RU2828787C2 - Methods for producing allogenic t-cells with car - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed are: a cell growth medium for T-cell propagation, comprising: first stimulator and second cell proliferation stimulator, each independently selected from a group consisting of IL-7 and IL-15, and an extracellular modulator of cell metabolism, which is extracellular potassium, in a concentration of not less than about 10 mM, where the first stimulant and the second stimulator are present at a concentration ratio of about 250:1 to about 10:1 IU/ml, and methods of producing a population of T-cells in vitro. Also disclosed is a cell growth medium for T-cell propagation, comprising: a first stimulator and a second cell proliferation stimulator, each of which is independently selected from the group consisting of IL-7 and IL-15; and optionally an extracellular modulator of cell metabolism, which is extracellular potassium, in a concentration of not less than about 10 mM, where the first stimulant and the second stimulator are present at a concentration ratio of about 250:1 to about 10:1, and wherein the concentration is in IU/ml.

EFFECT: invention can be used to obtain a population of T-cells.

31 cl, 5 dwg, 2 tbl, 4 ex

Description

CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/839,449, filed April 26, 2019, the contents of which are incorporated herein by reference in their entirety.

AREA OF TECHNOLOGY

[0002] The present invention relates to cell culture media and methods for producing engineered immune cells, including cells containing chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs), as well as methods for treating cancer in a patient using them.

SEQUENCE LIST

[0003] This application contains a sequence listing that was submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on April 17, 2020 is named AT-025_02WO_ST25.txt and is 15,405 bytes in size.

LEVEL OF TECHNOLOGY

[0004] Adoptive transfer of immune cells genetically modified to recognize antigens associated with malignancy is a promising new approach to cancer treatment (see, e.g., Brenner et al., Current Opinion in Immunology, 22(2): 251–257 (2010); Rosenberg et al., Nature Reviews Cancer, 8(4): 299–308 (2008)). Immune cells can be genetically modified to express chimeric antigen receptors (CARs) (see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2): 720–724 (1993), and Sadelain et al., Curr. Opin. Immunol, 21(2): 215–223 (2009)). Immune cells containing CARs, such as CAR T cells (CAR-T), are engineered to have antigen specificity while maintaining or enhancing their ability to recognize and kill a target cell. Effective immune cell growth media and methods for using them may be particularly useful for producing engineered immune cells such as CAR-T. Provided herein are cell growth media and methods for producing them that address this need.

BRIEF DESCRIPTION

[0005] This document describes improved immune cell culture media and methods of use. For example, this document describes media that are particularly suitable for expanding T cells that can be used to produce cells useful in adoptive cell therapies, including chimeric antigen receptor therapies (e.g., CAR T cell therapies).

[0006] In one aspect, the present invention provides a cell growth medium for expanding T cells, comprising: a first stimulator and a second stimulator of cell proliferation, each independently selected from the group consisting of IL-4, IL-7, IL-10, IL-12, and IL-15, and wherein the first stimulator and the second stimulator are present at a concentration ratio of from about 1000:1 to about 4:1.

[0007] In one aspect, the present invention provides a cell culture medium for expansion of T cells comprising an extracellular modulator of cellular metabolism, which is extracellular potassium, at a concentration of from about 4 mM to about 40 mM.

[0008] In one aspect, the present invention provides a cell growth medium for expanding T cells, comprising a first stimulator and a second stimulator of cell proliferation, each independently selected from the group consisting of IL-4, IL-7, IL-10, IL-12, and IL-15; and an extracellular modulator of cellular metabolism, which is extracellular potassium, at a concentration of from about 4 mM to about 40 mM; and wherein the first stimulator and the second stimulator are present at a concentration ratio of from about 1000:1 to about 4:1.

[0009] In some embodiments, the first stimulator of cell proliferation is IL-7.

[0010] In some embodiments, the second stimulator of cell proliferation is IL-15.

[0011] In some embodiments, the first stimulant and the second stimulant are present at a concentration ratio of from about 500:1 to about 10:1, from about 250:1 to about 10:1, from about 200:1 to about 10:1, from about 150:1 to about 10:1, from about 100:1 to about 10:1, from about 500:1 to about 50:1, from about 250:1 to about 50:1, from about 200:1 to about 50:1, from about 150:1 to about 50:1, from about 100:1 to about 50:1, from about 500:1 to about 75:1, from about 250:1 to about 75:1, from about 200:1 to about 75:1, from about 150:1 to about 75:1, from about 100:1 to about 75:1, from about 10:1 to about 4:1, from about 8:1 to about 4:1, or from about 7:1 to about 6:1.

[0012] In some embodiments, the first stimulant and the second stimulant are present at a concentration ratio of about 150:1, about 140:1, about 130:1, about 120:1, about 110:1, about 100:1, about 90:1, about 80:1, or about 70:1.

[0013] In some embodiments, the first stimulant is IL-7 present at a concentration of from about 100 IU/mL to about 5000 IU/mL; and the second stimulant is IL-15 present at a concentration of from about 1 IU/mL to about 100 IU/mL.

[0014] In some embodiments, IL-7 is present at a concentration of about 300 IU/mL to about 5000 IU/mL, and IL-15 is present at a concentration of about 25 IU/mL to about 50 IU/mL.

[0015] In some embodiments, IL-7 is present at a concentration of about 5000 IU/mL and IL-15 is present at a concentration of about 50 IU/mL.

[0016] In some embodiments, the first stimulant and the second stimulant are added to the cell culture simultaneously or sequentially.

[0017] In some embodiments, extracellular potassium is present at a concentration of from about 4 mM to about 40 mM; from about 10 mM to about 35 mM; from about 10 mM to about 25 mM; from about 20 mM to about 35 mM; from about 20 mM to about 25 mM; or from about 20 mM to about 30 mM.

[0018] In some embodiments, extracellular potassium is present at a concentration of about 20 mM or about 25 mM.

[0019] In some embodiments, extracellular potassium is present at a concentration of less than 40 mM and greater than 4 mM; less than 40 mM and greater than 10 mM; less than 40 mM and greater than 20 mM; or less than 40 mM and equal to or greater than 25 mM.

[0020] In some embodiments, the extracellular potassium is present as KCl.

[0021] In some embodiments, the resulting T cell population is enriched for T _CM and/or T _SCM cells.

[0022] In another aspect, the present invention provides a method of producing a population of T cells in vitro, comprising culturing a starting population of T cells with a cell growth medium comprising a first stimulator and a second stimulator of cell proliferation, each independently selected from the group consisting of IL-4, IL-7, IL-10, IL-12, and IL-15; and an extracellular modulator of cellular metabolism, which is extracellular potassium, at a concentration of from about 4 mM to about 40 mM; and wherein the first stimulator and the second stimulator are present at a concentration ratio of from about 1000:1 to about 4:1, and wherein the resulting population of T cells is enriched in T _CM and/or T _SCM cells.

[0023] In some embodiments, the cell growth medium is the cell growth medium described above.

[0024] In some embodiments, the resulting T cell population comprises at least about 30%, 35%, 40%, or 45% T _SCM cells.

[0025] In some embodiments, the resulting T cell population comprises at least about 40% T _SCM cells.

[0026] In some embodiments, the resulting T cell population is from about 100 times to about 1000 times larger than the starting T cell population, as measured over a period of about 7-16 days.

[0027] In some embodiments, the resulting T cell population is from about 100 times to about 1000 times larger than the starting T cell population, as measured over a period of about 10-14 days.

[0028] In some embodiments, the resulting T cell population is at least about 100 times or about 200 times larger than the starting T cell population.

[0029] In some embodiments, the resulting T cell population is enriched in about 100-fold to about 1000-fold more T _CM and/or T _SCM cells than the number of T _CM and/or T _SCM cells in the original T cell population, as measured over a period of about 7-16 days.

[0030] In some embodiments, the resulting T cell population is enriched in from about 100-fold to about 1000-fold more T _CM and/or T _SCM cells than the number of T _CM and/or T _SCM cells in the original T cell population, as measured over a period of about 10-14 days.

[0031] In some embodiments, the resulting T cell population is enriched in at least about 100-fold or at least about 200-fold more T _CM and/or T _SCM cells than the number of T _CM and/or T _SCM cells in the original T cell population.

[0032] In some embodiments, the initial T cell population is a population of engineered T cells.

[0033] In some embodiments, the initial population of T cells is a population of T cells expressing one or more chimeric antigen receptors.

[0034] In some embodiments, the T cells are allogeneic T cells.

[0035] In some embodiments, the T cells are autologous T cells.

[0036] In one aspect, the present invention provides a population of engineered immune cells, wherein said population comprises T cells expressing one or more chimeric antigen receptors (CAR T cells), and wherein said CAR T cells are produced using the cell growth medium described above.

[0037] In one aspect, the present invention provides a population of engineered immune cells, wherein said population comprises T cells expressing one or more chimeric antigen receptors (CAR T cells), and wherein said CAR T cells are obtained using the method described above.

[0038] In some embodiments, the CAR T cells comprise at least about 30%, 35%, 40%, or 45% T _SCM cells.

[0039] In some embodiments, the CAR T cells comprise at least about 40% T _SCM cells.

[0040] In one aspect, the present invention provides a pharmaceutical composition comprising a population of engineered immune cells as described above.

[0041] In one aspect, the present invention provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an engineered immune cell as described above or a pharmaceutical composition as described above.

[0042] In some embodiments, the disease or disorder is cancer.

[0043] In one aspect, the present invention provides an article of manufacture comprising an engineered immune cell as described above or a pharmaceutical composition as described above.

[0044] All embodiments presented herein are applicable to all aspects disclosed in the present invention.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0045] Fig. 1A shows a comparison of CAR T ⁺ phenotypes using methods including: sequential stimulation with IL-2; stimulation with IL-2 followed by stimulation with IL-7 + IL-15; and sequential stimulation with IL-7 + IL-15. The IL-7 + IL-15-based methods provide for an expansion of CAR T _SCM T cells.

[0046] Fig. 1B shows the ability of CD19-specific CAR T cells to release cytokines upon exposure to target cells, wherein the CAR T cells were generated using generation methods comprising sequential stimulation with IL-2; stimulation with IL-2 followed by stimulation with IL-7 + IL-15; and sequential stimulation with IL-7 + IL-15.

[0047] Figure 2A shows the effect of elevated extracellular potassium (40 mM, open bars) on increasing T _SCM cell numbers in IL-2- and IL-7+15 processes compared to normal (4 mM) potassium concentrations (filled bars).

[0048] Fig. 2B shows the effect of elevated extracellular potassium (40 mM, open bars) on the expansion capacity of allogeneic CAR T cells in the IL-2- and IL-7+15-containing regimens compared to normal (4 mM) potassium concentrations (4 mM, filled bars).

