3 / TCR complex-related signaling and / or a ligand that stimulates a cell surface co-stimulatory molecule, as described herein. In one embodiment, the drug is an anti-CD3 antibody or a fragment thereof and / or beads conjugated with an anti-CD28 antibody or a fragment thereof. 【0176】 In one embodiment, the reaction mixture described herein comprises a population of T regulatory depletion cells containing less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% CD25+ cells. In another embodiment, the reaction mixture comprises a population of T regulatory depletion cells containing less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% CD25+ cells and less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% CD25-expressing tumor cells, such as CLL cells. In one embodiment, the cell population comprises 15%, 10%, 5%, 4%, 3%, 2%, and less than 1% CD25+ cells and tumor cells, e.g., CD25-expressing tumor cells, e.g., CLL cells, in proportion to 15%, 10%, 5%, 4%, 3%, 2%, and less than 1%. In one embodiment, the cell population comprises 10%, 5%, 4%, 3%, 2%, and less than 1% CD25+ cells and tumor cells, e.g., CD25-expressing tumor cells, e.g., CLL cells, in proportion to 10%, 5%, 4%, 3%, 2%, and less than 1%. 【0177】 In one embodiment, the reaction mixture contains a population of T-regulatory depleted cells comprising 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and less than 1% of CD25+ cells and 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and less than 1% of checkpoint inhibitor-expressing cells, such as PD1+ cells, LAG3+ cells, or TIM3+ cells. The reaction mixture may further comprise buffers or other reagents, such as PBS-containing solutions. 【0178】 In one embodiment, the reaction mixture contains a population of T-regulated depleted cells comprising 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and less than 1% of CD25+ cells and 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and less than 1% of CD14+ cells. The reaction mixture may further comprise a buffer or other reagent, such as a PBS-containing solution. 【0179】 In one embodiment, the reaction mixture further comprises one or more factors for proliferation and / or viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-21, TGFβ and TNF-α, or any other additives for cell growth. In one embodiment, the reaction mixture further comprises IL-15 and / or IL-7. 【0180】 In one embodiment, a population of cells in the reaction mixture contains a nucleic acid molecule, for example, the nucleic acid molecule described herein, which contains a CAR-coding sequence, for example, the CD19 CAR-coding sequence described herein. 【0181】 In one embodiment, a population of cells in the reaction mixture includes a vector containing a nucleic acid sequence encoding a CAR, for example, the CAR described herein, for example, the CD19 CAR described herein. In one embodiment, the vector is a vector selected from the group consisting of the vectors described herein, for example, DNA, RNA, plasmid, lentiviral vector, adenovirus vector, or retroviral vector. 【0182】 In one embodiment, the reaction mixture further comprises, for example, saccharides, oligosaccharides, polysaccharides and polyols (e.g., trehalose, mannitol, sorbitol, lactose, sucrose, glucose and dextran), salts and cryoprotectants or stabilizers such as crown ethers. In one embodiment, the cryoprotectant is dextran. 【0183】 In one embodiment, the reaction mixture comprises a population of immunoeffector cells, where a plurality of cells in the population in the reaction mixture comprises nucleic acid molecules, e.g., 【0184】 In one embodiment, a population of cells in the reaction mixture includes a vector containing a nucleic acid sequence encoding a CAR, for example, the CAR described herein, for example, the CD19 CAR described herein. In one embodiment, the vector is a vector selected from the group consisting of the vectors described herein, for example, DNA, RNA, plasmid, lentiviral vector, adenovirus vector, or retroviral vector. 【0185】 Other characteristics, purposes, and advantages of the present invention are evident from the specification, drawings, and claims. 【0186】 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains. Similar or equivalent methods and materials may be used in carrying out or testing this invention, but suitable methods and materials are listed below. All publications, patent applications, patents and other citations mentioned herein are incorporated herein by reference in their entirety. Furthermore, materials, methods and examples are for illustrative purposes only and are not intended to limit them. [Brief explanation of the drawing] 【0187】 [Figure 1-1]Figures A-1D show the differential effects of γc cytokines and IL-18 on CAR-T cell accumulation. Figure 1A is a schematic diagram of the C4-27z CAR vector. Figure 1B is a graph showing the total accumulation of CAR-T cells in response to exposure to various cytokines. T cells were exposed to various exogenous cytokines at a final concentration of 10 ng / mL from the day after transduction (day 0). The number of CAR-T cells was calculated based on the number of T cells and the percentage of CAR expression. The curves are for 6 donors. *P<0.05, ***P<0.001. NC, no cytokines. Figure 1C is a histogram showing the proliferation of T cells in response to various cytokines. On day 7 after lentiviral transduction, T cells in the NC group were labeled with CFSE (2.5 μM) and then exposed to various cytokines. After 7 days, T cells were analyzed for CFSE dilution by flow cytometry. Figure 1D is a graph showing the viability of T cells 15 days after lentiviral transduction. T cells from various cytokine groups were stained with Annexin V and 7-AAD, and the proportion of viable cells was then analyzed (negative for both Annexin V and 7-AAD). *P<0.05, **P<0.01 vs. IL-2 group (n=6). [Figure 1-2]Figures A-1D show the differential effects of γc cytokines and IL-18 on CAR-T cell accumulation. Figure 1A is a schematic diagram of the C4-27z CAR vector. Figure 1B is a graph showing the total accumulation of CAR-T cells in response to exposure to various cytokines. T cells were exposed to various exogenous cytokines at a final concentration of 10 ng / mL from the day after transduction (day 0). The number of CAR-T cells was calculated based on the number of T cells and the percentage of CAR expression. The curves are for 6 donors. *P<0.05, ***P<0.001. NC, no cytokines. Figure 1C is a histogram showing the proliferation of T cells in response to various cytokines. On day 7 after lentiviral transduction, T cells in the NC group were labeled with CFSE (2.5 μM) and then exposed to various cytokines. After 7 days, T cells were analyzed for CFSE dilution by flow cytometry. Figure 1D is a graph showing the viability of T cells 15 days after lentiviral transduction. T cells from various cytokine groups were stained with Annexin V and 7-AAD, and the proportion of viable cells was then analyzed (negative for both Annexin V and 7-AAD). *P<0.05, **P<0.01 vs. IL-2 group (n=6). 【0188】 [Figure 2-1]Figures 2A-2F show the memory T cell subsets of CAR-T cells. Figure 2A shows CD95 expression in the CD45RA+CD62L+ T cell subset before transduction and in CAR-T cells 15 days after transduction. Figures 2B and 2C are graphs showing the increase in the proportion of memory stem T cells (Tscm) in CD4+ (Figure 2B) and CD8+ T cells (Figure 2C) after lentiviral transduction. Tscm are defined as the CD45RA+CD62L+CD95+CCR7+ T cell subset. Figure 2D is a graph showing the correlation between the amount of naive T cells (Tn, CD45RA+CD62L+CD95- subpopulation) in pre-transduction T cells and the proportion of Tscm in CAR-T cells after transduction (n=6). The bars on the left represent the percentage of Tn in CD4+ and CD8+ T cells before transduction, and the bars on the right represent the percentage of Tscm in CD4+ and CD8+ CAR-T cells. *P<0.05, **P<0.01. Figure 2E is a graph showing the self-renewal and differentiation of various subsets of CAR-T cells. FACS-selected CAR+ Tscm, Tcm, Tem, and Temra cells were cultured in IL-2 (10 ng / mL) for 3 days, and then the phenotype was analyzed based on CD45RA and CD62L expression (n=3). Figure 2F is a histogram plot showing the proliferation of various subsets of CAR-T cells in response to IL-2. FACS-selected CAR+ Tscm, Tcm, Tem, and Temra cells were labeled with CFSE (2.5 μM) and then cultured in IL-2 (10 ng / mL) for 3 days. Three days later, the T-cell CFSE dilution was analyzed. [Figure 2-2]Figures 2A-2F show the memory T cell subsets of CAR-T cells. Figure 2A shows CD95 expression in the CD45RA+CD62L+ T cell subset before transduction and in CAR-T cells 15 days after transduction. Figures 2B and 2C are graphs showing the increase in the proportion of memory stem T cells (Tscm) in CD4+ (Figure 2B) and CD8+ T cells (Figure 2C) after lentiviral transduction. Tscm are defined as the CD45RA+CD62L+CD95+CCR7+ T cell subset. Figure 2D is a graph showing the correlation between the amount of naive T cells (Tn, CD45RA+CD62L+CD95- subpopulation) in pre-transduction T cells and the proportion of Tscm in CAR-T cells after transduction (n=6). The bars on the left represent the percentage of Tn in CD4+ and CD8+ T cells before transduction, and the bars on the right represent the percentage of Tscm in CD4+ and CD8+ CAR-T cells. *P<0.05, **P<0.01. Figure 2E is a graph showing the self-renewal and differentiation of various subsets of CAR-T cells. FACS-selected CAR+ Tscm, Tcm, Tem, and Temra cells were cultured in IL-2 (10 ng / mL) for 3 days, and then the phenotype was analyzed based on CD45RA and CD62L expression (n=3). Figure 2F is a histogram plot showing the proliferation of various subsets of CAR-T cells in response to IL-2. FACS-selected CAR+ Tscm, Tcm, Tem, and Temra cells were labeled with CFSE (2.5 μM) and then cultured in IL-2 (10 ng / mL) for 3 days. Three days later, the T-cell CFSE dilution was analyzed. [Figure 2-3]Figures 2A-2F show the memory T cell subsets of CAR-T cells. Figure 2A shows CD95 expression in the CD45RA+CD62L+ T cell subset before transduction and in CAR-T cells 15 days after transduction. Figures 2B and 2C are graphs showing the increase in the proportion of memory stem T cells (Tscm) in CD4+ (Figure 2B) and CD8+ T cells (Figure 2C) after lentiviral transduction. Tscm are defined as the CD45RA+CD62L+CD95+CCR7+ T cell subset. Figure 2D is a graph showing the correlation between the amount of naive T cells (Tn, CD45RA+CD62L+CD95- subpopulation) in pre-transduction T cells and the proportion of Tscm in CAR-T cells after transduction (n=6). The bars on the left represent the percentage of Tn in CD4+ and CD8+ T cells before transduction, and the bars on the right represent the percentage of Tscm in CD4+ and CD8+ CAR-T cells. *P<0.05, **P<0.01. Figure 2E is a graph showing the self-renewal and differentiation of various subsets of CAR-T cells. FACS-selected CAR+ Tscm, Tcm, Tem, and Temra cells were cultured in IL-2 (10 ng / mL) for 3 days, and then the phenotype was analyzed based on CD45RA and CD62L expression (n=3). Figure 2F is a histogram plot showing the proliferation of various subsets of CAR-T cells in response to IL-2. FACS-selected CAR+ Tscm, Tcm, Tem, and Temra cells were labeled with CFSE (2.5 μM) and then cultured in IL-2 (10 ng / mL) for 3 days. Three days later, the T-cell CFSE dilution was analyzed. 【0189】 [Figure 3] Figures 3A and 3B show the correlation between CD45RA expression and CFSE intensity. Figure 3A shows that CD45RA expression is inversely correlated with CFSE intensity. Figure 3B shows that for all cytokine groups (IL-2, IL-7, IL-15, IL-18, and IL-21), CD45RA+ T cells exhibit much lower CFSE levels than CD45RA dim and negative T cells, indicating that CD45RA+ T cells have stronger proliferative activity than CD45RA- T cells. 【0190】 [Figure 4] Figure 4 shows the phenotypes of CAR-T cells resulting from exposure to various cytokines. Figure 4 is a series of graphs showing the quantification of CD45RA, CD62L, CCR7, CD27, CD28, and IL7Rα expression on the surface of CAR-T cells by FACS in the listed cytokine groups. The histograms show the mean ± SEM expression levels from six independent donors. *P<0.05, **P<0.01 vs. IL-2 group. 【0191】 [Figure 5-1] Figures 5A–5D show the functional analysis of CAR-T cells exposed to various cytokines. Figures 5A, 5B, and 5C are quantitative plots showing the percentage of cytokine-producing CAR-T cells in various cytokine groups (n=6) for the production of IFNγ (Figure 5A), TNF-α (Figure 5B), and IL-2 (Figure 5C). Lentiviral transdextrin T cells were exposed to the described cytokines for 14 days, then co-cultured with SKOV3 cells for 5 hours, and subsequently collected for flow cytometry analysis. Figure 5D is a graph showing the antigen-specific cytotoxic activity of CAR-T cells. After 14 days of exposure to the described cytokines, CAR-T cells were co-cultured with SKOV3 cells for 18 hours at the E / T ratio shown, and cell lysis was evaluated using a luciferase-based assay. Untransduced T cells (UNT) served as a negative effector control. The data shown are the mean ± SEM values ​​from six independent cell lysis assays. 【0192】 [Figure 5-2]Figures 5A–5D show the functional analysis of CAR-T cells exposed to various cytokines. Figures 5A, 5B, and 5C are quantitative plots showing the percentage of cytokine-producing CAR-T cells in various cytokine groups (n=6) for the production of IFNγ (Figure 5A), TNF-α (Figure 5B), and IL-2 (Figure 5C). Lentiviral transdextrin T cells were exposed to the described cytokines for 14 days, then co-cultured with SKOV3 cells for 5 hours, and subsequently collected for flow cytometry analysis. Figure 5D is a graph showing the antigen-specific cytotoxic activity of CAR-T cells. After 14 days of exposure to the described cytokines, CAR-T cells were co-cultured with SKOV3 cells for 18 hours at the E / T ratio shown, and cell lysis was evaluated using a luciferase-based assay. Untransduced T cells (UNT) served as a negative effector control. The data shown are the mean ± SEM values ​​from six independent cell lysis assays. [Figure 6] Figures 6A-6C show the phenotype and function of the CAR-T cells described in Figure 5. Figures 6A and 6B show that CD62L+ CAR-T cells (Tscm and Tcm) have lower cytokine production activity (Figures 6A and 6B) and weaker cytolytic ability (Figure 6C) compared to CD62L- CAR-T cells (Tem and Temra). 【0193】 [Figure 7-1] Figures 7A and 7B show the growth and phenotype of CAR-T cells exposed to antigen challenge. Figure 7A shows two graphs illustrating the overall accumulation and viability of CAR-T cells exposed to the cytokines described above, following antigen challenge. T cells exposed to the cytokines described were collected on day 15 and then co-cultured with SKOV3 at a 5:1 E / T ratio for 7 days. The growth of CAR-T cells was calculated on day 7 to evaluate T cell viability. Figure 7B shows two graphs illustrating the distribution of memory T subsets of CD4+ and CD8+ CAR-T cells in various cytokine groups. NS, no statistically significant differences. [Figure 7-2]Figures 7A and 7B show the growth and phenotype of CAR-T cells exposed to antigen challenge. Figure 7A shows two graphs illustrating the overall accumulation and viability of CAR-T cells exposed to the cytokines described above, following antigen challenge. T cells exposed to the cytokines described were collected on day 15 and then co-cultured with SKOV3 at a 5:1 E / T ratio for 7 days. The growth of CAR-T cells was calculated on day 7 to evaluate T cell viability. Figure 7B shows two graphs illustrating the distribution of memory T subsets of CD4+ and CD8+ CAR-T cells in various cytokine groups. NS, no statistically significant differences. 【0194】 [Figure 8-1] Figures 8A-8C show the antitumor activity of various CAR-T cells previously exposed to cytokines. Figure 8A shows tumor growth curves of mice treated with various cytokine-exposed C4-27z CAR-T cells, anti-CD19-27z CAR-T cells, and untransduced T cells. Data are shown as mean ± SEM. Arrows indicate the time of T cell injection. Figure 8B is a graph showing the quantification of circulating human CD4+ and CD8+ T cell counts in mouse peripheral blood 15 days after the first administration of CAR-T cell injection. Figure 8C is a graph showing the quantification of CAR expression in circulating human CD4+ and CD8+ T cells in mouse blood. [Figure 8-2] Figures 8A-8C show the antitumor activity of various CAR-T cells previously exposed to cytokines. Figure 8A shows tumor growth curves of mice treated with various cytokine-exposed C4-27z CAR-T cells, anti-CD19-27z CAR-T cells, and untransduced T cells. Data are shown as mean ± SEM. Arrows indicate the time of T cell injection. Figure 8B is a graph showing the quantification of circulating human CD4+ and CD8+ T cell counts in mouse peripheral blood 15 days after the first administration of CAR-T cell injection. Figure 8C is a graph showing the quantification of CAR expression in circulating human CD4+ and CD8+ T cells in mouse blood. 【0195】 [Figure 9]Figure 9 shows a series of FACS plots (top) illustrating CD3 and CD19 populations, and histograms (bottom) showing CD14 expression in cells from apheresis, cells selected with anti-CD3 / CD28, CD25-depleted cells, and CD25-enriched cells. 【0196】 [Figure 10] Figures 10A, 10B, and 10C show a comparison of the proliferation capacity of CD3 / CD28-selected cells and CD25-depleted cells. Figure 10A is a graph showing the total number of cells at the stated culture durations. Figure 10B is a graph showing the doubling of the quantified population at each of the stated culture durations. Figure 10C shows the percentage of viable cells at the stated culture durations. 【0197】 [Figure 11] Figure 11 is a series of FACs plots showing the distribution of CD3 and CD19 in untreated and CD25-depleted PBMCs after culture with the described cytokines, IL-7, IL-15, or IL-7 and IL-15. 【0198】 [Figure 12] Figure 12 is a graph showing the growth profiles in population doubling (Figure 17A) and mean size (fL) (Figure 17B) of PBMCs stimulated with anti-CD3 and CD28 beads, left untreated (UTD) or transduced with CD19 CAR (CD19.BBz), debeaded, and harvested on days 5 and 9. 【0199】 [Figure 13] Figure 13 is a graph showing cytotoxicity as lysis percentage of CD19-expressing K562 cells stimulated with anti-CD3 and CD28 beads, left untreated (UTD), or transduced with CD19 CAR (CD19.BBz), debeaded, and treated with PBMCs harvested on days 5 and 9. 【0200】 [Figure 14]Figure 14 is a graph representing the proliferation of PBMCs stimulated with anti-CD3 and CD28 beads (3x28 beads), wild-type K562 cells, CD19-expressing K562 cells, ALL cells (Nalm6) or CLL cells (PI14). PBMCs were either left untreated (UTD) or transduced with CD19 CAR (CART19), bead-depleted, and harvested on days 5 and 9. 【0201】 [Figure 15] Figure 15 is a schematic diagram of an example manufacturing scheme. 【0202】 [Figure 16] Figure 16 is a schematic diagram of an example manufacturing scheme. 【0203】 [Figure 17] Figure 17 is a graph representing the cell growth levels of two different manufacturing batches of donor cells transfected with CTL019 CAR, CHP959-115 and CHP959-121 and expanded for 0 - 9 days. 【0204】 [Figure 18] Figure 18 is a graph showing the production of pro-inflammatory cytokines, IFN-γ, GM-CSF, TNF-α and IL-4 in two different manufacturing batches of donor cells transfected with either CTL019 CAR, i.e. CHP959-115 or ss1-mesoCAR, i.e. CHP959-121 and expanded for 0 - 9 days after apheresis. 【0205】 [Figure 19] Figure 19 is a graph representing the production levels of IFN-γ, TNF-α, IL-6, IL-8, IL-2, IL-1β, GM-CSF and IL-4 in donor cells transfected with CTL019 CAR and stimulated with anti-CAR19-idiotype antibody beads or control beads and expanded for 5 - 9 days. No cytokines or low cytokine levels (<200 pg / ml) were detected with control beads. 【0206】 [Figure 20] Figure 20 is a graph showing cell death based on total lysate using a luciferase assay of Nalm6 (ALL) cells from PBBCs that were transduced either untreated (UTD) or with CD19 CAR (CART19), debeaded, and then harvested on days 5 and 9. PMBC vs. Nalm6 cells (effector (E):target (T)) were cultured in various ratios. As shown, CART19 cells harvested on day 5 exhibit good cytotoxicity. 【0207】 [Figure 21] Figure 21 is a graph showing the long-term in vivo cytotoxicity of PBMCs (Plant-Based Microorganisms) that were transduced either untreated (UTD) or with CD19 CAR (CART19), debeaded, and then harvested on days 5 and 9. PBMCs were introduced into non-obese diabetic / severely immunodeficient mice inoculated with Nalm6 cells. 【0208】 [Figure 22] Figure 22 is a schematic diagram illustrating the use of mesoserine-coated beads and mesoserine CART for cell growth. 【0209】 [Figure 23] Figure 23 is a schematic diagram of the experimental design for Example 4. 【0210】 [Figure 24] Figures 24A and 24B are graphs showing the population doubling of the cell types shown in Figure 23 (Figure 24A) and cell size (Figure 24B). 【0211】 [Figure 25-1] Figures 25A and 25B are graphs showing the transduction efficiency after 5 days (Figure 25A) and 11 days (Figure 25B). [Figure 25-2] Figures 25A and 25B are graphs showing the transduction efficiency after 5 days (Figure 25A) and 11 days (Figure 25B). 【0212】 [Figure 26] Figures 26A and 26B show the mesothelin CAR constructs and expression levels. Figure 26A is a schematic diagram of various CAR constructs used in Example 4. 【0213】 [Figure 27] Figures 27A-27C show the expansion of peripheral blood T cells and cord blood CD8 T cells in culture via mesothelin CAR stimulation. CD8 T cells are shown in Figure 27A. CD4 T cells are shown in Figure 27B. Cord blood CD8 T cells are shown in Figure 27C. 【0214】 [Figure 28] Figure 28 is a schematic diagram of a method of stimulation by its cognate antigen via a chimeric antigen receptor (CAR) transiently expressed on the T cell surface. 【0215】 [Figure 29] Figure 29 is a schematic diagram of the use of CAR for cell expansion with beads coated with cognate antigen. 【0216】 [Figure 30] Figures 30A and 30B are graphs representing the population doubling (Figure 30A) and cell size (Figure 30B) of mesothelin CAR-expressing cells after mesothelin bead exposure. 【0217】 [Figure 31-1] Figures 31A-31C are graphs representing the expansion of peripheral blood T cells stimulated with mesothelin CAR (Figure 31A) or CD19 CAR (Figure 31B) in culture and cord blood CD8 T cells stimulated with mesothelin CAR (Figure 31C). [Figure 31-2] Figures 31A-31C are graphs representing the expansion of peripheral blood T cells stimulated with mesothelin CAR (Figure 31A) or CD19 CAR (Figure 31B) in culture and cord blood CD8 T cells stimulated with mesothelin CAR (Figure 31C). 【0218】 [Figure 32-1]Figures 32A–32C show the CAR constructs and experimental design for Example 6. Figure 32A is a schematic diagram of the CAR constructs compared in Example 6. Both CARs are single-stranded variable fragments of SS1 scFv that bind to either the FMC63 antibody that recognizes human CD19 or human mesoserine. Transmembrane (TM) domain and intracellular domain are shown. Figure 32B is a graph showing flow cytometry analysis of CAR cell surface expression 1 day after electroporation compared to a control without CAR electroporation (mock). The right panel shows the mean fluorescence intensity (MFI) of the CAR detected with an anti-idiotype agent. The data are from independent experiments validated with cells from 25 independent healthy human donors. Figure 32C is a schematic diagram of the experimental design. CD8+ T cells are electroporated with in vitro transcription RNA. After resting the cells overnight, CAR expression is confirmed and in vitro culture is started in the presence of congener antigen-coated beads and cytokines. [Figure 32-2] Figures 32A–32C show the CAR constructs and experimental design for Example 6. Figure 32A is a schematic diagram of the CAR constructs compared in Example 6. Both CARs are single-stranded variable fragments of SS1 scFv that bind to either the FMC63 antibody that recognizes human CD19 or human mesoserine. Transmembrane (TM) domain and intracellular domain are shown. Figure 32B is a graph showing flow cytometry analysis of CAR cell surface expression 1 day after electroporation compared to a control without CAR electroporation (mock). The right panel shows the mean fluorescence intensity (MFI) of the CAR detected with an anti-idiotype agent. The data are from independent experiments validated with cells from 25 independent healthy human donors. Figure 32C is a schematic diagram of the experimental design. CD8+ T cells are electroporated with in vitro transcription RNA. After resting the cells overnight, CAR expression is confirmed and in vitro culture is started in the presence of congener antigen-coated beads and cytokines. 