[0049] Figure 3 shows the effect of 25 mM and 10 mM extracellular potassium concentrations: these concentrations did not have a negative effect on human T cell expansion during the generation of allogeneic CAR T cells compared to higher concentrations.

[0050] Figure 4 shows that maximal preservation of T _SCM cells was achieved at 25 mM extracellular potassium.

[0051] Figure 5 shows that improved potency of Flt3-specific CAR T cells (Allo-819) was obtained using a combination of the IL-7+15-based method with the addition of 25 mM extracellular potassium.

DETAILED DESCRIPTION

[0052] This document describes cell culture media and methods particularly useful for culturing immune cells.

[0053] Adoptive cell therapy using engineered immune cells is a type of immunotherapy that has emerged as a particularly promising new approach to the treatment of cancer. The production and manufacture of engineered immune cells involves harvesting immune cells (e.g., T cells) from a subject and then expanding the cells in vitro. Successful in vitro expansion of immune cells (e.g., T cells) is characterized by both sufficient cell proliferation and the generation of the desired cell phenotypes.

[0054] For example, expansion of T cells can result in the formation of a mixture of T cells that includes subsets of naive T cells (T _N ), memory T cells (including stem cell-like memory T cells ( _TSCM ), central memory T cells ( _TCM ), and effector memory T cells ( _TCM )), and effector ( _TEFF ) T cells. Varying amounts of different T cell subsets can affect the therapeutic profile and efficacy of the resulting engineered T cells. In particular, methods for expanding T cells that provide increased numbers of T cells at early stages of differentiation are therapeutically desirable.

[0055] Accordingly, the cell culture media described herein may be particularly useful for expanding T cells that have an increased proportion of T _SCM and/or T _CM cells. T _SCM cells are the least differentiated type of memory T cell and may be particularly useful in adoptive T cell therapy to provide prolonged T cell proliferation in vivo following administration of engineered cells to a patient. Accordingly, the use of such cell culture media and immune cells (e.g., T cells) produced using such media may provide more effective adoptive cell transfer-based cell therapies, including the CAR T cell therapies described herein.

[0056] In particular, cell culture media containing combinations of various cell proliferation stimulators (e.g., T cell growth factor I and II) in combination with an extracellular modulator of cellular metabolism can result in desired cell proliferation and cell phenotypes.

[0057] In one aspect, the present invention provides a cell growth medium (e.g., a medium suitable for expansion of T cells) comprising:

the first stimulator of cell proliferation (eg, the first cytokine that stimulates T-cell proliferation);

a second stimulator of cell proliferation (eg, a second cytokine that stimulates T cell proliferation) and

extracellular modulator of cellular metabolism (eg, T-cell metabolism).

[0058] In particular, the combinations and concentrations of cytokine and metabolic modulators described herein provide novel T cell expansion environments that can result in increased efficacy of immune cell-based cell therapies, including genetically modified allogeneic cell therapies and autologous cell therapies. In one embodiment, using a combination of IL-7 and IL-15 stimulators together with increased extracellular potassium, improvements can be achieved in the production of desired allogeneic CAR T cell phenotypes (e.g., an increased population of TSCM cells) and/or autologous cell therapies compared to classical IL-2-based expansion conditions.

1. Immune cells

[0059] Cells suitable for culture using the media and methods described herein include immune cells.

[0060] Prior to in vitro manipulation or genetic modification (e.g., as described herein), cells for use in the methods described herein (e.g., immune cells) can be obtained from a subject. The cells can be obtained from a variety of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, immune cells derived from stem cells or iPSCs, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines that are available and known to those skilled in the art can be used. In some embodiments, the cells can be obtained from a healthy donor, from a patient diagnosed with cancer, or from a patient diagnosed with an infection. In some embodiments, the cells can be part of a mixed population of cells that have different phenotypic characteristics.

[0061] In some embodiments, the immune cells are obtained from a subject who will ultimately receive the engineered immune cells. In some embodiments, the immune cells are obtained from a donor that is an individual different from the subject who will receive the engineered immune cells.

[0062] In some embodiments, the immune cells comprise T cells. The T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, stem cell-derived T cells or iPSCs, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some specific embodiments, the T cells can be obtained from a volume of blood drawn from a subject using any number of techniques known in the art, such as FICOLL™ separation.

[0063] Cells can be obtained from the circulating blood of an individual via apheresis. The product obtained via apheresis typically comprises lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In certain specific embodiments, cells collected via apheresis can be washed to remove the plasma fraction and placed in appropriate buffer or media for subsequent processing.

[0064] PBMCs can be used directly for genetic modification by immune cells (such as CAR or TCR) using the methods described herein. In some specific embodiments, after PBMCs are isolated, T lymphocytes can be further isolated, and both cytotoxic and helper T lymphocytes can be sorted into naive, memory T cell, and effector T cell subsets either before or after genetic modification and/or expansion.

[0065] In certain specific embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes, such as by PERCOLL™ gradient centrifugation. Certain T cell subpopulations, such as CCR7+, CD95+, CD122, CD27+, CD69+, CD127+, CD28+, CD3+, CD4+, CD8+, CD25+, CD62L+, CD45RA+, and CD45RO+ T cells, can be further isolated using positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies directed to surface markers unique to the negatively selected cells. One method of use herein is cell sorting and/or selection using negative magnetic immunoadhesion or flow cytometry, which uses a cocktail of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the cocktail of monoclonal antibodies typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present invention.

[0066] In some embodiments, the T cell population is enriched for C4+ cells.

[0067] In some embodiments, the T cell population is enriched for CD8+ cells.

[0068] In some embodiments, the CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of these CD8+ cell types. In some embodiments, expression of phenotypic markers of central memory T cells comprises expression of CD45RO, CD62L, CCR7, CD28, CD3, and CD127 and is negative for granzyme B. In some embodiments, the central memory T cells are CD45RO+, CD62L+, and/or CD8+ T cells. In some embodiments, the effector T cells are negative for CD62L, CCR7, CD28, and/or CD127 and positive for granzyme B and perforin. In some certain embodiments, the CD4+ T cells are further sorted into subsets. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that share cell surface antigens.

[0069] It should be appreciated that the PBMCs may further comprise other cytotoxic lymphocytes, such as NK cells or NKT cells. An expression vector carrying the coding sequence of a chimeric receptor as disclosed herein can be introduced into a population of donor human T cells, NK cells, or NKT cells. Successfully transduced T cells carrying the expression vector can be sorted using flow cytometry to isolate CD3-positive T cells and then expanded to increase the number of these CAR-expressing T cells, in addition to activating the cells using CD3 antibodies and IL-2 or other methods known in the art, as described elsewhere herein. Standard procedures are used to cryopreserve the CAR-expressing T cells for storage and/or recovery for use in a human subject. In one embodiment, transduction, culturing and/or expansion of T cells in vitro is carried out in the absence of non-human animal derived products, such as fetal calf serum and fetal bovine serum.

2. Engineered immune cells

[0070] The cell culture media and methods of use described herein may be particularly useful in the in vitro expansion of immune cells, including engineered immune cells (e.g., CAR T cells).

[0071] Engineered immune cells may be allogeneic or autologous.

[0072] In some embodiments, the engineered immune cell is a T cell (e.g., an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, a helper T lymphocyte, a tumor infiltrating lymphocyte (TIL)), an NK cell, an NK T cell, a TCR-expressing cell, a dendritic cell, a dendritic killer cell, a mast cell, or a B cell. In some embodiments, the cell can be obtained from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. In some exemplary embodiments, the engineered immune cell is a T cell.

[0073] In some embodiments, the engineered immune cell can be derived, for example, but not limited to, from a stem cell. The stem cells can be adult stem cells that are not human embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells.

[0074] In some embodiments, the cell is obtained or isolated from peripheral blood. In some embodiments, the cell is obtained or isolated from peripheral blood mononuclear cells (PBMCs). In some embodiments, the cell is obtained or isolated from bone marrow. In some embodiments, the cell is obtained or isolated from umbilical cord blood. In some embodiments, the cell is a human cell. In some embodiments, the cell is transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., gene gun), lipid transfection, polymeric transfection, nanoparticles, or polyplexes.

a. Binding agents (including antibodies)

[0075] In some embodiments, the engineered immune cells comprise an antigen-binding agent (e.g., comprising an antigen-binding domain or comprising an antibody or fragment thereof).

[0076] As used herein, the term "antibody" refers to a polypeptide that includes elements of a canonical immunoglobulin sequence sufficient to provide specific binding to a particular target antigen. As is known in the art, intact antibodies produced in nature are tetrameric agents of approximately 150 kDa, consisting of two identical heavy chain polypeptides (approximately 50 kDa each) and two identical light chain polypeptides (approximately 25 kDa each) that associate with each other in what is commonly referred to as a "Y-shaped" structure. Each heavy chain consists of at least four domains (each approximately 110 amino acids long) - an amino-terminal variable (VH) domain (located at the ends of the Y-shaped structure) followed by three constant domains: CHI, CH2, and the carboxyl-terminal CH3 (located at the base of the "stem" of the Y). A short region known as the "switch" connects the variable and constant regions of the heavy chain. The "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in the intact antibody. Each light chain consists of two domains - an amino-terminal variable (VL) domain followed by a carboxyl-terminal constant (CL) domain - separated from each other by another "switch". Those skilled in the art are familiar with antibody structure and sequence elements, recognize "variable" and "constant" regions in the sequences shown, and understand that there may be some flexibility in defining the "boundary" between such domains, such that different representations of the same antibody chain sequence may, for example, point to such a boundary at a position that is shifted by one or more residues relative to another representation of the same antibody chain sequence.

[0077] Intact antibody tetramers consist of two heavy chain-light chain dimers in which the heavy and light chains are linked to each other by one disulfide bond; two other disulfide bonds connect the hinge regions of the heavy chain to each other, so that the dimers are linked to each other to form a tetramer. Naturally produced antibodies are also glycosylated, typically at the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed by two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other into a compressed beta barrel in an antiparallel orientation. Each variable domain contains three hypervariable loops, known as "complementarity determining regions" (CDR1, CDR2, and CDR3), and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4). When natural antibodies fold, the FR regions form beta sheets that provide the structural framework for the domains, and the CDR loop regions of both the heavy and light chains are combined together in three-dimensional space so that they create a single hypervariable antigen-binding site located at the apex of a Y-shaped structure. The Fc region of naturally occurring antibodies binds to elements of the complement system as well as to receptors on effector cells, including, for example, on effector cells that mediate cytotoxicity. As is known in the art, the affinity and/or other binding properties of Fc regions for Fc receptors can be modulated by glycosylation or other modification. In some embodiments, antibodies produced and/or used in accordance with the present invention comprise glycosylated Fc domains, including Fc domains modified or engineered by such glycosylation.

[0078] In the context of the present invention, in certain specific embodiments, any polypeptide or complex of polypeptides that includes sufficient sequences of the immunoglobulin domains found in natural antibodies may be referred to and/or used as an "antibody," whether such polypeptide is produced naturally (e.g., created by an organism responding to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial system or technique. In some embodiments, the antibody is polyclonal; in some embodiments, the antibody is monoclonal. In some embodiments, the antibody has constant region sequences that are characteristic of antibodies from a mouse, rabbit, primate, or human. In some embodiments, the sequence elements of the antibody are humanized, primatized, chimeric, etc., as known in the art.

[0079] Moreover, the term "antibody" as used herein may refer in appropriate embodiments (unless otherwise indicated or clear from the context) to any of the constructs or formats known in the art or developed to utilize the structural and functional features of an antibody in an alternative presentation. For example, in some embodiments, an antibody of the present invention has a format selected from, but is not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single-chain Fv; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); camel antibodies; masked antibodies (e.g., Probodies®); small modular protein immunopharmaceuticals (SMIP™); single-chain or tandem diabodies (TandAb®); VHH; Anticalin®; Nanobodies® minibodies; BiTE®; ankyrin repeat proteins or DARPIN®; Avimer®; DART; TCR-like antibodies; Adnectin®; Affilin®; Trans-bodies®; Affibodies®; TrimerX®; microprotein; Fynomer®, Centyrin®, and KALBITOR®. In some embodiments, an antibody may lack a covalent modification (e.g., a glycan attachment) that it would have if it were naturally produced. In some embodiments, the antibody may comprise a covalent modification (e.g., glycan attachment), a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or another side group (e.g., polyethylene glycol, etc.).

[0080] In some embodiments, an antibody or binding agent may be "symmetric." By "symmetric" is meant that the antibody or binding agent has the same type of Fv regions (e.g., the antibody has two Fab regions). In some embodiments, an antibody or binding agent may be "asymmetric." By "asymmetric" is meant that the antibody or binding agent has at least two different types of Fv regions (e.g., the antibody has Fab and scFv regions, Fab and scFv2 regions, or Fab-VHH regions). Various asymmetric antibody or binding agent structures are known in the art (Brinkman and Kontermann et al. 2017 Mabs (9) (2): 182-212).

[0081] As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to provide specific binding. Exemplary antibody agents include, but are not limited to, monoclonal antibodies or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements that are humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody agent" is used in reference to one or more constructs or formats known in the art or developed to utilize the structural and functional features of an antibody in an alternative presentation. For example, an antibody agent useful in accordance with the present invention has a format selected from, but is not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (e.g., ^Zybodies® , etc.); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single-chain Fv; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); camel antibodies; masked antibodies (e.g., ^Probodies® ); immunopharmaceuticals based on small modular protein (SMIP™); single-chain or tandem diabodies (TandAb ^® ); VHH; Anticalin ^® ; Nanobodies ^® minibodies; BiTE ^® ; ankyrin repeat proteins or DARPIN ^® ; Avimer ^® ; DART; TCR-like antibodies; Adnectin ^® ; Affilin ^® ; Trans-bodies ^® ; Affibodies ^® ; TrimerX ^® ; microproteins; Fynomer ^® , Centyrin ^® and KALBITOR ^® .

[0082] An antibody or antigen-binding molecule encoded in accordance with the present invention may be single-chain or double-chain. In some embodiments, the antibody or antigen-binding molecule is single-chain. In some specific embodiments, the antigen-binding molecule is selected from the group consisting of scFv, Fab, Fab', Fv, F(ab') ₂ , dAb, and any combination thereof.

[0083] Antibodies include antibody fragments. An antibody fragment comprises a portion of an intact antibody, such as an antigen-binding or variable region of an intact antibody. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, diabodies, linear antibodies, multispecific antibodies formed from antibody fragments, and scFv fragments, as well as other fragments described below. Antibodies also include, but are not limited to, polyclonal, monoclonal, chimeric dAb (domain antibody), single chain, F _ab , _Fa , F _(ab) 2 fragments, scFv, and F _ab expression libraries. An antibody can be a whole antibody or an immunoglobulin or an antibody fragment. In some embodiments, the antibody is a full-length antibody, such as an intact IgG1 antibody or another class or isotype of antibodies described herein. (See, e.g., Hudson et al., Nat. Med., 9: 129-134 (2003); Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, pp. 269-315 (1994); Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993); WO93/01161 and U.S. Patent Nos. 5,571,894, 5,869,046, 6,248,516, and 5,587,458). A full-length antibody, intact antibody, or whole antibody is an antibody having a structure substantially similar to that of a native antibody or having heavy chains that contain an Fc region as defined herein. Antibody fragments can be produced by a variety of techniques, including but not limited to proteolytic cleavage of an intact antibody, as well as production by recombinant host cells (e.g., E. coli or phage), as is known in the art.

[0084] In some embodiments, the antibody is or includes a monoclonal antibody, including a chimeric, humanized, or human antibody.

[0085] In some embodiments, an antigen-specific antibody agent provided herein can be a chimeric antibody (see, e.g., U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). A chimeric antibody can be an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species and the remainder of the heavy and/or light chain is derived from another source or species. In one example, a chimeric antibody can comprise a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody may be a "class-switched" antibody, the class or subclass of which has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0086] In some embodiments, the chimeric antibody can be a humanized antibody (see, e.g., Almagro and Fransson, Front. Biosci., 13: 1619-1633 (2008); Riechmann et al., Nature, 332:323-329 (1988); Queen et al., Proc. Natl Acad. Sci. USA 86: 10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005); Padlan, Mol. Immunol, 28:489-498 (1991); Dall'Acqua et al., Methods, 36:43-60 (2005); Osbourn et al., Methods, 36:61-68 (2005); and Klimka et al., Br. J. Cancer, 83:252-260 (2000)). A humanized antibody is a chimeric antibody comprising amino acid residues from non-human hypervariable regions and amino acid residues from human FRs. In certain specific embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable regions (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody.

[0087] A non-human antibody can be humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the original non-human antibody. A humanized antibody can comprise one or more variable domains comprising one or more CDRs, or portions thereof, derived from a non-human antibody. A humanized antibody can comprise one or more variable domains comprising one or more FRs, or portions thereof, derived from human antibody sequences. A humanized antibody can optionally comprise at least a portion of a human constant region. In some embodiments, one or more FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived) to restore or improve the specificity or affinity of the antibody.

[0088] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using a "best match" method; framework regions derived from a human antibody consensus sequence of a particular subset of light or heavy chain variable regions; mature (somatically mutated) human framework regions or human germline framework regions, as well as framework regions obtained from FR screening libraries (see, e.g., Sims et al., J. Immunol. 151:2296 (1993); Carter et al., Proc. Natl. Acad. Sci. USA. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993); Baca et al., J. Biol. Chem., 272: 10678-10684 (1997); and Rosok et al., J. Biol. Chem., 271: 22611-22618 (1996)).

[0089] In some embodiments, an antibody agent provided herein is a human antibody. Human antibodies can be produced using a variety of techniques known in the art (see, e.g., van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001); and Lonberg, Curr. Opin. Immunol, 20: 450-459 (2008)). A human antibody can be an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or obtained from a non-human source that utilizes a human antibody repertoire or other human antibody encoding sequences. This definition of a human antibody specifically excludes a humanized antibody that contains non-human antigen-binding residues. Human antibodies can be produced by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen stimulation (see, e.g., Lonberg, Nat. Biotech., 23: 1117-1125 (2005); U.S. Patent Nos. 6,075,181, 6,150,584, 5,770,429, and 7,041,870, and U.S. Patent Application Publication No. US 2007/0061900). The human variable regions of intact antibodies produced by such animals can be further modified, e.g., by combining with another human constant region.

[0090] Human antibodies can also be produced by methods based on the use of hybridomas. For example, human antibodies can be produced from human myeloma and mouse-human heteromyeloma cell lines using human B-cell hybridoma technology and other methods (see, e.g., Kozbor, J. Immunol, 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (1987); Boerner et al., J. Immunol, 147: 86 (1991); Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006); U.S. Patent No. 7,189,826; Ni, Xiandai Mianyixue, 26(4): 265-268 (2006); Vollmers and Brandlein, Histology and Histopathology, 20(3): 927–937 (2005); and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185–91 (2005)). Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant region.

[0091] Modifications of an oligosaccharide in an antibody can be made, for example, to create antibody variants with certain improved properties. For example, glycosylation variants of antibodies can have improved CDC function. In some embodiments, the present invention may provide an antibody variant that has some, but not all, effector functions, making it a desirable candidate for applications in which the in vivo half-life of the antibody is important, but some effector functions (such as complement) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/depletion of CDC activity.

[0092] In some embodiments, the antibody-based agent provided herein can be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatization of the antibody can include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers may include, but are not limited to, polyethylene glycol (PEG), ethylene glycol-propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide-ethylene oxide copolymers, polyoxyethylated polyhydric alcohols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in preparation due to its stability in water.

[0093] The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if two or more polymers are attached, they may be the same or different molecules.

[0094] In some embodiments, conjugates of an antibody and a non-proteinaceous moiety are provided that can be selectively heated by exposure to radiation. In some embodiments, the non-proteinaceous moiety can be a carbon nanotube (see, e.g., Kam et al., Proc. Natl. Acad. Sci. USA, 102: 11600-11605 (2005)). The radiation can be of any wavelength and can be characterized by, but is not limited to, wavelengths that do not cause damage to normal cells but do heat the non-proteinaceous moiety to a temperature that kills cells proximal to the antibody-non-proteinaceous moiety.

b. Chimeric antigen receptors

[0095] In some embodiments, the engineered immune cell comprises a population of CARs, wherein each CAR comprises an extracellular antigen-binding domain. In some embodiments, the immune cell comprises a population of CARs, wherein each CAR comprises extracellular antigen-binding domains.

[0096] As used herein, chimeric antigen receptors (CARs) are proteins that specifically recognize target antigens (e.g., target antigens on cancer cells). When bound to a target antigen, a CAR can activate an immune cell to attack and destroy the cell bearing that antigen (e.g., a cancer cell). CARs can include costimulatory or signaling domains to enhance their effectiveness. See Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619,626); Finney et al., Journal of Immunology, 1998, 161: 2791,2797, Song et al., Blood 119:696-706 (2012); Kalos et al., Sci. Transl. Med. 3:95 (2011); Porter et al, N. Engl J. Med. 365:725–33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59–83 (2016); U.S. Patent Nos. 7,741,465 and 6,319,494.

[0097] The chimeric antigen receptors described herein comprise an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antigen-binding domain that specifically binds to a target.