【0219】 [Figure 33-1]Figures 33A–33E show that BBz ICD provides a survival and proliferation advantage to CD8 T cells in vitro. Figure 33A shows CD69 levels measured on the cell surface 24 hours after co-culture with congener antigens. Figure 33B shows CD19 CAR T cell proliferation, and CD4+ and CD8+ T cells were stimulated as shown in Figure 33A and as described in Example 6. Data are from at least 10 different healthy donors. Figure 33C shows mesoserine CAR T cell proliferation of bulk CD8+ T cells (left) or naive (CD45RO-CD62L+CD8+) T cells (right). CAR T cells were stimulated using mesoserine-Fc coated beads. Figure 33D shows representative plots of cell surface expression CCR7 and CD45RO of CAR T cells at specific time points during culture (from at least 6 donors). The cells shown are pre-gated against viable CD3+CD8+ T cells. The numbers listed represent the percentage of cells detected at each gate. Figure 33E shows the relative changes in Tcm and Tem subsets in 28z and BBzCD19 CAR T cell cultures at various time points. The absolute number of viable cells was calculated for each population at the identified time point. The graphs show the relative magnification changes in Tcm or Tem in BBz CAR T cells normalized to 28z CAR T cells. Data are plotted as mean ± SEM (****, p<0.0001, **, p≦0.01). [Figure 33-2]Figures 33A–33E show that BBz ICD provides a survival and proliferation advantage to CD8 T cells in vitro. Figure 33A shows CD69 levels measured on the cell surface 24 hours after co-culture with congener antigens. Figure 33B shows CD19 CAR T cell proliferation, and CD4+ and CD8+ T cells were stimulated as shown in Figure 33A and as described in Example 6. Data are from at least 10 different healthy donors. Figure 33C shows mesoserine CAR T cell proliferation of bulk CD8+ T cells (left) or naive (CD45RO-CD62L+CD8+) T cells (right). CAR T cells were stimulated using mesoserine-Fc coated beads. Figure 33D shows representative plots of cell surface expression CCR7 and CD45RO of CAR T cells at specific time points during culture (from at least 6 donors). The cells shown are pre-gated against viable CD3+CD8+ T cells. The numbers listed represent the percentage of cells detected at each gate. Figure 33E shows the relative changes in Tcm and Tem subsets in 28z and BBzCD19 CAR T cell cultures at various time points. The absolute number of viable cells was calculated for each population at the identified time point. The graphs show the relative magnification changes in Tcm or Tem in BBz CAR T cells normalized to 28z CAR T cells. Data are plotted as mean ± SEM (****, p<0.0001, **, p≦0.01). [Figure 33-3]Figures 33A–33E show that BBz ICD provides a survival and proliferation advantage to CD8 T cells in vitro. Figure 33A shows CD69 levels measured on the cell surface 24 hours after co-culture with congener antigens. Figure 33B shows CD19 CAR T cell proliferation, and CD4+ and CD8+ T cells were stimulated as shown in Figure 33A and as described in Example 6. Data are from at least 10 different healthy donors. Figure 33C shows mesoserine CAR T cell proliferation of bulk CD8+ T cells (left) or naive (CD45RO-CD62L+CD8+) T cells (right). CAR T cells were stimulated using mesoserine-Fc coated beads. Figure 33D shows representative plots of cell surface expression CCR7 and CD45RO of CAR T cells at specific time points during culture (from at least 6 donors). The cells shown are pre-gated against viable CD3+CD8+ T cells. The numbers listed represent the percentage of cells detected at each gate. Figure 33E shows the relative changes in Tcm and Tem subsets in 28z and BBzCD19 CAR T cell cultures at various time points. The absolute number of viable cells was calculated for each population at the identified time point. The graphs show the relative magnification changes in Tcm or Tem in BBz CAR T cells normalized to 28z CAR T cells. Data are plotted as mean ± SEM (****, p<0.0001, **, p≦0.01). [Figure 33-4]Figures 33A–33E show that BBz ICD provides a survival and proliferation advantage to CD8 T cells in vitro. Figure 33A shows CD69 levels measured on the cell surface 24 hours after co-culture with congener antigens. Figure 33B shows CD19 CAR T cell proliferation, and CD4+ and CD8+ T cells were stimulated as shown in Figure 33A and as described in Example 6. Data are from at least 10 different healthy donors. Figure 33C shows mesoserine CAR T cell proliferation of bulk CD8+ T cells (left) or naive (CD45RO-CD62L+CD8+) T cells (right). CAR T cells were stimulated using mesoserine-Fc coated beads. Figure 33D shows representative plots of cell surface expression CCR7 and CD45RO of CAR T cells at specific time points during culture (from at least 6 donors). The cells shown are pre-gated against viable CD3+CD8+ T cells. The numbers listed represent the percentage of cells detected at each gate. Figure 33E shows the relative changes in Tcm and Tem subsets in 28z and BBzCD19 CAR T cell cultures at various time points. The absolute number of viable cells was calculated for each population at the identified time point. The graphs show the relative magnification changes in Tcm or Tem in BBz CAR T cells normalized to 28z CAR T cells. Data are plotted as mean ± SEM (****, p<0.0001, **, p≦0.01). [Figure 33-5]Figures 33A–33E show that BBz ICD provides a survival and proliferation advantage to CD8 T cells in vitro. Figure 33A shows CD69 levels measured on the cell surface 24 hours after co-culture with congener antigens. Figure 33B shows CD19 CAR T cell proliferation, and CD4+ and CD8+ T cells were stimulated as shown in Figure 33A and as described in Example 6. Data are from at least 10 different healthy donors. Figure 33C shows mesoserine CAR T cell proliferation of bulk CD8+ T cells (left) or naive (CD45RO-CD62L+CD8+) T cells (right). CAR T cells were stimulated using mesoserine-Fc coated beads. Figure 33D shows representative plots of cell surface expression CCR7 and CD45RO of CAR T cells at specific time points during culture (from at least 6 donors). The cells shown are pre-gated against viable CD3+CD8+ T cells. The numbers listed represent the percentage of cells detected at each gate. Figure 33E shows the relative changes in Tcm and Tem subsets in 28z and BBzCD19 CAR T cell cultures at various time points. The absolute number of viable cells was calculated for each population at the identified time point. The graphs show the relative magnification changes in Tcm or Tem in BBz CAR T cells normalized to 28z CAR T cells. Data are plotted as mean ± SEM (****, p<0.0001, **, p≦0.01). 【0220】 [Figure 34-1]Figures 34A–34M show the effects of CAR signaling domains on cellular metabolism and the preferential dependence of CAR T cells on glycolysis or fatty acid oxidation. As shown in Figures 34A–34D, BBz CAR T cells show elevated levels of oxygen consumption and reserve respiratory capacity. Figure 34A shows the effect of antigen stimulation on mean cell volume after stimulation in CD19 CAR CD8+ T cells expressing 28z and BBz signaling domains along with anti-idiotype. As shown in this figure, 28z and BBz CAR T cells have comparable mean cell sizes when measured at 0, 7, and 20 days. Figure 34B shows the oxygen consumption rate (OCR) of 28z and BBz CAR T cells at baseline (after CAR mRNA electroporation and before stimulation) at 0 days and after stimulation at 7 and 21 days in culture in response to basal conditions and mitochondrial inhibitors specific to Example 6. Basal OCR levels (Figure 34C), basal OCR / ECAR ratio (Figure 34D), maximal respiratory levels (Figure 34F), and basal ECAR levels (Figure 34G) were measured on days 7 and 21 (to reveal the preferential elevation of OXPHOS in BBz CAR T cells). Data are from at least five independent experiments conducted with cells from at least five healthy human donors and are plotted as mean ± SEM (*, p<0.05). Figure 34E shows the relative mRNA expression levels of genes involved in glycolysis and lipid oxidation as assessed in 28z and BBz, CAR T cells. The plots represent data from at least three independent experiments using cells from four independent donors (**, p<0.01; *, p<0.05). Data are expressed as mean ± SEM. Figures 34H–34J show basal OCR levels measured in CAR T cells sorted for different memory phenotypes: central memory (CM; Figure 34H), naive memory (N; Figure 34I), and effector memory (EM; Figure 34J). Data are from at least three independent experiments conducted with cells obtained from at least three healthy human donors and are plotted as mean ± SEM. Figure 34K shows basal ECAR levels measured for three differently sorted memory subsets.The data are from at least three independent experiments conducted with cells obtained from at least three healthy human donors and are plotted as mean ± SEM (*, p<0.05). Figure 34L shows measurements of glucose uptake from extracellular medium and lactate release into medium over 48 hours. Figure 34M shows the percentage of labeled acetyl-CoA measured in T cells cultured with [13C16]palmitic acid to assess fatty acid uptake and degradation. [Figure 34-2]Figures 34A–34M show the effects of CAR signaling domains on cellular metabolism and the preferential dependence of CAR T cells on glycolysis or fatty acid oxidation. As shown in Figures 34A–34D, BBz CAR T cells show elevated levels of oxygen consumption and reserve respiratory capacity. Figure 34A shows the effect of antigen stimulation on mean cell volume after stimulation in CD19 CAR CD8+ T cells expressing 28z and BBz signaling domains along with anti-idiotype. As shown in this figure, 28z and BBz CAR T cells have comparable mean cell sizes when measured at 0, 7, and 20 days. Figure 34B shows the oxygen consumption rate (OCR) of 28z and BBz CAR T cells at baseline (after CAR mRNA electroporation and before stimulation) at 0 days and after stimulation at 7 and 21 days in culture in response to basal conditions and mitochondrial inhibitors specific to Example 6. Basal OCR levels (Figure 34C), basal OCR / ECAR ratio (Figure 34D), maximal respiratory levels (Figure 34F), and basal ECAR levels (Figure 34G) were measured on days 7 and 21 (to reveal the preferential elevation of OXPHOS in BBz CAR T cells). Data are from at least five independent experiments conducted with cells from at least five healthy human donors and are plotted as mean ± SEM (*, p<0.05). Figure 34E shows the relative mRNA expression levels of genes involved in glycolysis and lipid oxidation as assessed in 28z and BBz, CAR T cells. The plots represent data from at least three independent experiments using cells from four independent donors (**, p<0.01; *, p<0.05). Data are expressed as mean ± SEM. Figures 34H–34J show basal OCR levels measured in CAR T cells sorted for different memory phenotypes: central memory (CM; Figure 34H), naive memory (N; Figure 34I), and effector memory (EM; Figure 34J). Data are from at least three independent experiments conducted with cells obtained from at least three healthy human donors and are plotted as mean ± SEM. Figure 34K shows basal ECAR levels measured for three differently sorted memory subsets.The data are from at least three independent experiments conducted with cells obtained from at least three healthy human donors and are plotted as mean ± SEM (*, p<0.05). Figure 34L shows measurements of glucose uptake from extracellular medium and lactate release into medium over 48 hours. Figure 34M shows the percentage of labeled acetyl-CoA measured in T cells cultured with [13C16]palmitic acid to assess fatty acid uptake and degradation. [Figure 34-3]Figures 34A–34M show the effects of CAR signaling domains on cellular metabolism and the preferential dependence of CAR T cells on glycolysis or fatty acid oxidation. As shown in Figures 34A–34D, BBz CAR T cells show elevated levels of oxygen consumption and reserve respiratory capacity. Figure 34A shows the effect of antigen stimulation on mean cell volume after stimulation in CD19 CAR CD8+ T cells expressing 28z and BBz signaling domains along with anti-idiotype. As shown in this figure, 28z and BBz CAR T cells have comparable mean cell sizes when measured at 0, 7, and 20 days. Figure 34B shows the oxygen consumption rate (OCR) of 28z and BBz CAR T cells at baseline (after CAR mRNA electroporation and before stimulation) at 0 days and after stimulation at 7 and 21 days in culture in response to basal conditions and mitochondrial inhibitors specific to Example 6. Basal OCR levels (Figure 34C), basal OCR / ECAR ratio (Figure 34D), maximal respiratory levels (Figure 34F), and basal ECAR levels (Figure 34G) were measured on days 7 and 21 (to reveal the preferential elevation of OXPHOS in BBz CAR T cells). Data are from at least five independent experiments conducted with cells from at least five healthy human donors and are plotted as mean ± SEM (*, p<0.05). Figure 34E shows the relative mRNA expression levels of genes involved in glycolysis and lipid oxidation as assessed in 28z and BBz, CAR T cells. The plots represent data from at least three independent experiments using cells from four independent donors (**, p<0.01; *, p<0.05). Data are expressed as mean ± SEM. Figures 34H–34J show basal OCR levels measured in CAR T cells sorted for different memory phenotypes: central memory (CM; Figure 34H), naive memory (N; Figure 34I), and effector memory (EM; Figure 34J). Data are from at least three independent experiments conducted with cells obtained from at least three healthy human donors and are plotted as mean ± SEM. Figure 34K shows basal ECAR levels measured for three differently sorted memory subsets.The data are from at least three independent experiments conducted with cells obtained from at least three healthy human donors and are plotted as mean ± SEM (*, p<0.05). Figure 34L shows measurements of glucose uptake from extracellular medium and lactate release into medium over 48 hours. Figure 34M shows the percentage of labeled acetyl-CoA measured in T cells cultured with [13C16]palmitic acid to assess fatty acid uptake and degradation. [Figure 34-4]Figures 34A–34M show the effects of CAR signaling domains on cellular metabolism and the preferential dependence of CAR T cells on glycolysis or fatty acid oxidation. As shown in Figures 34A–34D, BBz CAR T cells show elevated levels of oxygen consumption and reserve respiratory capacity. Figure 34A shows the effect of antigen stimulation on mean cell volume after stimulation in CD19 CAR CD8+ T cells expressing 28z and BBz signaling domains along with anti-idiotype. As shown in this figure, 28z and BBz CAR T cells have comparable mean cell sizes when measured at 0, 7, and 20 days. Figure 34B shows the oxygen consumption rate (OCR) of 28z and BBz CAR T cells at baseline (after CAR mRNA electroporation and before stimulation) at 0 days and after stimulation at 7 and 21 days in culture in response to basal conditions and mitochondrial inhibitors specific to Example 6. Basal OCR levels (Figure 34C), basal OCR / ECAR ratio (Figure 34D), maximal respiratory levels (Figure 34F), and basal ECAR levels (Figure 34G) were measured on days 7 and 21 (to reveal the preferential elevation of OXPHOS in BBz CAR T cells). Data are from at least five independent experiments conducted with cells from at least five healthy human donors and are plotted as mean ± SEM (*, p<0.05). Figure 34E shows the relative mRNA expression levels of genes involved in glycolysis and lipid oxidation as assessed in 28z and BBz, CAR T cells. The plots represent data from at least three independent experiments using cells from four independent donors (**, p<0.01; *, p<0.05). Data are expressed as mean ± SEM. Figures 34H–34J show basal OCR levels measured in CAR T cells sorted for different memory phenotypes: central memory (CM; Figure 34H), naive memory (N; Figure 34I), and effector memory (EM; Figure 34J). Data are from at least three independent experiments conducted with cells obtained from at least three healthy human donors and are plotted as mean ± SEM. Figure 34K shows basal ECAR levels measured for three differently sorted memory subsets.The data are from at least three independent experiments conducted with cells obtained from at least three healthy human donors and are plotted as mean ± SEM (*, p<0.05). Figure 34L shows measurements of glucose uptake from extracellular medium and lactate release into medium over 48 hours. Figure 34M shows the percentage of labeled acetyl-CoA measured in T cells cultured with [13C16]palmitic acid to assess fatty acid uptake and degradation. [Figure 34-5]Figures 34A–34M show the effects of CAR signaling domains on cellular metabolism and the preferential dependence of CAR T cells on glycolysis or fatty acid oxidation. As shown in Figures 34A–34D, BBz CAR T cells show elevated levels of oxygen consumption and reserve respiratory capacity. Figure 34A shows the effect of antigen stimulation on mean cell volume after stimulation in CD19 CAR CD8+ T cells expressing 28z and BBz signaling domains along with anti-idiotype. As shown in this figure, 28z and BBz CAR T cells have comparable mean cell sizes when measured at 0, 7, and 20 days. Figure 34B shows the oxygen consumption rate (OCR) of 28z and BBz CAR T cells at baseline (after CAR mRNA electroporation and before stimulation) at 0 days and after stimulation at 7 and 21 days in culture in response to basal conditions and mitochondrial inhibitors specific to Example 6. Basal OCR levels (Figure 34C), basal OCR / ECAR ratio (Figure 34D), maximal respiratory levels (Figure 34F), and basal ECAR levels (Figure 34G) were measured on days 7 and 21 (to reveal the preferential elevation of OXPHOS in BBz CAR T cells). Data are from at least five independent experiments conducted with cells from at least five healthy human donors and are plotted as mean ± SEM (*, p<0.05). Figure 34E shows the relative mRNA expression levels of genes involved in glycolysis and lipid oxidation as assessed in 28z and BBz, CAR T cells. The plots represent data from at least three independent experiments using cells from four independent donors (**, p<0.01; *, p<0.05). Data are expressed as mean ± SEM. Figures 34H–34J show basal OCR levels measured in CAR T cells sorted for different memory phenotypes: central memory (CM; Figure 34H), naive memory (N; Figure 34I), and effector memory (EM; Figure 34J). Data are from at least three independent experiments conducted with cells obtained from at least three healthy human donors and are plotted as mean ± SEM. Figure 34K shows basal ECAR levels measured for three differently sorted memory subsets.The data are from at least three independent experiments conducted with cells obtained from at least three healthy human donors and are plotted as mean ± SEM (*, p<0.05). Figure 34L shows measurements of glucose uptake from extracellular medium and lactate release into medium over 48 hours. Figure 34M shows the percentage of labeled acetyl-CoA measured in T cells cultured with [13C16]palmitic acid to assess fatty acid uptake and degradation. [Figure 34-6]Figures 34A–34M show the effects of CAR signaling domains on cellular metabolism and the preferential dependence of CAR T cells on glycolysis or fatty acid oxidation. As shown in Figures 34A–34D, BBz CAR T cells show elevated levels of oxygen consumption and reserve respiratory capacity. Figure 34A shows the effect of antigen stimulation on mean cell volume after stimulation in CD19 CAR CD8+ T cells expressing 28z and BBz signaling domains along with anti-idiotype. As shown in this figure, 28z and BBz CAR T cells have comparable mean cell sizes when measured at 0, 7, and 20 days. Figure 34B shows the oxygen consumption rate (OCR) of 28z and BBz CAR T cells at baseline (after CAR mRNA electroporation and before stimulation) at 0 days and after stimulation at 7 and 21 days in culture in response to basal conditions and mitochondrial inhibitors specific to Example 6. Basal OCR levels (Figure 34C), basal OCR / ECAR ratio (Figure 34D), maximal respiratory levels (Figure 34F), and basal ECAR levels (Figure 34G) were measured on days 7 and 21 (to reveal the preferential elevation of OXPHOS in BBz CAR T cells). Data are from at least five independent experiments conducted with cells from at least five healthy human donors and are plotted as mean ± SEM (*, p<0.05). Figure 34E shows the relative mRNA expression levels of genes involved in glycolysis and lipid oxidation as assessed in 28z and BBz, CAR T cells. The plots represent data from at least three independent experiments using cells from four independent donors (**, p<0.01; *, p<0.05). Data are expressed as mean ± SEM. Figures 34H–34J show basal OCR levels measured in CAR T cells sorted for different memory phenotypes: central memory (CM; Figure 34H), naive memory (N; Figure 34I), and effector memory (EM; Figure 34J). Data are from at least three independent experiments conducted with cells obtained from at least three healthy human donors and are plotted as mean ± SEM. Figure 34K shows basal ECAR levels measured for three differently sorted memory subsets.The data are from at least three independent experiments conducted with cells obtained from at least three healthy human donors and are plotted as mean ± SEM (*, p<0.05). Figure 34L shows measurements of glucose uptake from extracellular medium and lactate release into medium over 48 hours. Figure 34M shows the percentage of labeled acetyl-CoA measured in T cells cultured with [13C16]palmitic acid to assess fatty acid uptake and degradation. [Figure 34-7]Figures 34A–34M show the effects of CAR signaling domains on cellular metabolism and the preferential dependence of CAR T cells on glycolysis or fatty acid oxidation. As shown in Figures 34A–34D, BBz CAR T cells show elevated levels of oxygen consumption and reserve respiratory capacity. Figure 34A shows the effect of antigen stimulation on mean cell volume after stimulation in CD19 CAR CD8+ T cells expressing 28z and BBz signaling domains along with anti-idiotype. As shown in this figure, 28z and BBz CAR T cells have comparable mean cell sizes when measured at 0, 7, and 20 days. Figure 34B shows the oxygen consumption rate (OCR) of 28z and BBz CAR T cells at baseline (after CAR mRNA electroporation and before stimulation) at 0 days and after stimulation at 7 and 21 days in culture in response to basal conditions and mitochondrial inhibitors specific to Example 6. Basal OCR levels (Figure 34C), basal OCR / ECAR ratio (Figure 34D), maximal respiratory levels (Figure 34F), and basal ECAR levels (Figure 34G) were measured on days 7 and 21 (to reveal the preferential elevation of OXPHOS in BBz CAR T cells). Data are from at least five independent experiments conducted with cells from at least five healthy human donors and are plotted as mean ± SEM (*, p<0.05). Figure 34E shows the relative mRNA expression levels of genes involved in glycolysis and lipid oxidation as assessed in 28z and BBz, CAR T cells. The plots represent data from at least three independent experiments using cells from four independent donors (**, p<0.01; *, p<0.05). Data are expressed as mean ± SEM. Figures 34H–34J show basal OCR levels measured in CAR T cells sorted for different memory phenotypes: central memory (CM; Figure 34H), naive memory (N; Figure 34I), and effector memory (EM; Figure 34J). Data are from at least three independent experiments conducted with cells obtained from at least three healthy human donors and are plotted as mean ± SEM. Figure 34K shows basal ECAR levels measured for three differently sorted memory subsets.The data are from at least three independent experiments conducted with cells obtained from at least three healthy human donors and are plotted as mean ± SEM (*, p<0.05). Figure 34L shows measurements of glucose uptake from extracellular medium and lactate release into medium over 48 hours. Figure 34M shows the percentage of labeled acetyl-CoA measured in T cells cultured with [13C16]palmitic acid to assess fatty acid uptake and degradation. [Figure 34-8]Figures 34A–34M show the effects of CAR signaling domains on cellular metabolism and the preferential dependence of CAR T cells on glycolysis or fatty acid oxidation. As shown in Figures 34A–34D, BBz CAR T cells show elevated levels of oxygen consumption and reserve respiratory capacity. Figure 34A shows the effect of antigen stimulation on mean cell volume after stimulation in CD19 CAR CD8+ T cells expressing 28z and BBz signaling domains along with anti-idiotype. As shown in this figure, 28z and BBz CAR T cells have comparable mean cell sizes when measured at 0, 7, and 20 days. Figure 34B shows the oxygen consumption rate (OCR) of 28z and BBz CAR T cells at baseline (after CAR mRNA electroporation and before stimulation) at 0 days and after stimulation at 7 and 21 days in culture in response to basal conditions and mitochondrial inhibitors specific to Example 6. Basal OCR levels (Figure 34C), basal OCR / ECAR ratio (Figure 34D), maximal respiratory levels (Figure 34F), and basal ECAR levels (Figure 34G) were measured on days 7 and 21 (to reveal the preferential elevation of OXPHOS in BBz CAR T cells). Data are from at least five independent experiments conducted with cells from at least five healthy human donors and are plotted as mean ± SEM (*, p<0.05). Figure 34E shows the relative mRNA expression levels of genes involved in glycolysis and lipid oxidation as assessed in 28z and BBz, CAR T cells. The plots represent data from at least three independent experiments using cells from four independent donors (**, p<0.01; *, p<0.05). Data are expressed as mean ± SEM. Figures 34H–34J show basal OCR levels measured in CAR T cells sorted for different memory phenotypes: central memory (CM; Figure 34H), naive memory (N; Figure 34I), and effector memory (EM; Figure 34J). Data are from at least three independent experiments conducted with cells obtained from at least three healthy human donors and are plotted as mean ± SEM. Figure 34K shows basal ECAR levels measured for three differently sorted memory subsets.The data are from at least three independent experiments conducted with cells obtained from at least three healthy human donors and are plotted as mean ± SEM (*, p<0.05). Figure 34L shows measurements of glucose uptake from extracellular medium and lactate release into medium over 48 hours. Figure 34M shows the percentage of labeled acetyl-CoA measured in T cells cultured with [13C16]palmitic acid to assess fatty acid uptake and degradation. 