[0098] In some embodiments, the antigen-specific CARs further comprise safety switches and/or one or more monoclonal antibody-specific epitopes.

i. Antigen-binding domains

[0099] As discussed above, the CARs described herein comprise an antigen-binding domain. As used herein, the term "antigen-binding domain" means any polypeptide that binds a specific target antigen. In some embodiments, the antigen-binding domain binds to an antigen on a tumor cell. In some embodiments, the antigen-binding domain binds to an antigen on a cell involved in a hyperproliferative disease.

[00100] In some embodiments, the antigen-binding domain comprises a variable heavy chain, a variable light chain, and/or one or more CDRs described herein. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) comprising the light chain CDRs CDR1, CDR2, and CDR3, and the heavy chain CDRs CDR1, CDR2, and CDR3.

[00101] Antigen-binding domain variants (e.g., CDR, VH, and/or VL variants) are also within the scope of the present invention, e.g., each of the variable light and/or variable heavy chains has at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or greater than 99% amino acid sequence identity to the antigen-binding domain sequences. In some cases, such molecules comprise at least one heavy chain and one light chain, while in other cases, variant forms comprise two variable light chains and two variable heavy chains (or subportions thereof). One of skill in the art will be able to determine suitable variants of the antigen-binding domains set forth herein using well-known techniques. In some specific embodiments, one of skill in the art can identify suitable regions of the molecule that can be altered without disrupting activity by targeting regions that are not believed to be important for activity.

[00102] In some specific embodiments, the polypeptide structure of the antigen-binding domains is based on antibodies, including but not limited to monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, fusion antibodies (sometimes referred to herein as "antibody conjugates"), and fragments thereof, respectively. In some embodiments, the antigen-binding domain comprises or consists of avimers.

[00103] An antigen-binding domain is said to be "selective" when it binds to one target more strongly than to a second target.

[00104] In some embodiments, the antigen binding domain is an scFv.

[00105] In some embodiments, the antigen-selective CAR comprises a leader or signal peptide.

[00106] In other embodiments, the present invention relates to isolated polynucleotides encoding any of the antigen-binding domains described herein. In some embodiments, the present invention relates to isolated polynucleotides encoding a CAR. Also provided herein are vectors comprising the polynucleotides and methods for producing the same.

ii. Safety switches and monoclonal antibody-specific epitopes

[00107] It will be appreciated that adverse events can be minimized by transducing immune cells (containing one or more CARs) with a "suicide" gene. It is also necessary to include an inducible "on" or "accelerate" switch in the immune cells. Suitable techniques include the use of inducible caspase-9 (US Patent Application No. 2011/0286980) or thymidine kinase before, after, or simultaneously with transduction of cells with the CAR construct of the present invention. Additional methods for introducing "suicide" genes and/or "on" switches include the use of TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other techniques known in the art.

[00108] In accordance with the present invention, additional on-off switches or other types of controlled switching techniques may be included herein. These techniques may employ dimerization domains and optional dimerization activators of such domains. These techniques include, for example, those described by Wu et al., Science 2014 350 (6258), using FKBP/Rapalog dimerization systems in certain cells, the contents of which are incorporated herein by reference in their entireties. Additional dimerization technology is described, for example, in Fegan et al. Chem. Rev. 2010, 110, 3315-3336, as well as U.S. Pat. Nos. 5,830,462, 5,834,266, 5,869,337, and 6,165,787, the contents of which are incorporated herein by reference in their entireties. Additional dimerization pairs may include cyclosporin A/cyclophilin receptor, estrogen/estrogen receptor (optionally using tamoxifen), glucocorticoids/glucocorticoid receptor, tetracycline/tetracycline receptor, vitamin D/vitamin D receptor. Additional examples of dimerization technology can be found, for example, in WO 2014/127261, WO 2015/090229, US2014/0286987, US2015/0266973, US2016/0046700, US Patent No. 8,486,693, US2014/0171649 and US2012/0130076, the contents of which are further incorporated herein by reference in their entireties.

[00109] In some embodiments, a CAR immune cell (e.g., a CAR T cell) of the present invention comprises a polynucleotide encoding a "suicide" polypeptide, such as, for example, RQR8. See, for example, WO2013153391A, which is hereby incorporated by reference in its entirety. In a CAR immune cell (e.g., a CAR T cell) comprising the polynucleotide, the "suicide" polypeptide is expressed on the surface of the CAR immune cell (e.g., a CAR T cell). In some embodiments, the "suicide" polypeptide comprises the amino acid sequence shown as SEQ ID NO: 1.

[00110] The "suicide" polypeptide may also comprise a signal peptide at the amino terminus, such as MGTSLLCWMALCLLGADHADA (SEQ ID NO: 2). In some embodiments, the "suicide" polypeptide comprises the amino acid sequence shown as SEQ ID NO: 3, which includes the signal sequence of SEQ ID NO: 2.

[00112] When the suicide polypeptide is expressed on the surface of a CAR immune cell (e.g., a CAR T cell), binding of rituximab to epitopes of the R polypeptide causes lysis of the cell. More than one rituximab molecule may be bound per polypeptide expressed on the cell surface. Each epitope of the R polypeptide may bind a separate rituximab molecule. Deletion of an antigen-specific CAR immune cell (e.g., a CAR T cell) may occur in vivo, such as upon administration of rituximab to a patient. The decision to delete the transferred cells may be based on the detection of undesirable effects in the patient associated with the transferred cells, such as, for example, detection of unacceptable levels of toxicity.

[00113] In some embodiments, the suicide polypeptide is expressed on the surface of a cell. In some embodiments, the suicide polypeptide is included in a CAR construct. In some embodiments, the suicide polypeptide is not part of a CAR construct.

[00114] In some embodiments, the extracellular domain of an antigen-specific CAR may comprise one or more epitopes that are specific for (i.e., specifically recognized by) a monoclonal antibody. These epitopes are also referred to herein as mAb-specific epitopes. Exemplary mAb-specific epitopes are disclosed in International Patent Publication No. WO 2016/120216, which is incorporated herein by reference in its entirety. In such embodiments, the extracellular domain of the CAR comprises antigen-binding domains that specifically bind an antigen and one or more epitopes that bind one or more monoclonal antibodies (mAbs). CARs comprising mAb-specific epitopes may be single-chain or multi-chain.

[00115] The inclusion of monoclonal antibody-specific epitopes in the extracellular domain of the CAR described herein allows for the sorting and depletion of engineered immune cells expressing the CAR. In some embodiments, this feature also facilitates the recovery of endogenous antigen-expressing cells that have been depleted by administration of engineered immune cells expressing the CAR. In some embodiments, the depletion capability provides a safety switch in the event of deleterious effects, such as when administered to a subject.

[00116] Therefore, in some embodiments, the present invention provides a method of sorting and/or depleting engineered immune cells having CARs containing mAb-specific epitopes and a method of ensuring recovery of endogenous antigen-expressing cells.

[00117] Several epitope-monoclonal antibody pairs can be used to create CARs containing epitopes specific for monoclonal antibodies; in particular, those that are already approved for medical use, such as the CD20/rituximab epitope, as a non-limiting example.

[00118] The antigen also encompasses methods for sorting engineered immune cells having antigen-specific CARs and expressing mAb-specific epitope(s) and therapeutic methods in which activation of engineered immune cells having these CARs is modulated by depleting the cells using an antibody that targets the outer ligand-binding domain of said CARs. Table 1 provides exemplary mimotope sequences that can be inserted into the extracellular domains of any of the CARs of the present invention.

[00119] In some embodiments, the extracellular binding domain of a CAR comprises the following sequence

V ₁ -L ₁ -V ₂ -(L) _x -epitope1-(L) _x -;

V ₁ -L ₁ -V ₂ -(L) _x -epitope1-(L) _x -epitope2-(L) _x -;

V ₁ -L ₁ -V ₂ -(L) _x -epitope1-(L)x-epitope2-(L) _x -epitope3-(L) _x -;

(L) _x -epitope1-(L) _x -V ₁ -L ₁ -V ₂ ;

(L) _x -epitope1-(L) _x -epitope2-(L) _x -V ₁ -L ₁ -V ₂ ;

epitope1-(L) _x -epitope2-(L) _x -epitope3-(L) _x -V ₁ -L ₁ -V ₂ ;

(L) _x -epitope1-(L) _x -V ₁ -L ₁ -V ₂ -(L) _x -epitope2-(L) _x ;

(L) _x -epitope1-(L) _x -V ₁ -L ₁ -V ₂ -(L) _x -epitope2-(L) _x -epitope3-(L) _x -;

(L) _x -epitope1-(L) _x -V ₁ -L ₁ -V ₂ -(L) _x -epitope2-(L) _x -epitope3-(L) _x -epitope4-(L)x-;

(L) _x -epitope1-(L) _x -epitope2-(L) _x -V ₁ -L ₁ -V ₂ -(L) _x -epitope3-(L) _x -;

(L) _x -epitope1-(L) _x -epitope2-(L) _x -V ₁ -L ₁ -V ₂ -(L) _x -epitope3-(L) _x -epitope4-(L) _x -;

V ₁ -(L) _x -epitope1-(L) _x -V ₂ ;

V ₁ -(L) _x -epitope1-(L) _x -V ₂ -(L) _x -epitope2-(L) _x ;

V ₁ -(L) _x -epitope1-(L) _x -V ₂ -(L) _x -epitope2-(L) _x -epitope3-(L) _x ;

V ₁ -(L) _x -epitope1-(L) _x -V ₂ -(L) _x -epitope2-(L) _x -epitope3-(L) _x -epitope4-(L)x;

(L) _x -epitope1-(L) _x -V ₁ -(L) _x -epitope2-(L) _x -V ₂ or

(L) _x -epitope1-(L) _x -V ₁ -(L) _x -epitope2-(L) _x -V ₂ -(L) _x -epitope3-(L) _x ;

Where

V ₁ is V _L , and V ₂ is V _H , or V ₁ is V _H , and V ₂ is V _L ;

L ₁ is a linker suitable for linking the V _H chain to the V _L chain;

epitope 1, epitope 2, epitope 3 and epitope 4 are mAb-specific epitopes and may be identical or different, and V _H is a variable fragment of the heavy chain and V _L is a variable fragment of the light chain.

iii. Hinge domain

[00120] The extracellular domain of the CAR of the present invention may include a "hinge" domain (or hinge region). The term generally refers to any polypeptide that functions to link the transmembrane domain of the CAR to the extracellular antigen-binding domain of the CAR. In particular, hinge domains can be used to provide greater flexibility and accessibility to the extracellular antigen-binding domain.

[00121] The hinge domain may comprise up to 300 amino acids, in some embodiments, from 10 to 100 amino acids, or in some embodiments, from 25 to 50 amino acids. The hinge domain may be derived from all or a portion of naturally occurring molecules, such as from all or a portion of the extracellular region of CD8, CD4, CD28, 4-1BB, or IgG (particularly the hinge region of IgG; it should be appreciated that the hinge region may comprise part or all of the immunoglobulin family members such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof), or from all or a portion of the constant region of an antibody heavy chain. Alternatively, domain A may be a synthetic sequence that corresponds to a naturally occurring sequence A, or may be a completely synthetic sequence A. In some embodiments, said domain A is part of a human CD8α chain (e.g., NP 001139345.1). In another specific embodiment, said hinge and transmembrane domains are part of a human CD8α chain. In some embodiments, the hinge domain of a CAR described herein comprises a subsequence of CD8α, IgG1, IgG4, PD-1, or FcγRIIIα, in particular a hinge region of any of CD8α, IgG1, IgG4, PD-1, or FcγRIIIα. In some embodiments, the hinge domain comprises a human CD8α hinge, a human IgG1 hinge, a human IgG4 hinge, a human PD-1, or a human FcγRIIIα. In some embodiments, the CARs disclosed herein comprise an scFv, a human CD8α hinge and transmembrane domains, a CD3ζ signaling domain, and a 4-1BB signaling domain. Table 2 provides amino acid sequences for exemplary hinges provided herein.

iv. Transmembrane domain

[00122] The CARs of the present invention are engineered with a transmembrane domain that is fused to an extracellular domain of the CAR. Likewise, it can be fused to an intracellular domain of the CAR. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid association of such domains with transmembrane domains of the same or different surface membrane proteins in order to minimize levels of interaction with other members of the receptor complex. In some embodiments, short linkers can form links between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

[00123] Suitable transmembrane domains for CARs disclosed herein have the ability to (a) be expressed on the surface of an immune cell, such as, for example, but not limited to, a lymphocyte, such as a helper ( _Th ) T cell, a cytotoxic ( _Tc ) T cell, a regulatory (T _reg ) T cell, or a natural killer (NK) cell, and/or (L) interact with an extracellular antigen binding domain and an intracellular signaling domain to direct a cellular response of the immune cell against a target cell.

[00124] The transmembrane domain may be obtained from either a natural or a synthetic source. If the source is natural, the domain may be obtained from any membrane-associated or transmembrane protein.

[00125] Transmembrane regions particularly useful in the present invention can be derived from CD28, OX-40.4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death protein 1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor proteins, immunoglobulin protein, cytokine receptor, integrins, signaling lymphocyte activating molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll-like receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof (may be contained in or may correspond to).

[00126] As non-limiting examples, the transmembrane region can be derived from or can be part of a T cell receptor, such as α, β, γ or δ, a polypeptide comprising a CD3 complex, an IL-2 receptor p55 (chain), p75 (β chain) or γ chain, a subunit chain of Fc receptors, in particular Fcγ receptor III, or CD proteins. Alternatively, the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues, such as leucine and valine. In some embodiments, the transmembrane domain is derived from the human CD8α chain (e.g., NP_001139345.1).

[00127] In some embodiments, the transmembrane domain of a CAR of the present invention is a CD8α transmembrane domain. In some embodiments, the transmembrane domain of a CAR of the present invention is a CD8α transmembrane domain comprising the amino acid sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 17). In some embodiments, the CD8α transmembrane domain comprises a nucleic acid sequence that encodes the transmembrane amino acid sequence of SEQ ID NO: 17. In some embodiments, the hinge and the transmembrane domain of a CAR of the present invention are a CD8α hinge and a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 17.

[00128] In some embodiments, a transmembrane domain in a CAR of the present invention is a CD28 transmembrane domain. In some embodiments, a transmembrane domain in a CAR of the present invention is a CD28 transmembrane domain comprising the amino acid sequence FWVLVWGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 18). In some embodiments, a CD28 transmembrane domain comprises a nucleic acid sequence that encodes the transmembrane amino acid sequence of SEQ ID NO: 18.

v. Intracellular domain

[00129] The intracellular (cytoplasmic) domain of a CAR of the present invention may provide for the activation of at least one of the normal effector functions of an immune cell comprising the CAR. The effector function of a T cell, for example, may relate to cytolytic activity or helper activity, including cytokine secretion.

[00130] In some embodiments, an activating intracellular signaling domain for use in a CAR may be cytoplasmic sequences, such as, but not limited to, a T cell receptor and coreceptors that act together to initiate signaling upon binding to an antigen receptor, as well as any derivative or variant of these sequences, and any synthetic sequence that has the same functionality.

[00131] It should be appreciated that suitable (e.g., activating) intracellular domains include, but are not limited to, signaling domains derived from CD28, OX-40.4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death protein 1 (PD-1), inducible T-cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor proteins, immunoglobulin protein, cytokine receptor, integrins, lymphocyte signaling activating molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll-like receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, 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, a ligand that specifically binds to CD83, or any combination thereof (or corresponding thereto).

[00132] The intracellular domains of the CARs of the present invention may include, in addition to the activating domains described above, costimulatory signaling domains (interchangeably referred to herein as costimulatory molecules) to enhance their effectiveness. Costimulatory domains may provide a signal in addition to the primary signal provided by the activating molecule as described herein.

[00133] It will be appreciated that suitable costimulatory domains within the scope of the present invention can be derived from, for example, CD28, OX40, 4-1BB/CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte functional-associated antigen-1 (LFA-1 (CD1 1a/CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNFR, integrin, signaling lymphocyte activating molecule, BTLA, Toll-like receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1-1d, ITGAE, CD103, ITGAL, CD1-1a, LFA-1, ITGAM, CD1-1b, ITGAX, CD1-1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, 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, CD83 ligand, or fragments or combinations thereof (or may correspond to such). It should be appreciated that additional costimulatory molecules or fragments thereof not listed above are within the scope of the present invention.

[00134] In some embodiments,

the intracellular/cytoplasmic domain of the CAR may be engineered to contain the 4-1BB/CD137 domain alone or in combination with any other necessary intracellular domain(s) useful in the context of the CAR of the present invention. The complete native amino acid sequence of 4-1BB/CD137 is described in the NCBI Reference Sequence Database: NP 001552.2. The complete native nucleic acid sequence of 4-1BB/CD137 is described in the NCBI Reference Sequence Database: NM_001561.5.

[00135] In some embodiments,

The intracellular/cytoplasmic domain of the CAR may be engineered to contain the CD28 domain alone or in combination with any other necessary intracellular domain(s) useful in the context of the CAR of the present invention. The complete native amino acid sequence of CD28 is described in the NCBI Reference Sequence Database: NP 006130.1. The complete native nucleic acid sequence of CD28 is described in the NCBI Reference Sequence Database: NM 006139.1.

[00136] In some embodiments, the intracellular/cytoplasmic domain of the CAR may be engineered to comprise a CD3 zeta domain alone or in combination with any other necessary intracellular domain(s) useful in the context of a CAR of the present invention. In some embodiments, the intracellular signaling domain of the CAR may provide a CD3ζ signaling domain that has an amino acid sequence with at least about 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence shown as SEQ ID NO: 19 or SEQ ID NO: 26.

For example, the intracellular domain of a CAR may comprise a portion of a CD3 zeta chain and a portion of a costimulatory signaling molecule. The intracellular signaling sequences in the intracellular signaling portion of a CAR of the present invention may be linked to each other in a random or specific order. In some embodiments, the intracellular domain is engineered to comprise a CD3 zeta activation domain and a CD28 signaling domain. In some embodiments, the intracellular domain is engineered to comprise a CD3 zeta activation domain and a 4-1BB signaling domain.

[00137] In some embodiments, 4-1BB (intracellular domain) comprises the amino acid sequence

(SEQ ID NO: 20). In some embodiments, 4-1BB (intracellular domain) is encoded by a nucleic acid sequence

[00138] In some embodiments, the intracellular domain of the CAR is engineered to contain a portion of CD28 and CD3 zeta, wherein the intracellular CD28 comprises the nucleic acid sequence set forth in SEQ ID NO: 22.

[00139] In some embodiments, the intracellular domain of a CAR is engineered to contain an amino acid sequence . The amino acid sequence of CD3 zeta may comprise SEQ ID NO: 23, and the nucleic acid sequence may comprise SEQ ID NO: 24:

[00140] In some embodiments, the intracellular signaling domain of a CAR of the present invention comprises a costimulatory molecule domain. In some embodiments, the intracellular signaling domain of a CAR of the present invention comprises a portion of a costimulatory molecule selected from the group consisting of the 4-1BB fragment (GenBank: AAA53133) and CD28 (NP 006130.1). In some embodiments, the intracellular signaling domain of a CAR of the present invention comprises an amino acid sequence that has at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence shown under SEQ ID NO: 20 and SEQ ID NO: 22. In some embodiments, the intracellular signaling domain of a CAR of the present invention comprises an amino acid sequence that has at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence shown under SEQ ID NO: 20 and/or at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence shown under SEQ ID NO: 22.

c. CAR-containing immune cells

[00141] Disclosed herein are engineered immune cells expressing the CAR of the present invention (e.g., CAR T cells).

[00142] In some embodiments, the engineered immune cell comprises a population of CARs, wherein each CAR comprises extracellular antigen-binding domains. In some embodiments, the engineered immune cell comprises a population of CARs, wherein each CAR comprises different extracellular antigen-binding domains. In some embodiments, the immune cell comprises a population of CARs, wherein each CAR comprises extracellular antigen-binding domains.

[00143] Engineered immune cells may be allogeneic or autologous.

[00144] In some embodiments, the engineered immune cell is a T cell (e.g., an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, a helper T lymphocyte, a tumor infiltrating lymphocyte (TIL)), an NK cell, an NK T cell, a TCR-expressing cell, a dendritic cell, a dendritic killer cell, a mast cell, or a B cell. In some embodiments, the cell can be obtained from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. In some exemplary embodiments, the engineered immune cell is a T cell. In some exemplary embodiments, the engineered immune cell is a gamma delta T cell. In some exemplary embodiments, the engineered immune cell is a macrophage.

[00145] In some embodiments, the engineered immune cell can be derived, for example, but not limited to, from a stem cell. The stem cells can be adult stem cells that are not human embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells.

[00146] In some embodiments, the cell is obtained or isolated from peripheral blood. In some embodiments, the cell is obtained or isolated from peripheral blood mononuclear cells (PBMCs). In some embodiments, the cell is obtained or isolated from bone marrow. In some embodiments, the cell is obtained or isolated from umbilical cord blood. In some embodiments, the cell is a human cell. In some embodiments, the cell is transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., gene gun), lipid transfection, polymeric transfection, nanoparticles, viral transfection (e.g., using a retrovirus, lentivirus, AAV), or polyplexes.