【0221】 [Figure 35] Figures 35A–35C show enhanced BBz CAR T cell reserve respiratory capacity (SRC). Figure 35A shows SRC as the ratio of the maximum OCR level after cell treatment with FCCP to the basal OCR level at steady state during culture. Data represent three independent donors tested (*p<0.05). Figure 35B shows transmission electron microscope images of 28z and BBz CAR CD8+ T cells taken at three different time points. Scale bar represents 2 μm. Figure 35C shows the count of individual mitochondria per cell. Data represent 20 randomly selected cells (out of at least 75 cells analyzed per condition) and are expressed as mean ± SEM (***, p<0.001). 【0222】 [Figure 36-1]Figures 36A–36D show that BBz CAR signaling imprints genetic alterations in T cells to enhance mitochondrial nasogenesis. Figure 36A shows confocal images stained with Mitotracker (green), DAPI (blue), and cell membrane dye DiI (red). Scale bars represent 2 μm. Figure 36B shows quantification of the percentage of cytoplasm occupied by mitochondria, measured as the percentage of Mitotracker (green) within the region enclosed by the cell membrane (red). Data are shown as mean ± SEM of at least three independent images at each identified time point, with at least 15 independent cells scored per image (****, p<0.0001). Figure 36C shows the relative mRNA expression of mitochondrial cytochrome c oxidase 1 (MT-CO1) and mitochondrial transcription factor A (TFAM) in BBz CAR T cells normalized to the expression levels of 28z CAR T cells at identified time points. Data were compiled from at least three independent experiments in four independent donors (*, p<0.05) and are expressed as mean ± SEM. Figure 36D shows the normalized mRNA expression levels of nuclear respiratory factor 1 (NRF1) and GA-binding protein (NRF2) in BBz CAR T cells compared to 28z CAR T cells at identified time points. Data were compiled from at least three independent experiments in four independent donors (*, p<0.05) and are expressed as mean ± SEM. [Figure 36-2]Figures 36A–36D show that BBz CAR signaling imprints genetic alterations in T cells to enhance mitochondrial nasogenesis. Figure 36A shows confocal images stained with Mitotracker (green), DAPI (blue), and cell membrane dye DiI (red). Scale bars represent 2 μm. Figure 36B shows quantification of the percentage of cytoplasm occupied by mitochondria, measured as the percentage of Mitotracker (green) within the region enclosed by the cell membrane (red). Data are shown as mean ± SEM of at least three independent images at each identified time point, with at least 15 independent cells scored per image (****, p<0.0001). Figure 36C shows the relative mRNA expression of mitochondrial cytochrome c oxidase 1 (MT-CO1) and mitochondrial transcription factor A (TFAM) in BBz CAR T cells normalized to the expression levels of 28z CAR T cells at identified time points. Data were compiled from at least three independent experiments in four independent donors (*, p<0.05) and are expressed as mean ± SEM. Figure 36D shows the normalized mRNA expression levels of nuclear respiratory factor 1 (NRF1) and GA-binding protein (NRF2) in BBz CAR T cells compared to 28z CAR T cells at identified time points. Data were compiled from at least three independent experiments in four independent donors (*, p<0.05) and are expressed as mean ± SEM. [Figure 36-3]Figures 36A–36D show that BBz CAR signaling imprints genetic alterations in T cells to enhance mitochondrial nasogenesis. Figure 36A shows confocal images stained with Mitotracker (green), DAPI (blue), and cell membrane dye DiI (red). Scale bars represent 2 μm. Figure 36B shows quantification of the percentage of cytoplasm occupied by mitochondria, measured as the percentage of Mitotracker (green) within the region enclosed by the cell membrane (red). Data are shown as mean ± SEM of at least three independent images at each identified time point, with at least 15 independent cells scored per image (****, p<0.0001). Figure 36C shows the relative mRNA expression of mitochondrial cytochrome c oxidase 1 (MT-CO1) and mitochondrial transcription factor A (TFAM) in BBz CAR T cells normalized to the expression levels of 28z CAR T cells at identified time points. Data were compiled from at least three independent experiments in four independent donors (*, p<0.05) and are expressed as mean ± SEM. Figure 36D shows the normalized mRNA expression levels of nuclear respiratory factor 1 (NRF1) and GA-binding protein (NRF2) in BBz CAR T cells compared to 28z CAR T cells at identified time points. Data were compiled from at least three independent experiments in four independent donors (*, p<0.05) and are expressed as mean ± SEM. 【0223】 [Figure 37] Figure 37 shows the growth profiles of CD19-28z and CD19-BBz CAR T cells from two other independent donors. BBz CAR T cells are consistently observed to proliferate in culture and survive for extended periods. 【0224】 [Figure 38] Figure 38 shows the growth profiles of mesoserine-specific CAR T cells for two other independent donors. BBz CAR T cells are consistently observed to continue proliferating in culture and to survive for extended periods. 【0225】 [Figure 39] Figure 39 shows the oxygen consumption rate (OCR) in 28z and BBz CAR T cells at 7 and 21 days of culture, before stimulation (day 0), under basal conditions, and in the presence of mitochondrial inhibitors specific to Example 6. Metabolic assays performed with mesoserine-specific CARs reveal high oxygen consumption rates in BBz-CAR stimulated cells. 【0226】 [Figure 40] Figure 40 shows the total population doubling between the two CAR constructs (CD19 CAR n=10, p**=<0.01, mesoserine CAR n=6, p*=<0.05) shown in Figures 37 and 38. CD19 or SS1 CAR T cells were stimulated with anti-idiotype antibodies against CD19 scFv or mesoserine-Fc immobilized on beads, respectively. 【0227】 [Figure 41] Figure 41 shows the expression levels of CARs and major cytokine receptors on the cell surface after antigen exposure. The upper panel shows that no detectable CAR expression levels remain on the T cell surface after binding to CD19 scFv immobilized on beads of anti-idiotype antibody. These plots are from the same cell population assayed in Figure 32B that expressed CARs before antigen stimulation. The lower panel shows the levels of cytokine receptors, IL-2Rα, IL-7Rα, and IL-15Rα, on the cell surface assimilated by flow cytometry. 【0228】 [Figure 42] Figure 42 shows the changes in mitochondrial contents in 28z and BBz CAR T cells measured on day 21. Representative transmission electron microscope images of 28z and BBz CAR CD8+ T cells were acquired on day 21. The scale bar represents 2 μm. [Modes for carrying out the invention] 【0229】 Detailed description The methods described herein are based, at least in part, on the discovery that activation of CARs expressed on the surface of immune effector cells (e.g., transiently) provides an effective means of increasing and / or activating immune effector cell populations. Activation of CARs on the surface of immune effector cells by congenital antigens or anti-idiotype antibodies, as described herein, can result in cell enlargement. In some embodiments, such cell enlargement can be achieved without substantially altering the genotype or phenotype of the cells by transient expression of CARs (e.g., by in vitro transcription RNA). The methods described herein offer significant advantages over previously used methods for immune effector cell enlargement. 【0230】 In addition to its applicability to primary human T cells, the method described herein can be used to augment specific subsets of T lymphocytes, including naive cells, T regulatory cells, Th-17 cells, anergized T cells, and stem cell T cells or umbilical cord blood cells. While we do not wish to be bound by any particular theory, the method and composition described herein represent an improvement over existing experimental systems, as repeated stimulation via the TCR can be lethal to antigen-inexperienced T cells. Single stimulation via transiently expressed surface receptors can avoid this problem. Furthermore, the method provided herein enables T cell immunotherapy without interfering with the TCR, resulting in less rapid differentiation and promotion of “immature” T cells in culture. In other embodiments, the method described herein enables highly efficient transduction using vectors such as lentiviral vectors. 【0231】 Advantageously, other cell types lacking or with impaired T cell receptors can be enlarged. For example, all types of hematopoietic stem cells can be enlarged without phenotypic modification, and anergized T cells, TH17, NK, NKT, and B cells can be enlarged. 【0232】 Viral-mediated gene transfer systems are widely used in preclinical and clinical immunotherapy trials. Current methods for viral-mediated gene transfer to T lymphocytes require cell activation followed by viral vector administration. This activation is also traditionally achieved via stimulation through the TCR. Using the CAR-based stimulation method described here, highly efficient transduction with vectors such as lentiviral vectors can be achieved. Stimulation via transiently expressed CARs to achieve initial activation allows cells to be transduced with lentiviral vectors encoding the same or different CAR constructs. 【0233】 In one embodiment, the method described herein is provided for in vitro augmentation of immune effector cells. In a further embodiment, the method described herein involves lymph node injection leading to in vivo augmentation of T cells or injection into a tumor following in vivo augmentation of TILs. 【0234】 Accordingly, in one embodiment, the method disclosed herein provides for a method of increasing a population of immune effector cells by contacting a population of immune effector cells with a nucleic acid encoding a CAR under conditions suitable for transient expression of the CAR (where the CAR targets a congeneral antigen molecule), and culturing the immune effector cell population in the presence of the congeneral antigen molecule. In one embodiment, the nucleic acid is RNA, for example, in vitro transcription RNA. In another embodiment, the congeneral antigen molecule is a cancer-associated antigen molecule. In one embodiment, the congeneral antigen molecule is bound to a substrate, for example, beads, and the immune effector cell population is increased in vitro. In another embodiment, the congeneral antigen is expressed in cells, for example, tumor cells, and the immune effector cell population is increased in vivo. In another embodiment, the present invention relates to a method of treating a subject having cancer or providing antitumor immunity, comprising administering an effective amount of an immune effector cell population to the subject, wherein the immune effector cell population is increased by the method described herein. 【0235】 definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains. 【0236】 The singular expression means one or more than one (i.e., at least one) of the object. For example, “element” means one element or more than one element. 【0237】 The term “approximately” means, when relating to measurable values ​​such as quantity or duration, to include deviations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, insofar as such deviations are substantially appropriate to the method of disclosure. 【0238】 To the extent that the term is used herein, “acquisition” or “to obtain” means the acquisition of an object (e.g., a sample, cells or a population of cells, a polypeptide, nucleic acid or sequence) or a value (e.g., a numerical value) by “direct acquisition” or “indirect acquisition” of said object or value. In some embodiments, acquisition means obtaining or taking out cells or a population of cells (e.g., immunoeffector cells or populations as described herein). “Direct acquisition” means the implementation of a method for obtaining an object or value (e.g., the implementation of a synthesis or analysis or purification method). “Indirect acquisition” means obtaining an object or value from another entity or source (e.g., a third-party laboratory that directly acquired the object or value). Direct acquisition of an object includes the implementation of a method that involves a physical change of a substance, e.g., a starting material. Examples of changes include the production of an object from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, mixing two or more substances into a mixture, and performing chemical reactions that involve the breaking or formation of covalent or non-covalent bonds. Direct acquisition of values ​​includes the implementation of methods that involve physical changes to a sample or other substance, for example, the implementation of analytical steps that involve physical changes to a substance, for example, a sample, specimen or reagent (sometimes referred to here as “physical analysis”), analytical methods, for example, the separation or purification of a substance, for example, a specimen or its fragment or other derivative from other substances, the mixing of a specimen or its fragment or other derivative with other substances, for example, a buffer, solvent or reactant, or structural modification of a specimen or its fragment or other derivative by, for example, the breaking or formation of covalent or non-covalent bonds between the first and second atoms of the specimen, or structural modification of a reagent or its fragment or other derivative by, for example, the breaking or formation of covalent or non-covalent bonds between the first and second atoms of the reagent. 【0239】 The term “bioequivalence” refers to the amount of a drug other than the control compound (e.g., RAD001) required to produce an effect equivalent to that produced by the control dose or control amount of the control compound (e.g., RAD001). In some embodiments, the effect refers to the mTOR inhibition level measured by the assay described herein, for example, the Boulay assay, or the measurement of phosphorylated S6 levels by Western blotting, measured, for example, by P70 S6 kinase inhibition and evaluated, for example, by an in vivo or in vitro assay. In some embodiments, the effect is a modification of the PD-1-positive / PD-1-negative T cell ratio, measured by cell sorting. In some embodiments, a bioequivalence amount or dose of the mTOR inhibitor is an amount or dose that achieves the same level of P70 S6 kinase inhibition as the control dose or control amount of the control compound. In some embodiments, a bioequivalence amount or dose of the mTOR inhibitor is an amount or dose that achieves the same level of modification of the PD-1-positive / PD-1-negative T cell ratio as the control dose or control amount of the control compound. 【0240】 The term “chimeric antigen receptor” or “CAR” generally refers, in its simplest embodiment, to a pair of two polypeptides that, when present in an immune effector cell, provide the cell with specificity to target cells, generally cancer cells, and involve intracellular signaling production. In some embodiments, the CAR includes at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (hereinafter also referred to as the “intracellular signaling domain”) that includes a functional signaling domain derived from a stimulating and / or co-stimulating molecule as defined below. In some embodiments, the pair of polypeptides are on the same polypeptide chain (e.g., including a chimeric fusion protein). In some embodiments, the pair of polypeptides are not contiguous with each other, for example, on different polypeptide chains. In some embodiments, the pair of polypeptides include a dimerization switch that, in the presence of a dimerizing molecule, can link the polypeptides together, for example, linking the antigen-binding domain to the intracellular signaling domain. In some embodiments, the stimulating molecule is a zeta chain that binds to a T cell receptor complex. In some embodiments, the cytoplasmic signaling domain includes a primary signaling domain (e.g., the primary signaling domain of CD3-zeta). In one embodiment, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from a co-stimulatory molecule as defined below. In one embodiment, the co-stimulatory molecule of the CAR is selected from the co-stimulatory molecules listed herein, e.g., 4-1BB (i.e., CD137), CD27, ICOS, and / or CD28. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule.In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In another embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In another embodiment, the CAR comprises an optional leader sequence at the amino terminus (N-ter) of the CAR fusion protein. In another embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, where the leader sequence is optionally cleaved from the antigen-binding domain (e.g., scFv) during cell processing and localization of the CAR to the cellular membrane. 【0241】 Depending on the context, "CAR molecule" can refer to CAR (e.g., CAR polypeptide), nucleic acid that codes for CAR, or both. 【0242】 CARs containing an antigen-binding domain that targets a specific tumor antigen X, such as those described here (e.g., scFv or TCR), are also called XCARs. For example, a CAR containing an antigen-binding domain that targets CD19 is called a CD19CAR. 【0243】 The term “signaling domain” refers to a functional portion of a protein that acts by transmitting information within a cell to regulate cellular activity via a defined signaling pathway, either by producing a second messenger or by functioning as an effector in response to such a messenger. 【0244】 The term "antibody" as used herein refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and may be of natural or recombinant origin. Antibodies may be tetramers of immunoglobulin molecules. 【0245】 The term “antibody fragment” refers to at least a portion of an antibody that maintains the ability to specifically interact with an antigen epitope (e.g., by binding, steric hindrance, stabilization / destabilization, or spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs(sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single-domain antibodies (either VL or VH) such as sdAb, camel VHH domains, and bivalent fragments including 2Fab fragments linked by disulfide crosslinking at the hinge region. Examples of antibody fragments include, but are not limited to, isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, bispecific antibodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be transplanted onto polypeptide-based scaffolds such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199 describing fibronectin polypeptide minibodies). 【0246】 The term “inhibition” or “inhibitor” includes a reduction in a certain parameter of a molecule, e.g., CD19, CD20, CD10, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, mesoserine, or CD79a, e.g., activity. For example, inhibition of at least 5%, 10%, 20%, 30%, 40%, or more of the activity of CD20, CD10, CD19, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, mesoserine, or CD79a is included in this term. Therefore, inhibition does not need to be 100%. The activity of an inhibitor can be determined as described herein or by assays known in the art. 【0247】 The term “CD10” used herein refers to an antigen-determining factor known to be detectable in leukemia cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequences of human CD10 can be found at Accession Nos. NP_009218.2; NP_000893.2; NP_009219.2; NP_009220.2, and the encoding mRNA sequences can be found at Accession Nos. NM_007287.2 (variant 1bis); NM_000902.3 (variant 1); NM_007288.2 (variant 2a); NM_007289.2 (variant 2b). In one embodiment, the antigen-binding portion of the CAR recognizes and binds to the antigen within the extracellular domain of the CD10 protein. In one embodiment, the CD10 protein is expressed in cancer cells. The term "CD10" used herein includes proteins containing mutations in the full-length wild-type CD10, such as point mutations, fragments, insertions, deletions, and splice variants. 【0248】 The term “CD19” used herein refers to the surface antigen classification 19 protein, an antigen-determining factor detectable in leukemia precursor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found in UniProt / Swiss-Prot Accession No. P15391, and the nucleotide sequence encoding human CD19 can be found in Accession No. NM_001178098. As used herein, “CD19” includes proteins containing mutations in the full-length wild-type CD19, such as point mutations, fragments, insertions, deletions, and splice variants. 【0249】 CD19 is expressed in most B-cell lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. Other cells that express CD19 provide for the definition of “Diseases Associated with CD19 Expression” below. It is also an early marker of B-cell progenitor cells. See, e.g., Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one embodiment, the antigen-binding portion T of CAR recognizes and binds to antigens within the extracellular domain of the CD19 protein. In one embodiment, the CD19 protein is expressed in cancer cells. 