[00147] In some embodiments, engineered immune cells expressing an antigen-specific CAR of the present invention on their cell membrane surface comprise a percentage of memory stem cells and central memory cells that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[00148] In some embodiments, engineered immune cells expressing a membrane antigen-specific CAR of the present invention on their cell membrane surface comprise a percentage of memory stem cells and central memory cells of from about 10% to about 100%, from about 10% to about 90%, from about 10% to about 80%, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 20%, from about 15% to about 100%, from about 15% to about 90%, from about 15% to about 80%, from about 15% to about 70%, from about 15% to about 60%, from about 15% to about 50%, from about 15% to about 40%, from about 15% to about 30%, from about 20% to about 100%, from about 20% to about 90%, from about 20% to about 80%, from about 20% to about 70%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, from about 30% to about 100%, from about 30% to about 90%, from about 30% to about 80%, from about 30% to about 70%, from about 30% to about 60%, from about 30% to about 50%, from about 30% to about 40%, from about 40% to about 100%, from about 40% to about 90%, from about 40% to about 80%, from about 40% to about 70%, from about 40% to about 60%, from about 40% to about 50%, from about 50% to about 100%, from about 50% to about 90%, from about 50% to about 80%, from about 50% to about 70%, from about 50% to about 60%, from about 60% to about 100%, from about 60% to about 90%, from about 60% to about 80%, from about 60% to about 70%, from about 70% to about 90%, from about 70% to about 80%, from about 80% to about 100%, from about 80% to about 90%, from about 90% to about 100%, from about 25% to about 50%, from about 75% to about 100%, or from about 50% to about 75%.

[00149] In some embodiments, engineered immune cells expressing an antigen-specific CAR of the present invention on their cell membrane surface comprise a percentage of memory stem cells and central memory cells greater than 10%, 20%, 30%, 40%, 50%, or 60%.

[00150] In some embodiments, engineered immune cells expressing an antigen-specific CAR of the present invention on their cell membrane surface comprise a percentage of memory stem cells and central memory cells of from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 15% to about 50%, from about 15% to about 40%, from about 20% to about 60%, or from about 20% to about 70%.

[00151] In some embodiments, engineered immune cells expressing an antigen-specific CAR of the invention on the surface of their cell membrane are enriched in T _CM and/or T _SCM cells, such that the engineered immune cells comprise at least about 60%, 65%, 70%, 75%, or 80% combined T _CM and T _SCM cells. In some embodiments, engineered immune cells expressing an antigen-specific CAR of the invention on the surface of their cell membrane are enriched in T _CM and/or T _SCM cells, such that the engineered immune cells comprise at least about 70% combined T _CM and T _SCM cells. In some embodiments, engineered immune cells expressing an antigen-specific CAR of the present invention on their cell membrane surface are enriched in T _CM and/or T _SCM cells, such that the engineered immune cells comprise at least about 75% combined T _CM and/or T _SCM cells.

[00152] In some embodiments, engineered immune cells expressing an antigen-specific CAR of the invention on the surface of their cell membrane are enriched in T _CM and/or T _SCM cells, such that the engineered immune cells comprise at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% T _SCM cells. In some embodiments, engineered immune cells expressing an antigen-specific CAR of the invention on the surface of their cell membrane are enriched in T _CM and/or T _SCM cells, such that the engineered immune cells comprise at least about 30%, 35%, 40%, or 45% TSCM cells.

[00153] In some embodiments, the immune cell is an inflammatory T lymphocyte that expresses any of the CARs described herein. In some embodiments, the immune cell is a cytotoxic T lymphocyte that expresses any of the CARs described herein. In some embodiments, the immune cell is a regulatory T lymphocyte that expresses any of the CARs described herein. In some embodiments, the immune cell is a helper T lymphocyte that expresses any of the CARs described herein.

[00154] Also provided herein are cell lines derived from a transformed immune cell (e.g., a T cell) according to any of the methods described above. Also provided herein are modified cells that are resistant to immunosuppressive treatment. In some embodiments, an isolated cell of the present invention comprises a polynucleotide encoding a CAR.

[00155] In some embodiments, an engineered immune cell of the present invention may comprise one or more disrupted or inactivated genes. In some embodiments, an engineered immune cell of the present invention comprises one disrupted or inactivated gene selected from the group consisting of CD52, DLL3, GR, PD-1, CTLA-4, LAG3, TIM3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, HLA, TCRα, and TCRβ, and/or expresses a CAR, a multi-chain CAR, and/or a pTa transgene. In some embodiments, the isolated cell comprises polynucleotides encoding polypeptides comprising a multi-chain CAR. In some embodiments, an isolated cell of the present invention comprises two disrupted or inactivated genes selected from the group consisting of CD52 and GR, CD52 and TCRα, CDR52 and TCRβ, DLL3 and CD52, DLL3 and TCRα, DLL3 and TCRβ, GR and TCRα, GR and TCRβ, TCRα and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, TIM3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ, and/or expresses a CAR, a multi-chain CAR, and a pTa transgene. In some embodiments, the method comprises disrupting or inactivating one or more genes by introducing into the cells an endonuclease capable of selectively inactivating a gene by selectively cleaving DNA. In some embodiments, the endonuclease can be, for example, a zinc finger nuclease (ZFN), a megaTAL nuclease, a meganuclease, a transcription activator-like effector nuclease (TALE nuclease), or a CRIPR (e.g., Cas9) endonuclease.

[00156] In some embodiments, the TCR is rendered dysfunctional in the cells of the present invention due to disruption or inactivation of the TCRα gene and/or the TCRβ gene(s). In some embodiments, a method is provided for producing modified cells obtained from an individual, wherein the cells can proliferate independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells that can proliferate independently of the MHC signaling pathway that can be obtained by this method are within the scope of the present invention. The modified cells disclosed herein can be used to treat patients in need thereof from host versus graft (HvG) rejection and graft versus host disease (GvHD); therefore, a method for treating patients in need thereof against host versus graft (HvG) rejection and graft versus host disease (GvHD), comprising treating said patient by administering to said patient an effective amount of modified cells containing disrupted or inactivated TCRα and/or TCRβ genes, is within the scope of the present invention.

[00157] In some embodiments, the immune cells are engineered to be resistant to one or more chemotherapeutic drugs. The chemotherapeutic drug can be, for example, a purine nucleotide analog (PNA), thereby making the immune cell suitable for cancer treatment that combines adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, cyclophosphamide, and cytarabine, alone or in combination. PNAs are metabolized by deoxycytidine kinase (dCK) into mono-, di-, and triphosphate PNAs. Their triphosphate forms compete with ATP for DNA synthesis, act as pro-apoptotic agents, and are potent inhibitors of ribonucleotide reductase (RNR), which is involved in the production of trinucleotides.

[00158] In some embodiments, the isolated cells or cell lines of the present invention may comprise pTα or a functional variant thereof. In some embodiments, the isolated cell or cell line may be further genetically modified by disrupting or inactivating the TCRα gene.

[00159] The present invention also provides engineered immune cells comprising any of the CAR polynucleotides described herein. In some embodiments, a CAR may be introduced into an immune cell as a transgene using a plasmid vector. In some embodiments, the plasmid vector may also contain, for example, a selection marker that allows for identification and/or selection of cells that have received the vector.

[00160] The CAR polypeptides can be synthesized in situ in a cell after introducing polynucleotides encoding the CAR polypeptides into the cell. Alternatively, the CAR polypeptides can be produced outside the cells and then introduced into the cells. Methods for introducing a polynucleotide construct into cells are known in the art. In some embodiments, stable transformation methods (e.g., using a lentiviral vector) can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, transient transformation methods can be used to transiently express the polynucleotide construct, and the polynucleotide construct is not integrated into the genome of the cell. In other embodiments, methods can be used that utilize virally mediated effects. The polynucleotides can be introduced into the cell using any suitable means, such as, for example, recombinant viral vectors (e.g., retroviral, adenoviral), liposomes, and the like. Transient transformation methods include, for example, but are not limited to, microinjection, electroporation, or particle bombardment. Polynucleotides can be included in vectors, such as, for example, plasmid vectors or viral vectors.

[00161] In some embodiments, isolated nucleic acids are provided that comprise a promoter operably linked to a first polynucleotide encoding an antigen-binding domain, at least one costimulatory molecule, and an activating domain. In some embodiments, the nucleic acid construct is contained in a viral vector. In some embodiments, the viral vector is selected from the group consisting of retroviral vectors, murine leukemia virus vectors, SFG vectors, adenoviral vectors, lentiviral vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, and vaccinia virus vectors. In some embodiments, the nucleic acid is contained in a plasmid.

3. Cell culture media for in vitro propagation of immune cells

[00162] In vitro cell expansion (including expansion of immune cells such as genetically modified T cells) can be achieved using a cell growth medium containing:

the first stimulator of cell proliferation (eg, the first cytokine that stimulates T-cell proliferation);

a second stimulator of cell proliferation (eg, a second cytokine that stimulates T cell proliferation) and

extracellular modulator of cellular metabolism (eg, T-cell metabolism).

[00163] The immune cells of the present invention may be activated and expanded before or after genetic modification of the immune cells using methods that are well known and generally described herein. In some embodiments, the immune cells (e.g., T cells) are activated and expanded before genetic modification of the immune cells. In some embodiments, the immune cells (e.g., T cells) are activated and expanded after genetic modification of the immune cells (e.g., engineered immune cells, including the cells described herein).

[00164] Accordingly, conditions suitable for expanding T cells include suitable media (e.g., minimal essential media, or RPMI 1640 media, or X-vivo 15, (Lonza)) that contain first and second stimulators of cell proliferation (e.g., IL-7 and IL-15), as well as an extracellular modulator of cellular metabolism (e.g., extracellular potassium), and which may contain additional factors necessary for proliferation and viability, including blood serum (e.g., fetal bovine or human serum), IL-2, insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGF beta and TNF, or any other cell growth additives known to one of skill in the art. In some embodiments, the T cell culture medium does not include exogenous IL-2. Other cell culture supplements include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. Media may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer with added amino acids, sodium pyruvate, and vitamins, either without serum or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and/or an amount of cytokine(s) sufficient to support T cell growth and expansion (e.g., IL-7 and/or IL-15). Antibiotics such as penicillin and streptomycin are included only in experimental cultures and not in cell cultures to be administered to the subject. The target cells are maintained under conditions necessary to support growth, such as appropriate temperature (e.g. 37°C) and atmosphere (e.g. air plus 5% _CO2 ).

[00165] T cells exposed to stimulation at varying times may exhibit different characteristics.

[00166] The methods described herein may further include contacting the cells with an agent that stimulates the CD3 TCR complex and a costimulatory molecule on the surface of the T cells to create an activation signal for the T cell. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to create an activation signal for the T cell.