【0250】 The term “CD20” as used herein refers to an antigen-determinant known to be detectable in B cells. Human CD20 is also known as the membrane-transmissible 4-domain, subfamily A, member 1 (MS4A1). Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD20 can be found at Accession Nos. NP_690605.1 and NP_068769.2, and the nucleotide sequences encoding transcript variants 1 and 3 of human CD20 can be found at Accession Nos. NM_152866.2 and NM_021950.3, respectively. In one embodiment, the antigen-binding portion of the CAR recognizes and binds to an antigen within the extracellular domain of the CD20 protein. In one embodiment, the CD20 protein is expressed in cancer cells. As used herein, “CD20” includes proteins containing mutations in the full-length wild-type CD20, e.g., point mutations, fragments, insertions, deletions, and splice variants. 【0251】 The term “CD22” used herein refers to an antigenic determinant known to be detectable in leukemia precursor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequences of isoforms 1–5 of human CD22 can be found at Accession Nos. NP 001762.2, NP 001172028.1, NP 001172029.1, NP 001172030.1, and NP 001265346.1, respectively, and the nucleotide sequences encoding variants 1–5 of human CD22 can be found at Accession Nos. NM 001771.3, NM 001185099.1, NM 001185100.1, NM 001185101.1, and NM 001278417.1, respectively. In one embodiment, the antigen-binding portion of the CAR recognizes and binds to an antigen within the extracellular domain of the CD22 protein. In another embodiment, the CD22 protein is expressed in cancer cells. As used herein, “CD22” includes proteins containing mutations in the full-length wild-type CD22, such as point mutations, fragments, insertions, deletions, and splice variants. 【0252】 The term “CD34” as used herein refers to an antigen-determinant known to be detectable in hematopoietic stem cells and certain cancer cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD34 can be found at Accession Nos. NP_001020280.1 (isoform a precursor); NP_001764.1 (isoform b precursor), and the encoding mRNA sequence can be found at Accession Nos. NM_001025109.1 (variant 1); NM_001773.2 (variant 2). In one embodiment, the antigen-binding portion of the CAR recognizes and binds to the antigen within the extracellular domain of the CD34 protein. In one embodiment, the CD34 protein is expressed in cancer cells. As used herein, “CD34” includes the protein containing mutations in the full-length wild-type CD34, e.g., point mutations, fragments, insertions, deletions, and splice variants. 【0253】 The term “CD123” used herein refers to an antigen-determining factor known to be detectable in certain malignant hematological cancer cells, such as leukemia cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD123 can be found at Accession Nos. NP_002174.1 (isoform 1 precursor); NP_001254642.1 (isoform 2 precursor), and the encoding mRNA sequence can be found at Accession Nos. NM_002183.3 (variant 1); NM_001267713.1 (variant 2). In one embodiment, the antigen-binding portion of the CAR recognizes and binds to the antigen within the extracellular domain of the CD123 protein. In one embodiment, the CD123 protein is expressed in cancer cells. The term "CD123" used herein includes proteins containing mutations in the full-length wild-type CD123, such as point mutations, fragments, insertions, deletions, and splice variants. 【0254】 The term “CD79b” used herein refers to an antigenic determinant known to be detectable in certain malignant hematological cancer cells, such as leukemia cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD79b can be found in Accession Nos. NP_000617.1 (isoform 1 precursor), NP_067613.1 (isoform 2 precursor), or NP_001035022.1 (isoform 3 precursor), and the encoding mRNA sequence can be found in Accession Nos. NM_000626.2 (transcript variant 1), NM_021602.2 (transcript variant 2), or NM_001039933.1 (transcript variant 3). In one embodiment, the antigen-binding portion of the CAR recognizes and binds to the antigen within the extracellular domain of the CD79b protein. In one embodiment, the CD79b protein is expressed in cancer cells. As used herein, “CD79b” includes proteins containing mutations in the full-length wild-type CD79b, such as point mutations, fragments, insertions, deletions, and splice variants. 【0255】 The term “CD79a” used herein refers to an antigen-determining factor known to be detectable in certain malignant hematological cancer cells, such as leukemia cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD79a can be found in Accession Nos. NP_001774.1 (isoform 1 precursor) or NP_067612.1 (isoform 2 precursor), and the encoding mRNA sequence can be found in Accession Nos. NM_001783.3 (transcript variant 1) or NM_021601.3 (transcript variant 2). In one embodiment, the antigen-binding portion of the CAR recognizes and binds to the antigen within the extracellular domain of the CD79a protein. In another embodiment, the CD79a protein is expressed in cancer cells. The term "CD79a" used herein includes proteins containing mutations in the full-length wild-type CD79a, such as point mutations, fragments, insertions, deletions, and splice variants. 【0256】 The term “CD179b” used herein refers to an antigen-determining factor known to be detectable in certain malignant hematological cancer cells, such as leukemia cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD179b can be found at Accession Nos. NP_064455.1 (isoform a precursor) or NP_690594.1 (isoform b precursor), and the encoding mRNA sequence can be found at Accession Nos. NM_020070.3 (transcript variant 1) or NM_152855.2 (transcript variant 2). In one embodiment, the antigen-binding portion of the CAR recognizes and binds to the antigen within the extracellular domain of the CD179b protein. In another embodiment, the CD179b protein is expressed in cancer cells. The term "CD179b" used herein includes proteins containing mutations in the full-length wild-type CD179b, such as point mutations, fragments, insertions, deletions, and splice variants. 【0257】 The term “FLT-3” as used herein refers to an antigenic determinant known to be detectable in hematopoietic progenitor cells and certain cancer cells, such as leukemia cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human FLT-3 can be found at Accession No. NP_004110.2, and the encoding mRNA sequence can be found at Accession No. NM_004119.2. In one embodiment, the antigen-binding portion of the CAR recognizes and binds to the antigen within the extracellular domain of the FLT-3 protein. In another embodiment, the FLT-3 protein is expressed in cancer cells. As used herein, “FLT-3” includes proteins containing mutations in the full-length wild-type FLT-3, such as point mutations, fragments, insertions, deletions, and splice variants. 【0258】 The term “ROR1” as used herein refers to an antigen-determining factor known to be detectable in leukemia precursor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequences of human ROR1 isoforms 1 and 2 precursors can be found at Accession Nos. NP_005003.2 and NP_001077061.1, respectively, and the encoding mRNA sequences can be found at Accession Nos. NM_005012.3 and NM_001083592.1, respectively. In one embodiment, the antigen-binding portion of the CAR recognizes and binds to the antigen within the extracellular domain of the ROR1 protein. In one embodiment, the ROR1 protein is expressed in cancer cells. As used herein, “ROR1” includes the protein containing mutations in the full-length wild-type ROR1, e.g., point mutations, fragments, insertions, deletions, and splice variants. 【0259】 The term “mesoserin” as used herein refers to the 40kDa protein mesoserin, which is fixed to the cell membrane by glycosylphosphatidylinositol (GPI) binding and an amino-terminal 31kDa shed fragment called megakaryocyte-enhancing factor (MPF). Both fragments contain an N-glycosylation site. The term also includes soluble splice variants of the 40kDa carboxyl-terminal fragment, also referred to as “soluble mesoserin / MPF-related.” Preferably, the term includes human mesoserin of GenBank accession number AAH03512.1 and its native cleavage portion, for example, expressed on cell membranes, e.g., cancer cell membranes. As used herein, “mesoserin” includes proteins containing mutations of full-length wild-type mesoserin, e.g., point mutations, fragments, insertions, deletions, and splice variants. 【0260】 The term “scFv” refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain and heavy chain variable regions are adjacently linked via, for example, a synthetic linker, such as a short-mobility polypeptide linker, and can be expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, the scFv used herein may contain the VL and VH variable regions in either order, for example, with respect to the N-terminus and C-terminus of the polypeptide, the scFv may contain either a VL-linker-VH or a VH-linker-VL. 【0261】 The portion of the CAR containing an antibody or an antibody fragment can exist in a variety of forms in which the antigen-binding domain is expressed as part of an adjacent polypeptide chain, including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), and a humanized antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen-binding domain of the CAR contains an antibody fragment. In a further embodiment, the CAR contains an antibody fragment containing an scFv. The terms “binding domain” or “antibody molecule” as used herein refer to a protein, such as an immunoglobulin chain or fragment thereof, containing at least one immunoglobulin variable domain sequence. The terms “binding domain” or “antibody molecule” encompass antibodies and antibody fragments. In some embodiments, the antibody molecule is a multispecific antibody molecule, for example, comprising multiple immunoglobulin variable domain sequences, where the first immunoglobulin variable domain sequence has binding specificity to the first epitope and the second immunoglobulin variable domain sequence has binding specificity to the second epitope. In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity to no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity to the first epitope and a second immunoglobulin variable domain sequence having binding specificity to the second epitope. 【0262】 The term “complementarity-determining region” or “CDR” as used herein refers to the sequence of amino acids within the antibody variable region that confers antigen specificity and binding affinity. For example, generally, each heavy chain variable region has 3 CDRs (e.g., HCDR1, HCDR2, and HCDR3) and each light chain variable region has 3 CDRs (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using any of the many well-known schemes, including those described in Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), or combinations thereof. Under the Kabat numbering scheme, in one embodiment, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in one embodiment, the CDR amino acids of VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combined Kabat and Chothia numbering scheme, in one embodiment, a CDR corresponds to an amino acid residue that is part of a Kabat CDR, a Chothia CDR, or both.For example, in one embodiment, CDR corresponds to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in VH, e.g., mammalian VH, e.g., human VH, and VL, e.g., amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in mammalian VL, e.g., human VL. 【0263】 The portion of the CAR of the present invention, which includes an antibody or an antibody fragment thereof, can exist in a variety of forms in which the antigen-binding domain is expressed as part of an adjacent polypeptide chain, for example, including a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen-binding domain of the CAR composition of the present invention includes an antibody fragment. In a further embodiment, the CAR includes an antibody fragment containing an scFv. 【0264】 The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in the naturally occurring three-dimensional structure of an antibody molecule, and this usually determines the class to which the antibody belongs. 【0265】 The term "antibody light chain" refers to the smaller of two types of polypeptide chains present in the naturally occurring three-dimensional structure of antibody molecules. Kappa (κ) and lambda (λ) light chains are the two main antibody light chain isotypes. 【0266】 The term “recombinant antibody” refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage or yeast expression system. This term should also be interpreted as meaning an antibody produced by the synthesis of an antibody-encoding DNA molecule, which expresses an antibody protein or amino acid sequence that identifies the antibody, where the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence technologies available and well-known in this field. 【0267】 The terms “antigen,” “Ag,” or “antigen molecule” refer to a molecule that elicits an immune response. This immune response may involve antibody production, activation of specific immunologically qualified cells, or both. In some embodiments, an antigen may be any macromolecule, including whole proteins or peptides. In other embodiments, an antigen may be recombinant or derived from genomic DNA. Any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as used herein. An antigen does not necessarily have to be encoded by a full-length nucleotide sequence of a gene. In some embodiments, an antigen may comprise one or more partial nucleotide sequences of genes, arranged in various combinations to encode a polypeptide that elicits a desired immune response. In some embodiments, an antigen does not have to be encoded by a “gene” at all. In some embodiments, an antigen may be produced by synthesis, derived from a biological sample, or may be a macromolecule other than a polypeptide. Such a biological sample may include, but is not limited to, a tissue sample, tumor sample, cells, or fluids with other biological components. In one embodiment, the antigen includes, for example, carbohydrates (e.g., monosaccharides, disaccharides, oligosaccharides, and polysaccharides). 【0268】 The term “homogeneic antigen molecule” includes all antigens described herein. In some embodiments, it refers to an antigen that is recognized, for example, targeted by a CAR molecule, for example, any of the CARs described herein. In other embodiments, it refers to a cancer-related antigen described herein. In some embodiments, the homogeneic antigen molecule is a recombinant molecule. 【0269】 The term “anti-idiotype (or idiotype) antibody molecule” or “anti-antigen idiotype (idiotype) antibody molecule” refers to an antibody molecule that binds to an antibody, for example, an antibody molecule that binds to the antigen-binding site or variable region of an antibody. In one embodiment, the anti-idiotype antibody molecule binds to an antibody epitope that comes into contact with an antigen, for example, the antigen described herein (for example, a homologous antigen molecule described herein). In one embodiment, the anti-idiotype antibody molecule binds to a CAR antigen-binding domain, for example, a portion of a CAR containing an antibody or antibody fragment (for example, the antigen-binding portion of a CAR). 【0270】 The term “ligand of a CAR molecule” as used herein refers to a CAR molecule or a molecule that binds to a portion of a CAR molecule. In one embodiment, the ligand binds to a portion of the CAR that contains a CAR antigen-binding domain, such as an antibody or antibody fragment. In another embodiment, the ligand is an antigen molecule, such as a congener antigen molecule as described herein. In yet another embodiment, the ligand is an anti-idiotype antibody molecule, such as an anti-antigen (e.g., CD19) idiotype antibody molecule as described herein. 【0271】 The term "self" refers to all substances originating from the same individual as the individual that will be reintroduced later. 【0272】 The term "homogeneous" refers to any substance originating from a different animal of the same species as the individual into which it is introduced. Two or more individuals can be considered homogeneous if they do not have identical genes at one or more loci. In some embodiments, homogeneous substances from individuals of the same species may be genetically sufficiently different to interact antigenically. 【0273】 The term "different species" refers to any substance derived from a different species of animal. 【0274】 The term “cancer” refers to a disease characterized by the uncontrolled proliferation of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are listed here, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer. The terms “tumor” and “cancer” are used interchangeably here, for example, both terms encompass solid and liquid tumors, e.g., generalized or circulating tumors. The terms “cancer” or “tumor” as used herein include pre-malignant and malignant cancers and tumors. 【0275】 The term "derived" used here refers to the relationship between the first and second molecules. Generally, it refers to the structural similarity between the first and second molecules and does not imply or include any process or limitation to the origin of the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from the CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure to have the necessary function, i.e., the ability to produce a signal under appropriate conditions. It does not imply or include any limitation to a specific process for producing the intracellular signaling domain; for example, it does not mean that, in order to provide an intracellular signaling domain, one must start from the CD3 zeta sequence, delete unwanted sequences or impose mutations, and then arrive at the intracellular signaling domain. 【0276】 The term “disease associated with the expression of tumor antigens” as used herein includes, but is not limited to, diseases associated with the expression of tumor antigens as described herein, or conditions associated with the expression of tumor antigens as described herein, including, for example, proliferative disorders of precancerous conditions such as cancer or malignant tumors or spinal dysplasia, myelodysplastic syndromes or preleukemic conditions, or non-cancer-related signs associated with cells expressing the tumor antigens described herein. In some embodiments, the cancer associated with the expression of tumor antigens described herein is a hematological cancer. In some embodiments, the cancer associated with the expression of tumor antigens described herein is a solid tumor. Furthermore, diseases associated with the expression of tumor antigens described herein include, but are not limited to, atypical and / or non-classical cancers, malignant tumors, precancerous conditions or proliferative diseases associated with the expression of tumor antigens described herein. Non-cancer-related signs associated with the expression of tumor antigens described herein include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplant rejection. In some embodiments, tumor antigen-expressing cells express, or express at some point in time, mRNA encoding the tumor antigen. In one embodiment, tumor antigen-expressing cells produce tumor antigen proteins (e.g., wild-type or mutant), which may be present at normal or low levels. In another embodiment, tumor antigen-expressing cells produce detectable levels of tumor antigen proteins at some point in time, and then substantially cease producing detectable levels of tumor antigen proteins thereafter. 【0277】 The term “diseases associated with CD19 expression” means diseases or conditions associated with cells expressing CD19 (e.g., wild-type or mutant CD19), including, for example, proliferative disorders such as cancer or malignant tumors or precancerous conditions such as spinal dysplasia, myelodysplastic syndromes or preleukemic states, or non-cancer-related signs associated with cells expressing CD19. To avoid doubt, diseases associated with CD19 expression may include conditions associated with cells that do not currently express CD19 but previously expressed CD19, for example, due to downregulation of CD19 expression by treatment with a CD19-targeting molecule, e.g., CD19 CAR. In some embodiments, cancers associated with CD19 expression are hematological malignancies. In some embodiments, hematological malignancies are leukemia or lymphoma. In one embodiment, cancers associated with CD19 expression include, but are not limited to, one or more acute leukemias, such as acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), and acute lymphoblastic leukemia (ALL); one or more chronic leukemias, such as, but are not limited to, chronic myeloid leukemia (CML) and chronic lymphoblastic leukemia (CLL); and cancers and malignancies, such as, but are not limited to.Further cancers or hematological conditions associated with CD19 expression include, for example, B-cell prelymphocytic leukemia, blastocyte plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, generalized large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, spinal dysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, Hodgkin lymphoma, and other conditions. This includes, but is not limited to, blastocyte lymphoma, plasmacytoid dendritic cell neoplasms, Waldenström hypergammaglobulinemia, myeloproliferative neoplasms, histiocytic disorders (e.g., mast cell disorders or blastocyte plasmacytoid dendritic cell neoplasms), mast cell disorders such as systemic mastocytosis or mast cell leukemia, B-cell prelymphocytic leukemia, plasmacytomyeloma, and “preleukemic states,” which are a diverse collection of blood conditions encompassing inability to produce (or malformation of) bone marrow blood cells. 【0278】 Furthermore, disease manifestations associated with CD19 expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or diseases associated with proliferative CD19 expression. Non-cancer-related manifestations associated with CD19 expression include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplant rejection. In some embodiments, CD19-expressing cells express CD19 mRNA or express it at some point in time. In some embodiments, CD19-expressing cells produce CD19 protein (e.g., wild-type or mutant), and the CD19 protein may be present at normal or low levels. In some embodiments, CD19-expressing cells produce detectable levels of CD19 protein at some point in time and then substantially cease producing detectable levels of CD19 protein. 【0279】 In one embodiment, tumor antigen-expressing cells express or express mRNA encoding the tumor antigen at some point in time. In one embodiment, tumor antigen-expressing cells produce tumor antigen protein (e.g., wild-type or mutant), which may be present at normal or low levels. In one embodiment, tumor antigen-expressing cells produce detectable levels of tumor antigen protein at some point in time, and subsequently produce substantially no detectable levels of tumor antigen protein. In another embodiment, the disease is CD19-negative cancer, e.g., CD19-negative recurrent cancer. In one embodiment, tumor antigen (e.g., CD19)-expressing cells express or express mRNA encoding the tumor antigen at some point in time. In one embodiment, tumor antigen (e.g., CD19)-expressing cells produce tumor antigen protein (e.g., wild-type or mutant), which may be present at normal or low levels. In one embodiment, tumor antigen (e.g., CD19)-expressing cells produce detectable levels of tumor antigen protein at some point in time, and subsequently produce substantially no detectable levels of tumor antigen protein. 【0280】 As used herein, the term “relapse” refers to the reappearance of the disease (e.g., cancer) after an initial responsive period, for example, after treatment with a prior therapeutic agent, e.g., an anti-cancer agent (e.g., a full response or partial response). The initial responsive period may include a decrease in cancer cell levels below a certain threshold, e.g., below 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. Reappearance refers to an increase in cancer cell levels above a certain threshold, e.g., above 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, in B-ALL, reappearance refers to the reappearance of blast cells in the blood, bone marrow (>5%), or anywhere outside the bone marrow, e.g., after a full response. A full response in this context may include <5% BM blast cells. More generally, in some embodiments, a response (e.g., a full response or partial response) may include the absence of detectable MRD (minimum residual disease). In one embodiment, the responsive initial period lasts for at least 1, 2, 3, 4, 5, or 6 days, at least 1, 2, 3, or 4 weeks, at least 1 month, 2, 3, 4, 6, 8, 10, or 12 months, or at least 1 year, 2, 3, 4, or 5 years. 【0281】 As used herein, "refractory" refers to a disease that does not respond to treatment, such as cancer. In one embodiment, refractory cancer may be resistant to treatment before or initially. In another embodiment, refractory cancer may become resistant during treatment. Refractory cancer is also called resistant cancer. 【0282】 The term “conservative sequence modification” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing an amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody or antibody fragment of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions involve the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CARs described herein can be replaced with other amino acid residues from the same side chain family, and the modified CARs can be tested using the functional assays described herein. 【0283】 The term “stimulus” refers to a primary response that is induced by the binding of a stimulating molecule (e.g., the TCR / CD3 complex or CAR) to its homologous ligand (e.g., an antigen molecule), thereby mediating a signaling event such as, but not limited to, signaling via the TCR / CD3 complex or signaling via the appropriate NK receptor or CAR signaling domain. Stimuli can also mediate the alteration of the expression of a molecule. 【0284】 The term “stimulating molecule” refers to a molecule expressed by immune cells (e.g., T cells, NK cells, B cells) that, in at least one aspect of an immune cell signaling pathway, provide cytoplasmic signaling sequences that regulate immune cell activation in a stimulating direction. In one aspect, the signal is a primary signal initiated, for example, by the binding of a TCR / CD3 complex to a peptide-laden MHC molecule, which leads to the mediation of a T cell response including, but not limited to, proliferation, activation, and differentiation. Stimulating primary cytoplasmic signaling sequences (also referred to as “primary signaling domains”) may include immune receptor tyrosine-based activation motifs or signaling motifs known as ITAMs. Examples of ITAM-containing cytoplasmic signaling sequences particularly useful in this invention include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In a particular CAR of the present invention, the intracellular signaling domain in any one or more CARS of the present invention comprises an intracellular signaling sequence, for example, the primary signaling sequence of CD3-zeta. In a particular CAR of the present invention, the primary signaling sequence of CD3-zeta is the sequence provided as SEQ ID NO: 9 or a corresponding residue from a non-human species, such as mouse, rodent, monkey, or ape. In a particular CAR of the present invention, the primary signaling sequence of CD3-zeta is the sequence provided as SEQ ID NO: 10 or a corresponding residue from a non-human species, such as mouse, rodent, monkey, or ape. 【0285】 The term “antigen-presenting cell” or “APC” refers to immune system cells, such as accessory cells (e.g., B cells, dendritic cells, etc.), that present foreign antigens on their surface, complexed with major histocompatibility complex (MHC). T cells can recognize these complexes using T cell receptors (TCRs). APCs process the antigens and present them to T cells. 【0286】 The term “intracellular signaling domain” as used herein refers to the intracellular portion of a molecule. Intracellular signaling domains can produce signals that promote immune effector functions in CAR-containing cells, such as CART cells. Examples of immune effector functions in CART cells include cytokine secretion, cytolytic activity, and helper activity. In some embodiments, the intracellular signaling domain is the portion of a protein that transmits effector function signals, instructing cells to perform specialized functions. While the entire intracellular signaling domain may be used, it is often not necessary to use the entire chain. To the extent that a cleavage portion of the intracellular signaling domain is used, such a cleavage portion can be used in place of the intact chain, as long as it transmits effector function signals. Therefore, the term “intracellular signaling domain” refers to any cleavage portion of an intracellular signaling domain sufficient for effector function signaling. 【0287】 In one embodiment, the intracellular signaling domain may include a primary intracellular signaling domain. Examples of primary intracellular signaling domains include those derived from molecules responsible for primary or antigen-dependent stimuli. In one embodiment, the intracellular signaling domain may include a co-stimulatory intracellular domain. Examples of co-stimulatory intracellular signaling domains include those derived from molecules responsible for co-stimulatory signals or antigen-independent stimuli. For example, in the case of CART, the primary intracellular signaling domain may include the cytoplasmic sequence of the T cell receptor, and the co-stimulatory intracellular signaling domain may include the cytoplasmic sequence from [the source]. 【0288】 The primary intracellular signaling domain may contain an immune receptor tyrosine-based activation motif or a signaling motif known as an ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI, CD66d, CD32, DAP10, and DAP12. 【0289】 The terms “zeta” or alternatively “zeta chain,” “CD3-zeta,” or “TCR-zeta” are defined as the protein provided as GenBank Acc. No. BAG36664.1 or corresponding residues from non-human species, e.g., mice, rodents, monkeys, apes, etc., and the “zeta-stimulating domain” or alternatively “CD3-zeta-stimulating domain” or “TCR-zeta-stimulating domain” are defined as amino acid residues from the cytoplasmic domain of the zeta chain sufficient for the functional transmission of the intended signal required for T cell activation. In one embodiment, the cytoplasmic domain of zeta includes residues 52-164 of GenBank Acc. No. BAG36664.1 or their functional orthologues from non-human species, e.g., mice, rodents, monkeys, apes, etc. In one embodiment, the “zeta-stimulating domain” or “CD3-zeta-stimulating domain” is the sequence provided as Sequence ID No. 9 (mutant CD3 zeta). In one embodiment, the “zeta-stimulating domain” or “CD3-zeta-stimulating domain” is the sequence provided as Sequence ID No. 10 (wild-type human CD3 zeta). 【0290】 The term "costimulatory molecule" refers to a T cell homologous binding partner that specifically binds to a costimulatory ligand and thereby mediates a T cell-mediated costimulatory response, including but not limited to proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or ligands necessary for an efficient immune response. Examples of costimulatory molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphoid activators (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), and B7-H3. CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 Alpha, CD8 Beta, IL2R Beta, IL2R Gamma, IL7R Alpha, ITGA4, VLA1, 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, LFA~1 , ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CR This includes, but is not limited to, ligands that specifically bind to TAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83. The co-stimulatory intracellular signaling domain refers to the intracellular portion of the co-stimulatory molecule.The intracellular signaling domain may include the entire intracellular portion of the derived molecule or the entire intrinsic intracellular signaling domain or its functional fragment. 【0291】 The intracellular signaling domain may include the entire intracellular portion of the derived molecule or the entire intrinsic intracellular signaling domain or its functional fragment. 【0292】 The term “4-1BB” refers to a member of the TNFR superfamily having an amino acid sequence provided in GenBank Acc. No. AAA62478.2 or corresponding residues from a non-human species, such as mouse, rodent, monkey, or ape, and the “4-1BB co-stimulatory domain” is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2 or corresponding residues from a non-human species, such as mouse, rodent, monkey, or ape. In one embodiment, the “4-1BB co-stimulatory domain” is the sequence provided as Sequence ID No. 7 or corresponding residues from a non-human species, such as mouse, rodent, monkey, or ape. 【0293】 The term “immune effector cells” as used herein refers to cells involved in the immune response, for example, in promoting the immune effector response. Examples of immune effector cells include T cells, such as alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytic cells. 【0294】 The term “immune effector function or immune effector response” as used herein refers to the function or response of immune effector cells, for example, that enhances or promotes the immune attack of target cells. For example, immune effector function or response refers to the properties of T or NK cells that promote the death or inhibition of growth or proliferation of target cells. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector function or response. 【0295】 The term “effector function” refers to a specialized function of a cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including cytokine secretion. The term “coding” refers to the inherent and derived biological properties of the specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, which has a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and acts as a template for the synthesis of other polymers and macromolecules in biological processes. Therefore, a gene, cDNA, or RNA codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, which has a nucleotide sequence identical to the mRNA sequence and is usually provided in the sequence listing, and the non-coding strand, which is used as a transcription template for the gene or cDNA, can be said to code for the protein or other products of that gene or cDNA. 【0296】 Unless otherwise specified, “nucleotide sequences encoding an amino acid sequence” include all nucleotide sequences encoding the same amino acid sequence, each being a degenerate version of the other. A nucleotide sequence or RNA encoding a protein may contain introns to the same extent that a protein-coding nucleotide sequence may contain introns in some versions. 【0297】 The term “endogenous” refers to any substance that originates from or is produced within an organism, cell, tissue, or system. 【0298】 The term "exogenous" refers to any substance introduced from outside an organism, cell, tissue, or system, or produced outside of that system. 【0299】 The term “expression” refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter. 【0300】 The term “transfer vector” refers to a composition containing isolated nucleic acids that can be used to deliver isolated nucleic acids into cells. Numerous vectors are known in this field and include, but are not limited to, linear polynucleotides, polynucleotides conjugated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “transfer vector” includes self-replicating plasmids or viruses. The term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the delivery of nucleic acids to cells, such as polylysine compounds and liposomes. Examples of viral transfer vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and lentiviral vectors. 【0301】 The term “expression vector” refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence manipulably ligated to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression may be shared from host cells or in an in vitro expression system. Expression vectors include all known in the art that incorporate recombinant polynucleotides, including cosmids, plasmids (e.g., naked or liposome-containing), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses). 【0302】 The term "lentivirus" refers to a genus within the retroviridae family. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells and deliver a significant amount of genetic information to the host cell's DNA, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. 【0303】 The term “lentiviral vector” refers to a vector derived from at least a portion of a lentiviral genome, particularly including the self-inactivating lentiviral vector provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors available for clinical use include, for example, Oxford BioMedica’s LENTIVECTOR® gene delivery technology and Lentigen’s LENTIMAX. TM This includes, but is not limited to, vector systems. Non-clinical lentiviral vectors are also available and are known to those skilled in the art. 【0304】 The term “homologous” or “identical” refers to the subunit sequence identity between two polymer molecules, between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When the subunit positions in both molecules are occupied by the same monomer subunit, for example, if a certain position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. Homologousness between two sequences is a direct function of the number of compatible or homologous positions. For example, if half of the positions of the two sequences are homologous (e.g., 5 positions out of 10 polymer subunit lengths), then the two sequences are 50% homologous, and if 90% of the positions (e.g., 9 out of 10) are compatible or homologous, then the two sequences are 90% homologous. 【0305】 The “humanized” form of a non-human (e.g., mouse) antibody refers to a chimeric immunoglobulin, immunoglobulin chain, or fragment (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody) containing minimal sequences derived from non-human immunoglobulins. Mostly, humanized antibodies and antibody fragments are human immunoglobulins (recipient antibodies or antibody fragments) in which residues from the recipient's complementarity-determining region (CDR) are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, possessing desired specificity, affinity, and capability. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments may contain residues not found in either the recipient antibody or the transferred CDR or framework sequence. These modifications further refine and optimize the performance of the antibody or antibody fragment. Generally, humanized antibodies or antibody fragments contain substantially all or at least one, generally two, variable domains, where all or substantially all CDR regions correspond to those of non-human immunoglobulins, and all or a substantial portion of the FR regions are human immunoglobulin sequences. Humanized antibodies or antibody fragments also contain at least a portion of the immunoglobulin constant region (Fc), which is generally that of human immunoglobulins. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992. 【0306】 "Completely human" refers to an immunoglobulin, such as an antibody or antibody fragment, whose entire molecule is of human origin or consists of the same amino acid sequence as the human form of the antibody or immunoglobulin. 【0307】 The term “isolation” means being altered or removed from its natural state. For example, nucleic acids or peptides that are naturally present in a living animal are not “isolated,” but the same nucleic acids or peptides that are partially or completely separated from substances coexisting in that natural state are “isolated.” Isolated nucleic acids or proteins can exist in a substantially purified form or in a non-natural environment, such as a host cell. 【0308】 In this invention, the following abbreviations for commonly existing nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine. 【0309】 The terms “manipulative linking” or “transcriptional regulation” refer to the functional linking of a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is manipulatively linked to a second nucleic acid sequence when the first nucleic acid sequence is positioned in a functional correlation with the second nucleic acid sequence. For example, a promoter is manipulatively linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Manipulatively linked DNA sequences can be adjacent to each other and, for example, within the same reading frame to link two protein coding regions if necessary. 【0310】 The term "non-enteral" administration of immunogenic compositions refers, for example, to subcutaneous (sc), intravenous (iv), intramuscular (im), intrasternal, or intratumoral injection or infusion methods. 【0311】 The terms “nucleic acid” or “polynucleotide” refer to single-stranded or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or combinations thereof of DNA or RNA, and polymers thereof. The term “nucleic acid” includes genes, cDNA, or mRNA. In some embodiments, nucleic acid molecules are synthetic (e.g., chemosynthesized) or recombinant. Unless otherwise specified, the terms include nucleic acids comprising analogs or derivatives of native nucleotides that have similar binding properties to control nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, particular nucleic acid sequences also imply that they include their conserved modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. In particular, degenerate codon substitution can be achieved by producing sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). 【0312】 The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide is any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, this term encompasses both short chains, commonly referred to in this field as peptides, oligopeptides, and oligomers, and long chains, commonly referred to in this field as proteins, of which many types exist. “Polypeptides” include, among other things, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, or combinations thereof. 【0313】 The term "promoter" refers to a DNA sequence that is recognized by the cell's synthetic or transgenic synthesis mechanism and is necessary for the initiation of specific transcription of a polynucleotide sequence. 【0314】 The term “promoter / regulatory sequence” refers to a nucleic acid sequence necessary for the expression of a gene product that is manipulably ligated to a promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, it may also include enhancer sequences and other regulatory elements necessary for the expression of the gene product. The promoter / regulatory sequence may, for example, express the gene product in a tissue-specific manner. 【0315】 The term “constitutive” promoter is a nucleotide sequence that, when manipulatively linked to a polynucleotide encoding or identifying a gene product, causes the production of that gene product in a cell under most or all physiological conditions. 【0316】 The term “inducible” promoter is a nucleotide sequence that, when manipulably ligated with a polynucleotide encoding or identifying a gene product, causes the production of a gene product in a cell only when a substantially promoter-corresponding inducer is present in the cell. 【0317】 The term “tissue-specific” promoter is a nucleotide sequence that, when manipulably linked to a polynucleotide encoding or identifying a gene product, causes the cell to produce the gene product only when the cell is a cell of the tissue type corresponding to the promoter. 【0318】 The term “cancer-associated antigen” or “tumor antigen” refers to a molecule (generally a protein, carbohydrate, or lipid) that is preferentially expressed on the surface of cancer cells, either whole or as a fragment (e.g., MHC / peptides), in a mutually interchangeable manner compared to normal cells, and is useful for preferential targeting of drugs to cancer cells. In some embodiments, the tumor antigen is a marker expressed on both normal and cancer cells, e.g., a cell lineage marker, e.g., CD19 on B cells. In some embodiments, the cancer-associated antigen is a cell surface molecule that is overexpressed on cancer cells compared to normal cells, e.g., 1x overexpression, 2x overexpression, 3x or more overexpression compared to normal cells. In some embodiments, the cancer-associated antigen is a cell surface molecule that is improperly synthesized on cancer cells, e.g., a molecule containing deletions, additions, or mutations compared to a molecule expressed on normal cells. In some embodiments, the cancer-associated antigen is exclusively expressed on the cell surface of cancer cells, either whole or as a fragment (e.g., MHC / peptides), and not synthesized or expressed on the surface of normal cells. In one embodiment, the CAR of the present invention comprises a CAR containing an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presenting peptide. Typically, endogenous protein-derived peptides fill the pocket of a major histocompatibility complex (MHC) class I molecule and are recognized by the T cell receptor (TCR) of CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy.TCR-like antibodies that target viral or tumor antigen-derived peptides in association with human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, for example, Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibodies can be identified by screening libraries such as human cFv phage display libraries. 【0319】 As used in the context of scFv, the term “mobile polypeptide linker” or “linker” refers to a peptide linker consisting of amino acids, such as glycine and / or serine residues, used alone or in combination to link variable heavy chain and variable light chain regions together. In one embodiment, the mobile polypeptide linker is a Gly / Ser linker comprising the amino acid sequence (Gly-Gly-Gly-Ser)n (SEQ ID NO: 22), where n is a positive integer greater than or equal to 1. For example, n=1, n=2, n=3; n=4, n=5, n=6, n=7, n=8, n=9, and n=10. In one embodiment, the mobile polypeptide linker comprises, but is not limited to, (Gly4Ser)4 (SEQ ID NO: 27) or (Gly4Ser)3 (SEQ ID NO: 28). In another embodiment, the linker comprises multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO: 29). Also included within the scope of this invention is the linker described in WO2012 / 138475, which is incorporated herein by reference. 【0320】 The 5' cap used here is an RNA cap (RNA 7-methylguanosine cap or RNA m 7 A 5' cap (also known as a G-cap) is a modified guanine nucleotide attached to the “front” or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of a terminal group that ligates to the first transcription nucleotide. Its presence is important for ribosome recognition and protection from RNases. Capping occurs cotranscribeally, linked to transcription and influencing each other. Immediately after transcription initiation, the 5' end of the synthesized mRNA is ligated by a cap synthesis complex bound to RNA polymerase. This enzyme complex catalyzes the chemical reactions necessary for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. Modifying the capping region can regulate mRNA functionality, such as stability or translation efficiency. 【0321】 In this context, "in vitro transcription RNA" refers to RNA synthesized in vitro, such as mRNA. Generally, in vitro transcription RNA is produced from an in vitro transcription vector. An in vitro transcription vector contains a template used for in vitro transcription RNA production. 【0322】 Herein, “poly(A)” refers to a sequence of adenosines linked by polyadenylation of mRNA. In one embodiment of the transient expression construct, polyA is 50 to 5000 (SEQ ID NO: 30), and poly(A) sequences of, for example, more than 64, for example more than 100, for example more than 300 or 400 may be chemically or enzymatically modified to regulate mRNA functionality such as localization, stability or translation efficiency. 