[00167] In some embodiments, populations of T cells can also be stimulated in vitro by contacting, for example, with an anti-CD3 antibody or an antigen-binding fragment thereof, or with an anti-CD2 antibody immobilized on a surface, or by contacting with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. To co-stimulate an accessory molecule on the surface of T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. The anti-CD3 antibody and the anti-CD28 antibody can be located on a bead, plate, or other substrate.

[00168] In some embodiments, the cells of the present invention can be expanded by co-culture with tissue or cells. The cells can also be expanded in vivo, such as in the blood of a subject after administration of the cell to the subject.

[00169] In particular, cell culture media and methods of using the same may be particularly useful in producing T cells, including CAR T cells, such as allogeneic CAR T cells.

a. Stimulators of cell proliferation

[00170] In some embodiments, the first and second stimulators are selected from an agent that stimulates the CD3 TCR complex; an anti-CD3 antibody or antigen-binding fragment thereof; an anti-CD2 antibody or antigen-binding fragment thereof; an activator of protein kinase C or a growth factor (e.g., T-cell growth factor), or any combinations thereof.

[00171] In some embodiments, a stimulator of immune cells (e.g., T cells or engineered immune cells) is an agent that stimulates the CD3 TCR complex and a costimulatory molecule on the surface of T cells to create an activation signal for the T cell. For example, chemicals such as the calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to create an activation signal for the T cell.

[00172] In some embodiments, the immune cell (e.g., T cells or engineered immune cells) stimulator is an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. To co-stimulate an accessory molecule on the surface of T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. The anti-CD3 antibody and the anti-CD28 antibody can be located on a bead, plate, or other substrate.

[00173] In some embodiments, the cell stimulator is a growth factor. In some embodiments, the cell stimulator is a T cell growth factor. In some embodiments, the T cell growth factor is a cytokine. In some embodiments, the first stimulator is a first cytokine and the second stimulator is a second cytokine. In some embodiments, the cytokine is an interleukin (e.g., IL-2, IL-4, IL-7, IL-10, IL-12, or IL-15). In some embodiments, the first stimulator and the second stimulator are selected from the group consisting of IL-4, IL-7, IL-10, IL-12, and IL-15.

[00174] In some embodiments, the first stimulant is IL-7 and the second stimulant is IL-15.

[00175] In some embodiments, the cell growth medium excludes IL-2 (e.g., the cell growth medium excludes exogenous IL-2, and IL-2 is not present in the cell growth medium as a first stimulator and/or a second stimulator).

[00176] In some embodiments, the amounts of the first and second stimulants used are described in concentrations (e.g., using IU/mL). Accordingly, the phrase "concentration ratio x:y" can be used to describe the ratio of the concentration of the first stimulant (e.g., x IU/mL IL-7) to the concentration of the second stimulant (e.g., y IU/mL IL-15).

[00177] In some embodiments, the first stimulant (e.g., a first cytokine, such as IL-7) and the second stimulant (e.g., a second cytokine, such as IL-15) are present at a concentration ratio of about 10,000:1 to about 1:10,000, about 1,000:1 to about 1:1,000, about 100:1 to about 1:100, or about 10:1 to about 1:10.

[00178] In some embodiments, the first stimulant (e.g., a first cytokine, such as IL-7) is present in an amount that is greater than the amount of the second stimulant (e.g., a second cytokine, such as IL-15). In some embodiments, the first stimulant (e.g., a first cytokine, such as IL-7) and the second stimulant (e.g., a second cytokine, such as IL-15) are present at a concentration ratio of from about 1000:1 to about 10:1, from about 900:1 to about 10:1, from about 800:1 to about 10:1, from about 700:1 to about 10:1, from about 600:1 to about 10:1, from about 500:1 to about 10:1, from about 400:1 to about 10:1, from about 300:1 to about 10:1, from about 250:1 to about 10:1, from about 200:1 to about 10:1, from about 150:1 to about 10:1, or from about 100:1 to about 4:1. In some embodiments, the first stimulant (e.g., a first cytokine, such as IL-7) and the second stimulant (e.g., a second cytokine, such as IL-15) are present at a concentration ratio of from about 1000:1 to about 50:1, from about 900:1 to about 50:1, from about 800:1 to about 50:1, from about 700:1 to about 50:1, from about 600:1 to about 50:1, from about 500:1 to about 50:1, from about 400:1 to about 50:1, from about 300:1 to about 50:1, from about 250:1 to about 50:1, from about 200:1 to about 50:1, from about 150:1 to about 50:1, or from about 100:1 to about 50:1. In some embodiments, the first stimulant (e.g., a first cytokine, such as IL-7) and the second stimulant (e.g., a second cytokine, such as IL-15) are present at a concentration ratio of about 200:1 to about 10:1, about 150:1 to about 10:1, about 125:1 to about 10:1, or about 100:1 to about 4:1. In some embodiments, the first stimulant (e.g., a first cytokine, such as IL-7) and the second stimulant (e.g., a second cytokine, such as IL-15) are present at a concentration ratio of about 100:1. In some embodiments, the first stimulant (e.g., a first cytokine, such as IL-7) and the second stimulant (e.g., a second cytokine, such as IL-15) are present at a concentration ratio of about 5:1, about 6:1, or about 7.1. In some embodiments, the first stimulant (e.g., a first cytokine, such as IL-7) and the second stimulant (e.g., a second cytokine, such as IL-15) are present at a concentration ratio of 6.25:1.

[00179] In some embodiments, the first stimulant (e.g., a first cytokine, such as IL-7) is present at a concentration that is from about 100 IU/mL to about 20,000 IU/mL. In some embodiments, the second stimulant (e.g., a second cytokine, such as IL-15) is present at a concentration that is from about 1 IU/mL to about 200 IU/mL.

[00180] In some embodiments, the first stimulant (e.g., a first cytokine, such as IL-7) is present at a concentration that is from about 100 IU/mL to about 10,000 IU/mL. In some embodiments, the second stimulant (e.g., a second cytokine, such as IL-15) is present at a concentration that is from about 1 IU/mL to about 100 IU/mL.

[00181] In some embodiments, the first stimulant (e.g., a first cytokine, such as IL-7) is present at a concentration that is from about 100 IU/mL to about 1000 IU/mL, from about 300 IU/mL to about 5000 IU/mL, or about 3000 IU/mL. In some embodiments, the first stimulant (e.g., a first cytokine, such as IL-7) is present at a concentration that is about 3000 IU/mL or about 5000 IU/mL.

[00182] In some embodiments, the first stimulant (e.g., a first cytokine, such as IL-7) is present at a concentration that is from about 2500 IU/mL to about 7500 IU/mL. In some embodiments, the second stimulant (e.g., a second cytokine, such as IL-15) is present at a concentration that is from about 25 IU/mL to about 75 IU/mL.

[00183] In some embodiments, the first stimulant (e.g., a first cytokine, such as IL-7) is present at a concentration that is from about 4000 IU/mL to about 6000 IU/mL. In some embodiments, the second stimulant (e.g., a second cytokine, such as IL-15) is present at a concentration that is from about 40 IU/mL to about 60 IU/mL. In some embodiments, the second stimulant (e.g., a second cytokine, such as IL-15) is present at a concentration that is from about 25 IU/mL to about 100 IU/mL, or about 50 IU/mL.

[00184] In some embodiments, the first stimulant (e.g., a first cytokine, such as IL-7) is present at a concentration that is about 5000 IU/mL. In some embodiments, the second stimulant (e.g., a second cytokine, such as IL-15) is present at a concentration that is about 50 IU/mL.

[00185] In some embodiments, immune cells (e.g., T cells, such as genetically modified T cells) are contacted with a first stimulator (e.g., a first cytokine, such as IL-7) and a second stimulator (e.g., a second cytokine, such as IL-15) about once a day, two days, three days, four days, five days, six days, seven days, or eight days. In some embodiments, immune cells (e.g., T cells, such as genetically modified T cells) are contacted with a first stimulator (e.g., a first cytokine, such as IL-7) and a second stimulator (e.g., a second cytokine, such as IL-15) about once a week. In some embodiments, immune cells (e.g., T cells, such as genetically modified T cells) are contacted with a first stimulator (e.g., a first cytokine, such as IL-7) and a second stimulator (e.g., a second cytokine, such as IL-15) once, twice, three times, four times, five times, six times, seven times, or eight times during a propagation process that lasts about one day, two days, three days, four days, five days, six days, seven days (e.g., about one week), about two weeks, about three weeks, or about four weeks. In some embodiments, immune cells (e.g., T cells, such as genetically modified T cells) are contacted with a first stimulator (e.g., a first cytokine, such as IL-7) and a second stimulator (e.g., a second cytokine, such as IL-15) for about two weeks.

b. Extracellular modulators of cellular metabolism

[00186] In some embodiments, the cell growth medium further comprises an extracellular modulator of cellular metabolism in addition to the first stimulator (e.g., a first cytokine, such as IL-7) and the second stimulator (e.g., a second cytokine, such as IL-15), as described herein.

[00187] In some embodiments, the extracellular modulator is extracellular potassium (K ⁺ ). In some embodiments, the extracellular potassium is present at a concentration of about 4 mM to about 50 mM, about 4 mM to about 40 mM, about 4 mM to about 30 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 15 mM to about 40 mM, about 15 mM to about 35 mM, about 15 mM to about 30 mM, or about 20 mM to about 30 mM. In some embodiments, extracellular potassium is present at a concentration that is from about 10 mM to about 30 mM or from about 20 mM to about 30 mM.

[00188] In some embodiments, the extracellular potassium is present at a concentration that is about 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM.

[00189] In some embodiments, the extracellular potassium is present at a concentration greater than about 15 mM. In some embodiments, the extracellular potassium is present at a concentration greater than or equal to about 20 mM. In some embodiments, the extracellular potassium is present at a concentration that is about 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM.

[00190] In some embodiments, the extracellular potassium is present at a concentration that is no less than about 10 mM. In some embodiments, the extracellular potassium is present at a concentration that is no less than about 20 mM. In some embodiments, the extracellular potassium is present at a concentration that is no more than about 30 mM. In some embodiments, the extracellular potassium is present at a concentration that is about 25 mM.

[00191] In some embodiments, extracellular potassium is provided as KCl.

c. Additional features and components of cell culture media

[00192] The cell growth medium described herein (e.g., a cell growth medium comprising a first stimulator that is IL-7, a second stimulator that is IL-15, and an extracellular modulator that is extracellular potassium) can be used in various methods for activating and expanding T cells that are known in the art and described, for example, in U.S. Patent No. 6,905,874, U.S. Patent No. 6,867,041, U.S. Patent No. 6,797,514, and PCT WO2012/079000, the contents of which are incorporated herein by reference in their entireties.