【0323】 In this context, "polyadenylation" refers to the covalent bonding of a polyadenylyl portion or a modified variant to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at their 3' end. The 3' poly(A) tail is a long sequence (often hundreds) of adenine nucleotides added to premRNA by the action of the enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to the transcript containing a specific sequence, the polyadenylation signal. The poly(A) tail and the proteins that bind to it help protect mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, extrusion of mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA to RNA, but can also occur later in the cytoplasm. After transcription is stopped, the mRNA strand is cleaved by an endonuclease complex bound to RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. Once the mRNA is cleaved, an adenosine residue is added to the free 3' end of the cleavage site. 【0324】 As used herein, “transient” refers to the expression of a non-integrated transgene for a certain period of time, such as days or weeks, where the expression duration is shorter than the expression duration of the gene when it is integrated into the genome or contained within a stable plasmid replicon of the cell. In one embodiment, the CAR molecule is transiently expressed in cells, such as host cells, for a limited time or number of cell replications, such as less than 50 days (e.g., 40, 30, 25, 20, 15, 10, 5, 4, 3, 2, or less). In one embodiment, transient expression is performed using in vitro transcription RNA. 【0325】 Here, "stable" refers to the expression of a transgene for a longer period than transient expression. In one embodiment, the transgene is integrated into the genome of a cell, for example, a host cell, or contained within a stable plasmid replicon of the cell. In another embodiment, the transgene is integrated into the cell genome using a gene delivery vector, for example, a retroviral vector such as a lentiviral vector. 【0326】 Apheresis is a procedure in which whole blood is collected from an individual, separated into selective components, and the remainder is returned to circulation. Generally, there are two methods for separating blood components: centrifugation and non-centrifugation. Leukocyte apheresis results in the active selection and removal of the patient's white blood cells. 【0327】 As used herein, the terms “treatment” and “to treat” mean a reduction or improvement in the progression, severity and / or duration of a proliferative disorder or improvement in one or more symptoms of a proliferative disorder (e.g., one or more recognizable symptoms) resulting from the administration of one or more therapeutic agents (e.g., one or more therapeutic agents such as CAR in the present invention). In certain embodiments, the terms “treatment” and “to treat” mean an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which is not necessarily recognizable to the patient. In other embodiments, the terms “treatment” and “to treat” mean denial of the progression of a proliferative disorder, for example, by stabilization of a recognizable symptom, by physical means, by stabilization of a physical parameter, or by both. In other embodiments, the terms “treatment” and “to treat” mean a reduction or stabilization of tumor size or the number of cancerous cells. Treatment does not need to be 100%, and in some embodiment, a reduction or delay of at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of at least one symptom of the disease or disorder is sufficient to be considered within these terms. 【0328】 The term "signaling pathway" refers to the biochemical interactions between various signaling molecules that play a role in transmitting signals from one part of a cell to another. The term "cell surface receptor" refers to molecules and complexes of molecules that can receive and transmit signals across the cell membrane. 【0329】 The term “subject” is intended to include living organisms (e.g., mammals, e.g., humans) in which an immune response can be elicited. Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and their transgenic species. T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural exudate, splenic tissue, and tumors. 【0330】 The term “substantially purified” cells refer to cells that are essentially free from other cell types. Substantially purified cells also refer to cells that have been isolated from other cell types that normally bind to them in their natural state. In some cases, a population of substantially purified cells refers to a population of allogeneic cells. In other cases, the term simply refers to cells that have been isolated from cells that naturally bind to them in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro. 【0331】 In the present invention, “tumor antigen,” “hyperproliferative disorder antigen,” or “antigen associated with hyperproliferative disorder” refers to an antigen common to specific hyperproliferative disorders. In one embodiment, the tumor antigen is cancer-derived, including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, and pancreatic cancer. 【0332】 The terms “transfect,” “transform,” or “transfer” refer to the process by which exogenous nucleic acids are transmitted to or introduced into host cells. “Transfected,” “transformed,” or “transferred” cells are those that have been transfected, transformed, or transferred with exogenous nucleic acids. Cells include primary target cells and their offspring. 【0333】 The term "specifically binding" refers to an antibody or ligand that recognizes and binds to a homologous binding partner protein present in a sample, but that antibody or ligand does not substantially recognize or bind to other molecules in the sample. 【0334】 Scope: Throughout this specification, various aspects of the present invention may be provided in the form of a range. The use of a range is solely for convenience and brevity and should not be construed as a firm limitation of the scope of the present invention. Accordingly, a range description should be interpreted as specifically disclosing all possible subranges and the individual numerical values ​​within those ranges. For example, a range description such as 1-6 should be interpreted as specifically disclosing subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., and the individual numerical values ​​within those ranges, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity may include 95%, 96%, 97%, 98%, or 99% identity, and include subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the scope. 【0335】 Manufacturing and manufacturing method of immune effector cells A method for producing immunoeffector cells, which can be modified using CARs, for example, the CARs described herein, as well as reaction mixtures and compositions containing such cells, is provided herein. 【0336】 In one embodiment, the present invention relates to immunoeffector cells (e.g., T cells, NK cells) modified to express CAR, wherein the modified immunoeffector cells exhibit antitumor properties. Examples of antigens are cancer-associated antigens (i.e., tumor antigens) as described herein. In one embodiment, cells are transformed with CAR, and the CAR is expressed on the cell surface. In one embodiment, cells (e.g., T cells, NK cells) are transduced with a viral vector encoding CAR. In one embodiment, the viral vector is a retroviral vector. In one embodiment, the viral vector is a lentiviral vector. In one such embodiment, cells can stably express CAR. In another embodiment, cells (e.g., T cells, NK cells) are transfected with nucleic acids encoding CAR, such as mRNA, cDNA, or DNA. In one such embodiment, cells can transiently express CAR. 【0337】 Furthermore, the present invention provides CART compositions and their use in pharmaceuticals or methods for the treatment of cancer or any malignant tumor or autoimmune disease involving cells or tissues expressing the tumor antigens described herein. 【0338】 In one embodiment, the CAR of the present invention can be used to eradicate normal cells expressing the tumor antigen described herein, thereby making it applicable for use as cell conditioning before cell transplantation. 【0339】 Source of immune effector cells In one embodiment, cells, e.g., immune effector cells, e.g., a source of a population of immune effector cells, can be obtained from a subject before augmentation and genetic modification or other modifications. In one embodiment, the immune effector cells include T cells. In one embodiment, the T cells include CD4 T cells. In another embodiment, the T cells include CD8 T cells. In another embodiment, the T cells include regulatory T cells. In a further embodiment, the T cells include naive T cells. In one embodiment, the immune effector cells include hematopoietic stem cells (e.g., umbilical cord blood cells). In another embodiment, the immune effector cells include B cells. In a further embodiment, the immune effector cells include NK cells. In another embodiment, the immune effector cells include NKT cells. In another embodiment, the immune effector cells include Th-17 cells. In one embodiment, the immune effector cells do not have T cell receptors. In another embodiment, the immune effector cells have non-functional or substantially impaired T cell receptors. 【0340】 In one embodiment, a cell population, for example, a harvested cell population, includes, for example, T cells or a population of T cells at various stages of differentiation. The T cell differentiation stages, from lowest to highest differentiation, include naive T cells, stem central memory T cells, central memory T cells, effector memory T cells, and terminal effector T cells. After antigen exposure, naive T cells proliferate and differentiate into memory T cells, for example, stem central memory T cells and central memory T cells, which then differentiate into effector memory T cells. Upon receiving appropriate T cell receptors, co-stimuli, and inflammatory signals, memory T cells further differentiate into terminal effector T cells. See, for example, Restifo. Blood. 124.4(2014):476-77; and Joshi et al. J. Immunol. 180.3(2008):1309-15. 【0341】 Naive T cells (T NStem central memory T cells (T) are characterized by the following expression patterns of cell surface markers: CCR7+, CD62L+, CD45RO-, CD95-. SCM Central memory T cells (T) are characterized by the following expression patterns of cell surface markers: CCR7+, CD62L+, CD45RO-, CD95+. CM Effector memory T cells (T) are characterized by the following expression patterns of cell surface markers: CCR7+, CD62L+, CD45RO+, CD95+. EM Terminal effector T cells (T) are characterized by the following expression patterns of cell surface markers: CCR7-, CD62L-, CD45RO+, CD95+. Eff This condition is characterized by the following expression pattern of cell surface markers: CCR7-, CD62L-, CD45RO-, CD95+. See, for example, Gattinoni et al. Nat. Med. 17(2011):1290-7; and Flynn et al. Clin. Translat. Immunol. 3(2014):e20. 【0342】 In one embodiment, immune effector cells (e.g., a population of immune effector cells), such as T cells, are derived (e.g., differentiated) from stem cells, such as embryonic stem cells or pluripotent stem cells, such as induced pluripotent stem cells (iPSCs). In one embodiment, the cells are self or allogeneic. In one embodiment where the cells are allogeneic, for example, stem cell (e.g., iPSC)-derived cells are modified to reduce their alloreactivity. For example, cells can be modified to reduce alloreactivity by, for example, modification (e.g., destruction) of the T cell receptor. In one embodiment, site-specific nucleases can be used to destroy the T cell receptor after T cell differentiation, for example. In another example, cells, such as iPSC-derived T cells, can be produced from virus-specific T cells, which are less likely to cause graft-versus-host disease by recognizing pathogen-derived antigens. In yet another example, alloreactivity can be reduced, for example, minimized, by producing iPSCs from a common HLA haplotype so that they are histocompatibility with compatible, unrelated recipient subjects. In other examples, alloreactivity can be reduced, for example, minimized, by suppressing HLA expression through gene modification. For example, IPSC-derived T cells can be treated as described in Themeli et al. Nat. Biotechnol. 31.10(2013):928-35, which is incorporated herein by reference. In some examples, stem cell-derived immune effector cells, such as T cells, can be treated / produced using the method described in WO2014 / 165707, which is incorporated herein by reference. Further embodiments involving allogeneic cells are described herein, for example, in the “Allogeneic CAR Immunoeffector Cells” section herein. 【0343】 In one aspect of this specification, immune effector cells, such as T cells, are treated with Ficoll TMCells can be obtained from blood units collected from a subject using any number of techniques known to those skilled in the art, such as separation. In one embodiment, cells are obtained from the circulating blood of an individual by apheresis. Apheresis products generally include lymphocytes, including T cells, monocytes, granulocytes, B cells, and other nucleated leukocytes, as well as erythrocytes and platelets. In one embodiment, cells collected by apheresis may be washed to remove the plasma fraction, and optionally, the cells may be placed in a buffer or medium appropriate for subsequent processing steps. In one embodiment, the cells are washed with phosphate-buffered saline (PBS). In another embodiment, the washing solution may be calcium-deficient, magnesium-deficient, or most, if not all, divalent cations-deficient. 【0344】 The initial activation step in the absence of calcium can enhance activation. As will be readily apparent to those skilled in the art, the washing step can be achieved by methods known in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) in accordance with the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutions with or without buffers. Alternatively, undesirable components of the apheresis sample can be removed, and the cells can be directly resuspended in culture medium. 【0345】 In one aspect, T cells are lysed, red blood cells are lysed, and monocytes are lysed, for example, PERCOLL TM They are isolated from peripheral blood lymphocytes by depletion using gradient or countercurrent centrifugation. 【0346】 The methods described herein may include, for example, the selection of a subpopulation of T cells, such as a T regulatory cell depletion population, a specific immune effector cell, such as a subpopulation of T cells, which is a CD25+ depleted cell, using, for example, a negative selection technique described herein. In one embodiment, the T regulatory cell population contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% CD25+ cells. 【0347】 In one embodiment, T regulatory cells, such as CD25+ T cells, are removed from the population using an anti-CD25 antibody or its fragment or a CD25-binding ligand, such as IL-2. In one embodiment, the anti-CD25 antibody or its fragment or a CD25-binding ligand is conjugated to a substrate, such as beads, or coated to a substrate, such as beads, in another manner. In one embodiment, the anti-CD25 antibody or its fragment is conjugated to the substrate described herein. 【0348】 In one embodiment, T regulatory cells, for example, CD25+ T cells, are selected from a population of Miltenyi TM The CD25 is removed using a CD25 depletion reagent. In one embodiment, the cell-to-CD25 depletion agent ratio is 1e 7 Cells per 20 μL or 1 e 7 Cell pair 15 μL or 1 e 7 Cells per 10 μL or 1 e 7 Cells per 5 μL or 1 e 7 Cell pair 2.5 μL or 1e 7 The cell volume is 1.25 μL per cell. In one embodiment, for example, for T regulatory cells, e.g., CD25+ depletion, 500 million cells / ml is used. In a further embodiment, cell concentrations of 600 million, 700 million, 800 million or 900 million cells / ml are used. 【0349】 In one embodiment, the population of immune effector cells to be depleted is approximately 6 × 10⁶ 9 This includes CD25+ T cells. In another embodiment, the population of immune effector cells to be depleted is approximately 1 × 10⁶ 9 ~1 × 10 10This includes CD25+ T cells and any integer values ​​in between. In one embodiment, the resulting population of T regulatory depleted cells is 2 × 10⁻⁶ 9 The following T regulatory cells, e.g., CD25+ cells (e.g., 1 × 10⁻¹⁰ 9 , 5×10 8 , 1 x 10 8 , 5×10 7 , 1 x 10 7 Includes the following CD25+ cells. 【0350】 In one embodiment, T regulatory cells, such as CD25+ cells, are removed from a population using a CliniMAC system with a depletion tube set, such as tubing 162-01. In another embodiment, the CliniMAC system is driven in a depletion setting, such as DEPLETION2.1. 【0351】 While we do not wish to be bound by a specific theory, we are interested in the reduction of immune cell negative regulatory factor levels in subjects before apheresis or during the production of CAR-expressing cell products (e.g., unwanted immune cells, e.g., T REG A reduction in the number of cells significantly reduces the risk of target recurrence. For example, T REG Methods for depleting cells are known in this field. REG Methods for reducing cell populations include, but are not limited to, cyclophosphamide, anti-GITR antibodies (as described herein), CD25 depletion, mTOR inhibitors, and combinations thereof. 【0352】 In one embodiment, the manufacturing method involves T before the production of CAR-expressing cells. REG This includes a decrease in cells (e.g., depletion). For example, the manufacturing method involves CAR-expressing cells (e.g., T cells, NK cells) before the production of the product, for example, T REG For cell depletion, the procedure involves contacting a sample, such as an apheresis sample, with an anti-GITR antibody and / or an anti-CD25 antibody (or its fragment or CD25-binding ligand). 【0353】 While we do not wish to be bound by a specific theory, we do want to reduce the levels of negative regulatory factors in immune cells in subjects before apheresis or during the production of CAR-expressing cell products (e.g., unwanted immune cells, e.g., T REG A reduction in the number of cells can reduce the risk of relapse in the subject. In one embodiment, the subject undergoes T before cell harvesting for the production of CAR-expressing cell products. REG The cells are pretreated with one or more therapeutic agents that reduce the number of cells, thereby reducing the risk of target relapse in response to CAR-expressing cell treatment. In one embodiment, T REG Methods for reducing cells include, but are not limited to, the administration of one or more cyclophosphamide, anti-GITR antibodies, CD25 depletion, or a combination thereof to the target. In one embodiment, T REG Methods for reducing cells include, but are not limited to, the administration of one or more of the following to the target: cyclophosphamide, anti-GITR antibody, CD25 depletion, mTOR inhibitor, or a combination thereof. The administration of one or more of these may be before, during, or after CAR-expressing cell product injection. 【0354】 In one embodiment, the manufacturing method involves T before the production of CAR-expressing cells. REG This includes a decrease in the number of cells (e.g., depletion). For example, the manufacturing method involves CAR-expressing cells (e.g., T cells, NK cells) before product manufacturing, for example, T REG To deplete cells, the method involves contact between a sample, e.g., an apheresis sample, and an anti-GITR antibody and / or an anti-CD25 antibody (or its fragment or CD25-binding ligand). In one embodiment, the subject is pretreated with cyclophosphamide before cell harvesting for the production of a CAR-expressing cell product, thereby reducing the risk of subject relapse to CAR-expressing cell treatment (e.g., CTL019 treatment). In another embodiment, the subject is pretreated with an anti-GITR antibody before cell harvesting for the production of a CAR-expressing cell product (e.g., T cells or NK cells), thereby reducing the risk of subject relapse to CAR-expressing cell treatment. 【0355】 In one embodiment, before producing CAR-expressing cell (e.g., T cell, NK cell) products (e.g., CTL019 products), the method for producing CAR-expressing cells (e.g., T cells, NK cells) is modified, REG To deplete cells. In one embodiment, CD25 depletion is used before the production of CAR-expressing cell products (e.g., CTL019 products) from CAR-expressing cells (e.g., T cells, NK cells). REG It depletes the cells. 【0356】 In one embodiment, the population of cells to be removed is neither regulatory T cells nor tumor cells, but cells that otherwise negatively affect the growth and / or function of CART cells, such as cells expressing CD14, CD11b, CD33, CD15, or other markers that may be expressed by immunosuppressive cells. In one embodiment, such cells are intended to be removed simultaneously with or after the depletion of regulatory T cells and / or tumor cells, or in any other order. 【0357】 The methods described herein may include more than one selection step, e.g., more than one depletion step. Enrichment of T cell populations by negative selection is achieved, for example, by a combination of antibodies against surface markers unique to negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present in negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail may include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. 【0358】 The method described herein further includes removing cells from a population expressing tumor antigens, e.g., tumor antigens that do not contain CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14, or CD11b, thereby providing a population of T-regulatory depleted cells, e.g., CD25+ depleted and tumor antigen depleted cells, suitable for the expression of CARs, e.g., the CARs described herein. In one embodiment, tumor antigen-expressing cells are removed simultaneously with T-regulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody or a fragment thereof and an anti-tumor antigen antibody or a fragment thereof may be conjugated to the same substrate, e.g., beads, that can be used for cell removal, or the anti-CD25 antibody or a fragment thereof or an anti-tumor antigen antibody or a fragment thereof may be conjugated to separate beads, and a mixture thereof can be used for cell removal. In another embodiment, the removal of T-regulatory cells, e.g., CD25+ cells and tumor antigen-expressing cells is sequential and can be performed in any order, e.g. 【0359】 Also provided is a method for removing cells expressing one or more checkpoint inhibitors, such as the checkpoint inhibitors described herein, such as PD1+ cells, LAG3+ cells, and TIM3+ cells, from a population, thereby providing a population of T regulatory depletion, such as CD25+ depleted cells and checkpoint inhibitor depleted cells, such as PD1+, LAG3+, and / or TIM3+ depleted cells. Examples of checkpoint inhibitors include, for example, PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF (e.g., TGF beta). In one embodiment, checkpoint inhibitor-expressing cells are removed simultaneously with T-regulatory cells, e.g., CD25+ cells. For example, an anti-CD25 antibody or a fragment thereof and an anti-checkpoint inhibitor antibody or a fragment thereof may be conjugated to the same beads that can be used for cell removal, or the anti-CD25 antibody or a fragment thereof and an anti-checkpoint inhibitor antibody or a fragment thereof may be conjugated to separate beads, and the mixture thereof can be used for cell removal. In other embodiments, the removal of T regulatory cells, such as CD25+ cells, and the removal of checkpoint inhibitor-expressing cells are sequential and can be performed in any order, for example. 【0360】 The methods described herein may include a positive selection step. For example, T cells can be isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3x28) conjugate beads such as DynaBeads® M-450 CD3 / CD28 T for a time sufficient for positive selection of the desired T cells. In one embodiment, the time is approximately 30 minutes. In further embodiments, the time is in the range of 30 minutes to 36 hours or longer and any integer value in between. In further embodiments, the time is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. In yet another embodiment, the time is 10 to 24 hours, for example, 24 hours. Long incubation times can be used for T cell isolation in any situation where T cells are scarce compared to other cell types, such as the isolation of tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunodeficient individuals. Furthermore, the use of long incubation times may increase the CD8+ T cell capture efficiency. Therefore, by simply lengthening or shortening the time, T cells can bind to CD3 / CD28 beads and / or by increasing or decreasing the bead-to-T cell ratio (as further described here), a subpopulation of T cells can be preferentially selected at the start of culture or at any other point in time during processing. Furthermore, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, a subpopulation of T cells can be preferentially selected at the start of culture or at any other desired point in time. 【0361】 In one embodiment, a population of T cells expressing IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or one or more other suitable molecules, for example, other cytokines, can be selected. A method for screening cell expression can be determined, for example, by the method described in PCT Publication WO2013 / 126712. 【0362】 For the isolation of a desired cell population by positive or negative selection, the concentrations of cells and surfaces (e.g., beads or other particles) may vary. In some embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (e.g., increase the cell concentration) to ensure maximum contact between cells and beads. For example, in some embodiments, concentrations of 10 billion cells / ml, 9 billion / ml, 8 billion / ml, 7 billion / ml, 6 billion / ml, or 5 billion / ml are used. In some embodiments, a concentration of 1 billion cells / ml is used. In some embodiments, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml are used. In further embodiments, concentrations of 125 million or 150 million cells / ml can be used. 【0363】 The use of high concentrations can lead to increased cell yield, cell activation, and cell growth. Furthermore, the use of high cell concentrations allows for more efficient capture from samples containing many tumor cells, such as CD28-negative T cells, which may have weak expression of the target antigen of interest, or from samples containing many tumor cells (e.g., leukocytes, tumor tissue, etc.). Populations of such cells may have therapeutic value and are desirable to obtain. For example, the use of high cell concentrations allows for more efficient selection of CD8+ T cells, which typically have weak CD28 expression. 【0364】 In a related embodiment, it may be desirable to use low concentrations of cells. By considerably diluting the mixture of T cells and surface (e.g., bead-like particles), the interaction between particles and cells is minimized. This is selected from cells that express high levels of the desired antigen to bind to the particles. For example, CD4+ T cells express high levels of CD28 and are captured more efficiently than CD8+ T cells at dilution concentrations. In one embodiment, the concentration of cells used is 5 × 10⁻⁶ 6 In another embodiment, the concentration used is approximately 1 × 10⁻⁶ / ml. 5 / ml~1×10 6 / ml can be any integer value between / ml and / ml. 【0365】 In another embodiment, cells may be incubated with a rotor at various lengths and speeds at 2-10°C or room temperature. 【0366】 In one embodiment, a large number of immune effector cells in a population do not express diaglycerol kinase (DGK), for example, DGK-deficient. In another embodiment, a large number of immune effector cells in a population do not express Ikaros, for example, Ikaros-deficient. In yet another embodiment, a large number of immune effector cells in a population do not express both DGK and Ikaros, for example, both DGK and Ikaros-deficient. 【0367】 Stimulating T cells can be frozen again after the washing step. While we do not wish to be bound by theory, the freezing and subsequent thawing steps provide a more homogeneous product by removing granulocytes and, to some extent, monocytes from the cell population. After the washing step, which removes plasma and platelets, the cells may be suspended in a freezing solution. Many freezing solutions and parameters are known in the art and useful in this situation, but one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% 5% dextrose, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, then freezing the cells at -80°C at a rate of 1° / min and storing them in the vapor layer of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as uncontrolled freezing at -20°C or immediately in liquid nitrogen, can also be used. 【0368】 In one embodiment, cryopreserved cells are thawed, washed as described herein, and rapidly allowed to rise at room temperature for 1 hour before activation using the method of the present invention. 【0369】 Also intended in this invention is the collection of blood samples or apheresis products from subjects at a time prior to when the augmented cells described herein are needed. For example, a source of cells to be augmented is collected at any time as needed, desired cells such as T cells are isolated and frozen for subsequent use in immunoeffector cell therapy for any number of diseases or conditions that would benefit from such therapy, as described herein. In one embodiment, blood samples or apheresis are collected from generally healthy subjects. In one embodiment, blood samples or apheresis are collected from generally healthy subjects who are at risk of developing a disease but have not yet developed one, and the cells of interest are isolated and frozen for subsequent use. In one embodiment, T cells can be augmented, frozen, and used at a later time. In one embodiment, a sample is collected from a patient immediately after diagnosis of a specific disease described herein, but before any treatment. In a further embodiment, cells are isolated from blood samples or apheresis from subjects prior to treatment with any number of relevant treatment modalities, including but not limited to treatment with factors such as natalizumab, efalizumab, antivirals, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunodepletion agents such as CAMPATH, anti-CD3 antibodies, cytoxane, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228 and irradiation. 【0370】 In a further embodiment of the present invention, T cells are obtained directly from a patient after a procedure that leaves functional T cells in the subject. In this regard, it has been observed that after certain cancer procedures, particularly procedures that damage the immune system, the quality of the T cells obtained may be optimal or improved in terms of their ability to proliferate ex vivo for a period of time post-procedure, which is the period when the patient normally recovers from the procedure. Similarly, after ex vivo procedures using the methods described herein, these cells may be in a favorable state for transplantation and enhancement of in vivo proliferation. Therefore, in the present invention, blood cells, including T cells, dendritic cells, or other cells of the hematopoietic cell lineage, are intended to be harvested during this recovery period. Furthermore, in a further embodiment, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens may be used to create a state in the subject that is favorable for regrowth, recirculation, regeneration, and / or proliferation of specific cell types, particularly within a defined post-treatment timeframe. Examples of cell types include T cells, B cells, dendritic cells, and other cells of the immune system. 【0371】 In one embodiment, an immune effector CAR molecule, for example, cells expressing the CAR molecule described herein, is obtained from a subject receiving a low, immunoenhancing dose of an mTOR inhibitor. In another embodiment, a population of immune effector cells modified to express the CAR, for example, T cells, is collected in or from a subject, after a sufficient time or sufficient dose of a low, immunoenhancing dose of an mTOR inhibitor, such that the level of PD1-negative immune effector cells, for example, T cells, or the ratio of PD1-negative immune effector cells, for example, T cells to PD1-positive immune effector cells, for example, T cells, is increased at least transiently. 【0372】 In other embodiments, immunoeffector cells modified to express CARs, such as a population of T cells, may be treated ex vivo by contacting them with a certain amount of an mTOR inhibitor that increases the number of PD1-negative immunoeffector cells, such as T cells, or increases the ratio of PD1-negative immunoeffector cells, such as T cells, to PD1-positive immunoeffector cells, such as T cells. 【0373】 The application method utilizes culture medium conditions containing 5% or less, for example, 2%, of human AB serum, and can use known culture medium conditions and compositions, such as those described in Smith et al., “Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement” Clinical & Translational Immunology (2015) 4, e31; doi:10.1038 / cti.2014.31. 【0374】 In one embodiment, the method disclosed herein can utilize culture medium conditions including serum-free medium. In one embodiment, the serum-free medium is OpTmizer CTS (LifeTech), Immunocult XF (Stemcell Technologies), CellGro (CellGenix), TexMacs (Miltenyi), Stemline (Sigma), Xvivo15 (Lonza), PrimeXV (Irvine Scientific), or StemXVivo (RandD Systems). The serum-free medium may be supplemented with a serum substitute such as ICSR (Immune Cell Serum Replacement) from LifeTech. The level of the serum substitute (e.g., ICSR) is, for example, up to 5%, for example, about 1%, 2%, 3%, 4%, or 5%. In one embodiment, the T cell population is diglycerol kinase (DGK) deficient. DGK-deficient cells include cells that do not express DGK RNA or protein or have reduced or inhibited DGK activity. DGK-deficient cells can be produced by genetic methods, such as administering RNA interferants, e.g., siRNA, shRNA, or miRNA, to reduce or inhibit DGK expression. Alternatively, DGK-deficient cells can be produced by treatment with the DGK inhibitors described herein. 【0375】 In one embodiment, the T cell population is Ikaros-deficient. Ikaros-deficient cells include cells that do not express Ikaros RNA or protein, or whose Ikaros activity is reduced or inhibited, and Ikaros-deficient cells can be produced by genetic methods, for example, by administering RNA interferants for reducing or blocking Ikaros expression, such as siRNA, shRNA, or miRNA. Alternatively, Ikaros-deficient cells can be produced by treatment with an Ikaros inhibitor, such as lenalidomide. 【0376】 In one embodiment, the T cell population is DGK-deficient and Ikaros-deficient, for example, by not expressing DGK and Ikaros, or by having reduced or inhibited DGK and Ikaros activity. Such DGK and Ikaros-deficient cells can be produced by any of the methods described herein. 【0377】 In one embodiment, NK cells are obtained from a subject. In another embodiment, NK cells are an NK cell line, for example, the NK-92 cell line (Conkwest). 【0378】 In one embodiment, the method, for example, the manufacturing method, further comprises contact with IL-15 and / or IL-7, a cell population (e.g., a cell population depleted of T regulatory cells such as CD25+ T cells; or a cell population pre-contacted with an anti-CD25 antibody, its fragment, or a CD25-binding ligand). For example, the cell population (e.g., pre-contacted with an anti-CD25 antibody, its fragment, or a CD25-binding ligand) is augmented in the presence of IL-15 and / or IL-7. 【0379】 In one embodiment, the CAR-expressing cells described herein are contacted, for example, ex vivo with a composition containing interleukin-15 (IL-15) polypeptide, interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of both IL-15 polypeptide and IL-15Ra polypeptide, for example, hetIL-15, during the production of the CAR-expressing cells. In one embodiment, the CAR-expressing cells described herein are contacted, for example, ex vivo with a composition containing IL-15 polypeptide, during the production of the CAR-expressing cells. In one embodiment, the CAR-expressing cells described herein are contacted, for example, ex vivo with a composition containing both IL-15 polypeptide and IL-15Ra polypeptide, during the production of the CAR-expressing cells. In one embodiment, the CAR-expressing cells described herein are contacted, for example, ex vivo with a composition containing hetIL-15, during the production of the CAR-expressing cells. 【0380】 In one embodiment, the CAR-expressing cells described herein are contacted with a composition containing hetIL-15 during ex vivo augmentation. In another embodiment, the CAR-expressing cells described herein are contacted with a composition containing IL-15 polypeptide during ex vivo augmentation. In yet another embodiment, the CAR-expressing cells described herein are contacted with a composition containing both IL-15 polypeptide and IL-15Ra polypeptide during ex vivo augmentation. In yet another embodiment, the contact results in the survival and proliferation of a lymphocyte subpopulation, such as CD8+ T cells. 【0381】 Same type CAR In one embodiment described herein, the immune effector cells may be allogeneic immune effector cells, such as T cells or NK cells. For example, the cells may be allogeneic T cells, such as allogeneic T cells lacking the expression of functional T cell receptors (TCRs) and / or human leukocyte antigens (HLAs), such as HLA class I and / or HLA class II. 【0382】 T cells lacking a functional TCR can be modified, for example, to not express any functional TCR on their surface, to not express one or more subunits including a functional TCR (e.g., modified to not express (or show reduced expression of) TCR alpha, TCR beta, TCR gamma, TCR delta, TCR epsilon, and / or TCR zeta), or to produce a very small amount of functional TCR on their surface. Alternatively, T cells can express a substantially impaired TCR, for example, by expressing one or more mutations or cleavage forms of TCR subunits. The term “substantially impaired TCR” means that this TCR does not elicit a harmful immune response in the host. 【0383】 The T cells described herein may be modified, for example, to not express functional HLA on their surface. For example, the T cells described herein may be modified so that cell surface HLA expression, such as HLA class 1 and / or HLA class II, is downregulated. In one embodiment, downregulation of HLA may be achieved by reducing or eliminating beta-2 microglobulin (B2M) expression. In one embodiment, the T cells may lack a functional TCR and functional HLA, such as HLA class I and / or HLA class II. 【0384】 Modified T cells lacking functional TCR and / or HLA expression can be obtained by any suitable means, including knockout or knockdown of one or more subunits of the TCR or HLA. For example, T cells may undergo TCR and / or HLA knockdown using transcription activators such as siRNA, shRNA, clustered and regularly arranged short palindromic sequence repeats (CRISPR), effector nucleases (TALENs), or zinc finger endonucleases (ZFNs). 【0385】 In one embodiment, allogeneic cells may be cells that do not express or express at low levels the inhibitory molecule, for example, by the method described herein. For example, cells may be cells that do not express or express at low levels the inhibitory molecule, which may reduce the ability of CAR-expressing cells to initiate an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF (e.g., TGF beta). Inhibition by inhibitory molecules, for example at the DNA, RNA, or protein level, can optimize CAR-expressing cellular performance. In one embodiment, inhibitory nucleic acids, such as dsRNA, siRNA, or shRNA, or transcription activators such as clustered and regularly arranged short palindromic sequence repeats (CRISPR), effector nucleases (TALENs), or zinc finger endonucleases (ZFNs), can be used, as described herein. 【0386】 siRNAs and shRNAs for inhibiting TCR or HLA In one embodiment, TCR expression and / or HLA expression can be inhibited in cells, for example, T cells, using siRNA or shRNA that targets nucleic acids encoding TCR and / or HLA and / or the inhibitory molecules described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF beta). 【0387】 Expression systems for siRNA and shRNA, and examples of shRNAs, are described, for example, in paragraphs 649 and 650 of International Application WO2015 / 142675, filed March 13, 2015, which is incorporated herein by reference in its entirety. 【0388】 CRISPR to inhibit TCR or HLA The terms “CRISPR,” “CRISPR against TCR and / or HLA,” or “CRISPR for inhibiting TCR and / or HLA” used herein refer to a system containing a series of clustered, regularly arranged short palindromic sequence repeats or sets of such repeats. The term “Cas” used herein refers to CRISPR-related proteins. The “CRISPR / Cas” system refers to a CRISPR and Cas-derived system that can silence or mutate TCR and / or HLA genes and / or inhibitory molecules listed herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF beta) in cells, such as T cells. 【0389】 The CRISPR / Cas system and its use are described, for example, in paragraphs 651-658 of international application WO2015 / 142675 filed March 13, 2015, which is incorporated herein by reference in its entirety. 【0390】 TALEN for inhibiting TCR and / or HLA "TALEN" or "TALEN for HLA and / or TCR" or "TALEN for inhibiting HLA and / or TCR" refers to a transcription activator-like effector nuclease, which is an artificial nuclease that can be used to edit HLA and / or TCR genes and / or the inhibitory molecules listed herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF beta) in cells, e.g., T cells. 【0391】 TALEN and its use are described, for example, in paragraphs 659-665 of international application WO2015 / 142675 filed March 13, 2015, which are incorporated herein by reference in their entirety. 【0392】 Zinc finger nucleases for inhibiting HLA and / or TCR "ZFN" or "zinc finger nuclease" or "ZFN against HLA and / or TCR" or "ZFN for inhibiting HLA and / or TCR" refers to the inhibition of HLA and / or TCR genes and / or inhibitory molecules described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or) in cells, e.g., T cells). This refers to zinc finger nucleases, which are artificial nucleases that can be used for editing CEACAM-5, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF beta. 【0393】 The ZFN and its use are described, for example, in paragraphs 666-671 of international application WO2015 / 142675 filed March 13, 2015, which are incorporated herein by reference in their entirety. 【0394】 Telomerase expression Telomeres play a useful role in somatic cell persistence, and their length is maintained by telomerase (TERT). Telomere length in CLL cells may be extremely short (Roth et al., “Significantly shorter telomeres in T-cells of patients with ZAP-70+ / CD38 chronic lymphocytic leukaemia” British Journal of Haematology, 143, 383-386., August 28 2008), and even shorter in manufactured CAR-expressing cells, such as CART19 cells, which may limit their ability to proliferate after adoptive transfer to patients. Telomerase expression may help CAR-expressing cells from replication exhaustion. 【0395】 While we do not wish to be bound by any particular theory, in one embodiment, therapeutic T cells have short-term persistence in the patient due to shortened telomeres in the T cells, and therefore transfection with telomerase genes may lengthen the telomeres of T cells and improve T cell persistence in the patient. See Carl June, “Adoptive T cell therapy for cancer in the clinic”, Journal of Clinical Investigation, 117:1466-1476 (2007). Therefore, in one embodiment, immune effector cells, e.g., T cells, ectopically express telomerase subunits, e.g., catalytic subunits of telomerase, e.g., TERT, e.g., hTERT. In one embodiment, the present invention provides a method for producing CAR-expressing cells, comprising contacting cells with a nucleic acid encoding a telomerase subunit, e.g., catalytic subunit of telomerase, e.g., TERT, e.g., hTERT. The cells may be contacted simultaneously with, or after, contact with this nucleic acid and the construct encoding the CAR. 【0396】 Telomerase expression can be stable (e.g., the nucleic acid can be integrated into the cell's genome) or transient (e.g., the nucleic acid is not integrated, and expression decreases for a certain period, e.g., after a few days). Stable expression can be achieved by transfecting or transducing cells with DNA encoding a telomerase subunit and a selectable marker, and then selecting a stable construct. Separately or in combination with this, stable expression can be achieved by site-directed recombination, for example, using Cre / Lox or FLP / FRT systems. 【0397】 Transient expression may involve transfection or transduction with nucleic acids, such as DNA or RNA like mRNA. In one embodiment, transient mRNA transfection avoids the gene instability sometimes associated with stable transfection in TERT. Transient expression of exogenous telomerase activity is described, for example, in international application WO2014 / 130909, which is incorporated herein by whole reference. In one embodiment, mRNA-based transfection of telomerase subunits is commercialized by Moderna Therapeutics Messenger RNA Therapeutics. TM The method will be implemented according to the platform. For example, the method may be the method described in U.S. Patent Nos. 8710200, 8822663, 8680069, 8754062, 8664194, or 8680069. 【0398】 In one embodiment, hTERT has the amino acid sequence of GenBank Protein ID AAC51724.1 (Meyerson et al., “hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization” Cell Volume 90, Issue 4, 22 August 1997, Pages 785-795), which is provided herein as Sequence ID No. 5. 【0399】 In one embodiment, hTERT has a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 5. In one embodiment, hTERT has the sequence of SEQ ID NO: 5. In one embodiment, hTERT includes deletions (e.g., not exceeding 5, 10, 15, 20, or 30 amino acids) at the N-terminus, C-terminus, or both. In one embodiment, hTERT includes a transgenic amino acid sequence (e.g., not exceeding 5, 10, 15, 20, or 30 amino acids) at the N-terminus, C-terminus, or both. 【0400】 In one embodiment, hTERT has the nucleic acid sequence of GenBank Accession No. AF018167 (Meyerson et al., “hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization” Cell Volume 90, Issue 4, 22 August 1997, Pages 785-795), which is provided herein as Sequence ID No. 8. 【0401】 In one embodiment, hTERT is encoded by a nucleic acid having a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of sequence number 8. In one embodiment, hTERT is encoded by the nucleic acid of sequence number 8. 【0402】 RNA transfection Disclosed herein is a method for producing an in vitro transcribed RNA CAR. The method described herein may involve the introduction of a CAR-encoding RNA construct that can be directly transfected into cells. The method for producing mRNA for use in transfection involves in vitro transcription (IVT) of a specially designed primer template, followed by poly-A addition, to produce a construct generally 50 to 2000 nucleotides long, comprising 3' and 5' untranslated sequences ("UTR"), a 5' cap and / or an intra-sequence ribosome entry site (IRES), the nucleic acid to be expressed, and a poly-A tail (e.g., SEQ ID NO: 30). The RNA thus produced can be efficiently transfected in a variety of cells. In one embodiment, the template includes a sequence for the CAR. RNA CARs and methods using the same are described, for example, in paragraphs 553-570 of International Application WO2015 / 142675 of March 13, 2015, which are incorporated herein by reference in their entirety. 【0403】 Immune effector cells may contain CARs encoded by messenger RNA (mRNA). In one embodiment, mRNA encoding the CARs described herein is introduced into immune effector cells produced, for example, by the method described herein, for the purpose of producing CAR-expressing cells. 【0404】 In one embodiment, an in vitro transcription RNA CAR can be introduced into cells in the form of transient transfection. RNA is produced by in vitro transcription using a polymerase chain reaction (PCR) production template. Desired DNA from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The DNA source may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other suitable source of DNA. The desired template for in vitro transcription is the CAR described herein. For example, a template for an RNA CAR includes an extracellular region containing a single-strand variable domain of an antibody against a tumor-associated antigen described herein, a hinge region (e.g., the hinge region described herein), a transmembrane domain (e.g., the transmembrane domain of CD8a described herein), and an intracellular signaling domain, for example, the CD3-zeta signaling domain and the 4-1BB signaling domain, or a cytoplasmic region containing the intracellular signaling domain described herein. 【0405】 In one embodiment, the DNA for use in PCR includes an open reading frame. The DNA may be derived from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the nucleic acid may include some or all of the 5' and / or 3' untranslated regions (UTRs). The nucleic acid may include exons and introns. In one embodiment, the DNA for use in PCR is a human nucleic acid sequence. In another embodiment, the DNA for use in PCR is a human nucleic acid sequence including the 5' and 3' UTRs. The DNA may be an artificial DNA sequence that is not normally expressed in naturally occurring organisms. An example of an artificial DNA sequence is one that includes portions of genes ligated together to form an open reading frame encoding a fusion protein. The portions of DNA to be ligated may be from a single organism or from more than one organism. 【0406】 PCR is used to produce templates for in vitro transcription of mRNA to be used for transfection. Methods for performing PCR are well known in the art. Primers used for PCR are designed to have regions that are substantially complementary to the DNA region used as the template in PCR. Here, "substantially complementary" means a nucleotide sequence in which most or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary or inappropriate. Substantially complementary sequences can anneal or hybridize with the intended DNA target under the annealing conditions used in PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify a portion of nucleic acid (open reading frame) that is normally transcribed in cells, including the 5' and 3' UTRs. Primers can also be designed to amplify a portion of nucleic acid that codes for a specific domain of interest. In one embodiment, primers are designed to amplify the coding region of human cDNA, including all or part of the 5' and 3' UTRs. Primers useful for PCR can be produced by synthetic methods well known in the art. A “forward primer” is a primer that contains a region of nucleotides that are substantially complementary to the nucleotides of the DNA template upstream of the DNA sequence to be amplified. “Upstream” here refers to the 5' end of the DNA sequence to be amplified relative to the coding strand. A “reverse primer” is a primer that contains a region of nucleotides that are substantially complementary to the double-stranded DNA template downstream of the DNA sequence to be amplified. “Downstream” here refers to the 3' end of the DNA sequence to be amplified relative to the coding strand. 【0407】 Any DNA polymerase useful in PCR can be used in the method disclosed herein. Reagents and polymerases are commercially available from numerous suppliers. 【0408】 Chemical structures that have the ability to enhance stability and / or translation efficiency can also be used. In one embodiment, the RNA has 5' and 3' UTRs. In one embodiment, the 5' UTR is 1 to 3000 nucleotides long. The lengths of the 5' and 3' UTR sequences to be added to the coding region vary by various methods, including, but not limited to, the design of PCR primers that anneal to various regions of the UTR. Using this technique, those skilled in the art can modify the 5' and 3' UTR lengths necessary to achieve optimal post-transfection translation efficiency of the transcription RNA. 【0409】 The 5' and 3' UTRs may be naturally occurring, endogenous 5' and 3' UTRs of the nucleic acid of interest. Alternatively, non-endogenous UTR sequences can be added to the nucleic acid of interest by incorporating the UTR sequences into forward and reverse primers or by any other modification of the template. The use of non-endogenous UTR sequences in the nucleic acid of interest may be useful for modifying RNA stability and / or translation efficiency. For example, AU-rich elements in the 3' UTR sequence are known to reduce mRNA stability. Therefore, the 3' UTR can be selected or designed to increase the stability of the transcription RNA, based on the UTR properties well known in this art. 【0410】 In one embodiment, the 5' UTR may contain a Kozak sequence of an endogenous nucleic acid. Alternatively, when a non-endogenous 5' UTR is added to the nucleic acid of interest by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. While the Kozak sequence may increase the translation efficiency of some RNA transcripts, it does not appear to be necessary for efficient translation of all RNAs. The demand for Kozak sequences for many mRNAs is well known in this field. In another embodiment, the 5' UTR may be the 5' UTR of an RNA virus whose RNA genome is stable in cells. In yet another embodiment, various nucleotide analogs may be used in the 3' or 5' UTR to inhibit exonuclease degradation of mRNA. 【0411】 To enable RNA synthesis from a DNA template without the need for gene cloning, a transcription promoter should be added to the DNA template upstream of the sequence to be transcribed. When a sequence functioning as an RNA polymerase promoter is added to the 5' end of a forward primer, the RNA polymerase promoter begins to be incorporated into the PCR product upstream of the open reading frame to be transcribed. In one embodiment, the promoter is the T7 polymerase promoter, as described elsewhere in this specification. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences of the T7, T3, and SP6 promoters are known in the art. 【0412】 In one embodiment, mRNA has caps at both the 5' end and the 3' poly(A) tail, which determine ribosome binding, translation initiation, and stability of mRNA in cells. In circular DNA templates, such as plasmid DNA, RNA polymerase produces long-chain products unsuitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR end results in mRNA that, even after post-transcriptional polyadenylation, is classified as normal size and not effective for eukaryotic transfection. 【0413】 In a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)). 【0414】 The conventional method for incorporating polyA / T stretches into DNA templates is molecular cloning. However, integration of polyA / T sequences into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other abnormalities. This makes cloning methods not only cumbersome and time-consuming but also often unreliable. This is why a method that allows for the construction of DNA templates with polyA / T 3' stretches without cloning is highly desirable. 【0415】 The poly(A) tail of the transcription DNA template can be produced during PCR by using a poly(T) tail-containing reverse primer, such as a 100T tail (SEQ ID NO: 31) (sizes can range from 50 to 5000T (SEQ ID NO: 32)), or after PCR by any other method including, but not limited to, DNA ligation or in vitro recombination. The poly(A) tail also provides stability to the RNA and reduces degradation. Generally, the length of the poly(A) tail positively correlates with the stability of the transcription RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines (e.g., SEQ ID NO: 33). 【0416】 The poly(A) tail of RNA can be further extended in vitro after transcription using a poly(A) polymerase such as E. coli poly(A) polymerase (E-PAP). In one embodiment, extending the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides (SEQ ID NO: 34) approximately doubles the RNA translation efficiency. Furthermore, the attachment of various chemical groups to the 3' end can increase mRNA stability. Such attachments may include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be integrated into the poly(A) tail using a poly(A) polymerase. ATP analogs can further increase RNA stability. 【0417】 The 5' cap also provides stability to the RNA molecule. In one embodiment, RNA produced by the method disclosed herein includes a 5' cap. The 5' cap is provided using a method known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)). 【0418】 RNA produced by the methods disclosed herein may also include intra-sequence ribosome entry sites (IRES) sequences. IRES sequences are sequences of any virus, chromosomal, or artificially designed nature that initiate cap-independent ribosome binding to mRNA and promote translation initiation. Any solute suitable for cellular electroporation may be included, which may contain factors that promote cellular permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants. 【0419】 RNA can be introduced into target cells by any number of different methods, including but not limited to electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), ECM 830 (BTX) (Harvard Instruments, Boston, Mass.), or using particulate matter gun particle delivery systems such as lipofection, polymer encapsulation, peptide-mediated transfection, or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection, etc., using commercially available methods (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)). 【0420】 Activation and / or increase of immune effector cells In one embodiment, the present invention provides a method for increasing a population of immune effector cells by contacting a population of immune effector cells with a nucleic acid encoding a CAR (where the CAR targets a congener antigen molecule) under conditions suitable for CAR expression, e.g., transient expression, and culturing the immune effector cell population in the presence of a ligand, e.g., a congener antigen molecule. In one embodiment, the nucleic acid is RNA, e.g., in vitro transcription RNA. In another embodiment, the congener antigen molecule is a cancer-associated antigen molecule. 【0421】 The method provided herein increases a population of immune effector cells by bringing them into contact with a surface bound to a congener antigen molecule that stimulates CARs on the surface of the immune effector cells. In one embodiment, the congener antigen molecule may be bound to the surface in solution. In one embodiment, the congener antigen molecule providing the stimulating signal is bound to the cell surface. In one embodiment, the congener antigen molecule may be in solution. In one embodiment, the congener antigen molecule is in a soluble form and can then be crosslinked to the surface. 【0422】 In one embodiment, a congener antigen molecule binds to a substrate. In one embodiment, the substrate is a solid support. In one embodiment, the substrate is selected from a microtiter plate (e.g., an ELISA plate), a membrane (e.g., a nitrocellulose membrane, a PVDF membrane, a nylon membrane, an acetic acid derivative, and combinations thereof), a fiber matrix, a Sepharose matrix, a sugar matrix, a plastic chip, a glass chip, or any type of bead (e.g., Luminex beads, DynaBeads, magnetic beads, flow cytometry beads, and combinations thereof). In one embodiment, the substrate is an ELISA plate. In another embodiment, the substrate is a bead, for example, DynaBeads. 【0423】 Particle-to-cell ratios of integer values ​​between 1:500 and 500:1 can be used to stimulate immune effector cells, such as T cells or other target cells. As will be readily apparent to those skilled in the art, the particle-to-cell ratio may depend on the particle size relative to the target cells. For example, smaller beads can bind to fewer cells, while larger beads can bind to many. In one embodiment, the cell-to-particle ratio is in the range of integer values ​​between 1:100 and 100:1, and in a further embodiment, ratios including integer values ​​between 1:9 and 9:1 can also be used for T cell stimulation. The ratio of congeneral antigen-binding particles to immune effector cells, e.g., T cells, that induce T cell stimulation can vary as described above, however, some preferred values ​​include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with an example of a suitable ratio being at least 1:1 particle:T cell. In some embodiments, particle-to-cell ratios of 1:1 or less are used. In further embodiments, the particle-to-cell ratio may vary from day to day of stimulation. For example, in one embodiment, the particle-to-cell ratio is 1:1 to 10:1 on day one, with additional particles added daily or every other day for up to 10 days thereafter, resulting in a final ratio of 1:1 to 1:10 (based on the number of cells on the day of addition). In a particular embodiment, the particle-to-cell ratio is 1:1 on day one of stimulation and adjusted to 1:5 on days three and five of stimulation. In one embodiment, particles are added daily or every other day based on a final ratio of 1:1 on day one and 1:5 on days three and five of stimulation. In one embodiment, the particle-to-cell ratio is 2:1 on day one of stimulation and adjusted to 1:10 on days three and five of stimulation. In one embodiment, particles are added daily or every other day based on a final ratio of 1:1 on day one and 1:10 on days three and five of stimulation. In a particular embodiment, a suitable particle-to-cell ratio is 1:3. 【0424】 In a further embodiment, cells such as T cells are combined with homogeneous antigen molecule-coated beads, the beads and cells are then separated, and the cells are subsequently cultured. In another embodiment, the homogeneous antigen molecule-coated beads and cells are not separated before culturing, but are cultured together. In a further embodiment, the beads and cells are first enriched by the application of a force such as magnetic force to increase the ligation of cell surface markers, thereby inducing cell stimulation. 【0425】 As an example, cell surface proteins can be ligated by bringing paramagnetic beads (3x28 beads) to which congener antigen molecules are bound into contact with T cells. In one embodiment, cells (e.g., 10 4 ~10 9T cells and beads (e.g., DynaBeads® M-450 tosyl-activated paramagnetic beads, 1:3 ratio) are combined in a buffer, e.g., PBS (free of divalent cations such as calcium and magnesium). Other cell concentrations are intended. For example, target cells may be extremely rare in the sample, constituting only 0.01% of the sample, or the entire sample (i.e., 100%) may contain the target cells of interest. Thus, any cell number can be used in this invention. In some embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (e.g., increase the cell concentration) to ensure maximum contact between cells and beads. For example, in some embodiments, concentrations of approximately 10 billion cells / ml, 9 billion / ml, 8 billion / ml, 7 billion / ml, 6 billion / ml, 5 billion / ml, or 2 billion cells / ml are used. In some embodiments, more than 100 million cells / ml are used. In a further embodiment, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / ml are used. In one embodiment, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml are used. In a further embodiment, concentrations of 125 million or 150 million cells / ml can be used. The use of high concentrations may result in increased cell yield, cell activation, and cell growth. Furthermore, the use of high cell concentrations allows for efficient capture of cells that may weakly express CAR, for example, transiently. 【0426】 In one embodiment, cells transduced with a CAR, for example, the nucleic acid encoding the CAR described herein, for example, the CD19 CAR described herein, are enlarged, for example, by the method described herein. In one embodiment, cells are grown by culturing for a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 40 days (e.g., day 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, or 40). In one embodiment, cells are grown for a period of 4 to 9 days. In one embodiment, cells are enlarged for a period of 8 days or less, for example, 7, 6, or 5 days. In one embodiment, cells are enlarged by 5 days of culture, and the resulting cells are more potent than the same cells enlarged by 9 days of culture under the same culture conditions. The potency can be determined, for example, by various T cell functions, such as proliferation, target cell death, cytokine production, activatio...

Claims

[Claim 1] (a) A population of immune effector cells in which multiple immune effector cells simultaneously or sequentially express a first CAR molecule and a second CAR molecule, wherein the first CAR molecule is transiently expressed and the second CAR molecule is stably expressed, and the first CAR molecule and the second CAR molecule are directed to the same or different cancer-associated antigens; and (b) A ligand for the primary CAR binding domain of a primary CAR molecule selected from a congeneral antigen molecule or an anti-antigen idiotype antibody molecule. An immunoeffector cell preparation or reaction mixture containing the above. [Claim 2] (a) A population of immune effector cells comprising a nucleic acid encoding a first CAR molecule and a nucleic acid encoding a second CAR molecule, wherein the nucleic acid encoding the first CAR molecule is not integrated into the cell genome, further comprising an in vitro transcription RNA or synthetic RNA, and the nucleic acid encoding the second CAR molecule is integrated into the cell genome, further comprising a population of immune effector cells in which a plurality of immune effector cells simultaneously or sequentially express the first CAR and the second CAR molecules, wherein the first CAR molecule is transiently expressed and the second CAR molecule is stably expressed, and the first CAR and second CAR molecules are directed to the same or different cancer-associated antigens; and (b) Ligand of a primary CAR molecule selected from a congener antigen molecule or an anti-antigen idiotype antibody molecule A reaction mixture containing the following: [Claim 3] Cancer-related antigens include CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1 (CLECL1), CD33, EGFRvIII, GD2, GD3, BCMA, and Tn Ag, PSMA, ROR1, FLT3, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesoserine, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, Folate Receptor Alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, FAP, IGF-I Receptor, CAIX, LMP2, gp100, bcr-ab l, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variant, prostain, survivor and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras variant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG(TMPRSS2) ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, regmine, HPV E6, E7, intestinal carboxylesterase, mutA population of immunoeffector cells according to claim 1, selected from hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, or IGLL1, or a reaction mixture according to claim 2. [Claim 4] A population of immunoeffector cells according to claim 1 or a reaction mixture according to claim 2, wherein the first CAR and second CAR molecules are independently selected from CD19 CAR, BCMA CAR, CD33 CAR, CLL-1 CAR, EGFRvIII CAR, GFR alpha 4 CAR, ROR1 CAR, CD20 CAR, CD22 CAR, CD123 CAR, CD10 CAR, CD34 CAR, FLT-3 CAR, CD79b CAR, CD179b CAR, mesoserine CAR, or CD79a CAR. [Claim 5] A population of immunoeffector cells according to claim 1 or a reaction mixture according to claim 2, wherein the first CAR and second CAR molecules are a mesoserine CAR and a CD19 CAR molecule, respectively. [Claim 6] A population of immunoeffector cells according to claim 1 or a reaction mixture according to claim 2, wherein the first and / or second CAR-expressing immunoeffector cells include CD19 CAR, BCMA CAR, CD33 CAR, CLL-1 CAR, EGFRvIII CAR, GFR alpha 4 CAR, ROR1 CAR, CD19 CAR, CD20 CAR, CD22 CAR, CD123 CAR, CD10 CAR, CD34 CAR, FLT-3 CAR, CD79b CAR, CD179b CAR, mesoserine CAR, or CD79a CAR. [Claim 7] A population or reaction mixture of immunoeffector cells according to claim 6, wherein the first and / or second CAR-expressing immunoeffector cells contain CD19 CAR. [Claim 8] (a) CD19 CAR contains the amino acid sequence described in any of SEQ ID NOs: 39-51, 64-76, 78-89, or 107-119. (b) The CD19 CAR contains the amino acid sequence of the antigen-binding domain of CTL019 of SEQ ID NO: 51, or (c) CD19 CAR contains the amino acid sequence of SEQ ID NO: 89 (including the signal sequence) or CTL019 which does not have a signal sequence, or (d) CD19 CAR is encoded by a nucleotide sequence containing one of sequence numbers 52-63 or 90-102. A population or reaction mixture of immunoeffector cells according to claim 7. [Claim 9] In an immune effector cell population, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of immune effector cells express primary and / or secondary CAR molecules on their cell surface. A population of immunoeffector cells according to claim 1 or any of claims 3 to 8, or a reaction mixture according to any of claims 2 to 8. [Claim 10] A population of immune effector cells according to claim 1 or any of claims 3 to 9, or a reaction mixture according to any of claims 2 to 9, wherein the immune effector cells are obtained from human cancer patients. [Claim 11] (a) Cancer is B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell promyelocytic leukemia, blastocyte plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, generalized large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large It is a hematological cancer selected from one or more of the following: follicular lymphoma, malignant lymphoproliferative state, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, spinal dysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, and Waldenström hypergammaglobulinemia. (b) The cancer is a hematological cancer selected from leukemia or lymphoma, (c) The cancer is a CD25-expressing cancer. A population or reaction mixture of immunoeffector cells according to claim 10. [Claim 12] (a) The leukemia is chronic lymphoblastic leukemia (CLL) or acute lymphoblastic leukemia (ALL), (b) The lymphoma is mantle cell lymphoma (MCL), or (c) CD25-expressing cancer is chronic lymphocytic leukemia (CLL), A population or reaction mixture of immunoeffector cells according to claim 11. [Claim 13] The group of immune effector cells is the group of T regulatory depletion cells, (a) The population of T regulatory depleted cells contains less than 30% CD25+ cells, and / or (b) The population of T regulatory depleted cells contains less than 30% leukemia cells, A population of immunoeffector cells according to claim 1 or any of claims 3 to 12, or a reaction mixture according to any of claims 2 to 12. [Claim 14] A group of immune effector cells (a) A population of tumor antigen-depleted cells, and / or (b) Population of checkpoint inhibitor-depleted cells A population of immunoeffector cells according to claim 1 or any of claims 3 to 13, or a reaction mixture according to any of claims 2 to 13, comprising the above. [Claim 15] A population of immunoeffector cells or a reaction mixture according to claim 14, wherein the checkpoint inhibitor is one or more of PD-1, LAG-3, and TIM-3.

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