[00193] In some embodiments, the method further comprises contacting the PBMC or isolated T cells with an additional stimulatory molecule and a costimulatory molecule, such as anti-CD3 and anti-CD28 antibodies, typically attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2. The anti-CD3 and anti-CD28 antibodies attached to the same bead serve as a "mimicker" of an antigen-presenting cell (APC). One example is the Dynabeads® system, a CD3/CD28 activator/stimulator system for physiological activation of human T cells. In other embodiments, T cells can be activated and stimulated to proliferate by feeder cells and appropriate antibodies, as well as other cytokines (e.g., in addition to IL-7 and IL-15) using methods such as those described in U.S. Patent No. 6,040,177, U.S. Patent No. 5,827,642, and WO2012129514, the contents of which are incorporated herein by reference in their entireties. In some embodiments, cell growth media and methods of using the same exclude exogenous IL-2 as a T cell growth factor.

[00194] Other conditions suitable for culturing T cells include suitable media (e.g., minimal essential media, or RPMI 1640 media, or X-vivo ^™ medium (Lonza)), which may contain additional factors, such as the first and second stimulators for cell proliferation, for proliferation and viability, including blood serum (e.g., fetal bovine or human serum), insulin, IFN-γ, GM-CSF, TGFβ and TNF, or any other cell growth additives known to one of skill in the art. Other cell growth additives include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. Media may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo™ 10, X-Vivo™ 15 and X-Vivo™ 20, OpTmizer™, may contain added amino acids, sodium pyruvate and/or vitamins and may be serum-free or supplemented with an appropriate amount of serum (or plasma) and/or a defined set of hormones. Antibiotics such as penicillin and streptomycin are included only in experimental cultures and not in cell cultures to be administered to the subject. Target cells are maintained under conditions necessary to support growth, such as appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO ₂ ). T cells exposed to varying amounts of stimulation may exhibit different characteristics.

[00195] In some embodiments, the cells of the present invention can be expanded by co-culture with tissue or cells. The cells can also be expanded in vivo, such as in the blood of a subject after administration of the cell to the subject.

[00196] In some embodiments, the cell growth medium comprises blood serum. In some embodiments, the cell growth medium does not comprise blood serum. In some embodiments, the cell growth medium does not comprise xenogeneic components. In some embodiments, the cell growth medium is chemically defined.

d. Cell proliferation

[00197] In some embodiments, culturing cells (including immune cells such as T cells) for expansion in vitro using the cell growth medium described herein (e.g., a cell growth medium comprising a first stimulant that is IL-7, a second stimulant that is IL-15, and an extracellular modulator that is extracellular potassium) or any of the methods described herein has a duration of about one day, two days, three days, four days, five days, six days, seven days (e.g., about one week), about two weeks, about three weeks, or about four weeks. In some embodiments, the duration is from about one week to about three weeks, or from about one week to about two weeks (e.g., about 7-15 days or about 10-14 days).

[00198] In some embodiments, the cell growth medium described herein (e.g., a cell growth medium comprising a first stimulant that is IL-7, a second stimulant that is IL-15, and an extracellular modulator that is extracellular potassium), or any of the methods described herein, provide for cell expansion from about 10-fold to about 10,000-fold, as measured over a period of from about one week to about three weeks. In some embodiments, cell expansion from about 100-fold to about 1,000-fold is observed over about 7-15 days (e.g., over about 10-14 days or over about 2 weeks). In some embodiments, cell expansion of at least about 100-fold is observed over about 7-15 days (e.g., over about 10-14 days or over about 2 weeks). In some embodiments, cell proliferation of at least about 200-fold is observed within about 7-15 days (e.g., about 10-14 days or about 2 weeks).

[00199] In some embodiments, the cell growth medium described herein (e.g., a cell growth medium comprising a first stimulant that is IL-7, a second stimulant that is IL-15, and an extracellular modulator that is extracellular potassium) or any of the methods of expanding the cells provides for the production of a cell count of at least about 50×10 ⁶ to about 100×10 ⁶ over a period of about 8-16 days. In some embodiments, the cell count is at least about 100×10 ⁶ over a period of about 10-14 days (e.g., at least about 100×10 ⁶ , about 125×10 ⁶ , about 150×10 ⁶ , or about 100×10 ⁶ over a period of about 10-14 days).

e. Cellular phenotypes

[00200] In some embodiments, the cell growth medium described herein (e.g., a cell growth medium comprising a first stimulant that is IL-7, a second stimulant that is IL-15, and an extracellular modulator that is extracellular potassium), or any method of using it, provides for the formation of the desired phenotype for producing engineered immune cells.

[00201] In some embodiments, the cell growth medium described herein (e.g., a cell growth medium comprising a first stimulant that is IL-7, a second stimulant that is IL-15, and an extracellular modulator that is extracellular potassium), or any method of using it, provides for the formation of a T cell population (e.g., genetically modified T cells) enriched in T _CM and T _SCM cells.

[00202] In some embodiments, a population of T cells (e.g., genetically modified T cells) enriched in _TCM and/or _TSCM cells comprises at least about 60%, 65%, 70%, 75%, or 80% of combined _TCM and _TSCM cells. In some embodiments, a population of T cells enriched in _TCM and/or _TSCM cells comprises at least about 70% of combined _TCM and _TSCM cells. In some embodiments, a population of T cells (e.g., genetically modified T cells) enriched in _TCM and _TSCM cells comprises at least about 75% of combined _TCM and/or _TSCM cells.

[00203] In some embodiments, a population of T cells (e.g., genetically modified T cells) enriched in _TCM and/or _TSCM cells comprises at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% _TSCM cells. In some embodiments, a population of T cells enriched in _TCM and/or _TSCM cells comprises at least about 30%, 35%, 40%, or 45% _TSCM cells.

4. Obtaining engineered immune cells (including CAR T cells)

[00204] This document provides methods for using the cell growth media described herein to produce immune cells (including engineered immune cells such as CAR T cells).

[00205] A number of known techniques can be used in producing the polynucleotides, polypeptides, vectors, antigen-binding domains, immune cells, compositions, etc. of the present invention.

[00206] Prior to the in vitro manipulation or genetic modification of immune cells described herein, the cells may be obtained from a subject. The CAR-expressing cells may be obtained by an allogeneic or autologous method.

a. Source material

[0100] The cell culture media described herein (e.g., containing IL-7 + IL-15 and increased extracellular potassium) can be used to culture a variety of cells, including in vitro expansion of a variety of immune cells (e.g., genetically modified T cells). Exemplary cells are described herein.

[0101] In some embodiments, the immune cells include T cells. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some specific embodiments, T cells can be obtained from a volume of blood drawn from a subject using any number of techniques known to one of skill in the art, such as FICOLL™ separation.

[0102] Cells can be obtained from the circulating blood of an individual via apheresis. The product obtained via apheresis typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In some specific embodiments, cells collected via apheresis can be washed to remove the plasma fraction and then placed in appropriate buffer or media for subsequent processing.

[0103] In certain specific embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes, such as by PERCOLL™ gradient centrifugation. Certain subpopulations of T cells (e.g., CD28+, CD4+, CD45RA, and CD45RO+ T cells or CD28+, CD4+, CDS+, CD45RA- CD45RO+, and CD62L+ T cells) can be further isolated by positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies directed to surface markers unique to the negatively selected cells. One method of use herein is cell sorting and/or selection using negative magnetic immunoadhesion or flow cytometry, which uses a cocktail of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the cocktail of monoclonal antibodies typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present invention.

[0104] PBMCs can be used directly for genetic modification by immune cells (such as CAR or TCR) using the methods described herein. In some specific embodiments, after PBMCs are isolated, T lymphocytes can be further isolated, and both cytotoxic and helper T lymphocytes can be sorted into naive, memory T cell, and effector T cell subsets either before or after genetic modification and/or expansion.

[0105] In some embodiments, the CD8+ cells are further sorted into naive, memory stem cells, central memory cells, and effector cells by identifying cell surface antigens that are associated with each of these CD8+ cell types. In some embodiments, the expression of phenotypic markers of central memory T cells includes CD27, CD45RA, CD45RO, CD62L, CCR7, CD28, CD3, and CD127 and is negative for granzyme B. In some embodiments, the memory T stem cells are CD45RO-, CD62L+, CD8+ T cells. In some embodiments, the central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, the effector T cells are negative for CD62L, CCR7, CD28, and CD127 and positive for granzyme B and perforin. In some specific embodiments, CD4+ T cells are further sorted into subsets. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens.

b. Stem cell-derived immune cells

[0106] In some embodiments, immune cells can be derived from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. Suitable HSCs, mesenchymal cells, iPS cells, and other types of stem cells can be cultured immortalized cell lines or can be isolated directly from a patient. Various methods for isolating, developing, and/or culturing stem cells are known in the art and can be used to practice the present invention.

[0107] In some embodiments, the immune cell is an induced pluripotent stem cell (iPSC) derived from a reprogrammed T cell. In some embodiments, the starting material may be an induced pluripotent stem cell (iPSC) derived from a T cell or a cell other than a T cell. The starting material may be an embryonic stem cell. The starting material may be a B cell or any other cell from peripheral blood mononuclear cell isolates, a hematopoietic progenitor cell, a hematopoietic stem cell, a mesenchymal stem cell, an adipose stem cell, or any other type of somatic cell.

c. Genetic modification of isolated cells

[0108] Immune cells, such as T cells, can be genetically modified after isolation using known methods, or the immune cells can be activated and expanded (or differentiated in the case of progenitors) in vitro prior to genetic modification. In some embodiments, the isolated immune cells are genetically modified to reduce or eliminate expression of endogenous TCRa and/or CD52. In some embodiments, the cells are genetically modified using gene editing technology (e.g., CRISPR/Cas9, zinc finger nuclease (ZFN), TALEN, MegaTAL, meganuclease) to reduce or eliminate expression of endogenous proteins (e.g., TCRa and/or CD52). In another embodiment, immune cells, such as T cells, are genetically modified with the chimeric antigen receptors described herein (e.g., transduced with a viral vector containing one or more nucleotide sequences encoding a CAR) and then activated and/or expanded in vitro.

[0109] Certain methods for producing the constructs and engineered immune cells of the present invention are described in PCT application PCT/US15/14520, the contents of which are incorporated herein by reference in its entirety.

[0110] It should be appreciated that the PBMCs may further comprise other cytotoxic lymphocytes, such as NK cells or NKT cells. An expression vector carrying the coding sequence of a chimeric receptor as disclosed herein can be introduced into a population of donor human T cells, NK cells, or NKT cells. Successfully transduced T cells carrying the expression vector can be sorted using flow cytometry to isolate CD3-positive T cells and then expanded to increase the number of these CAR-expressing T cells, in addition to activating the cells using CD3 antibodies and IL-2 or other methods known in the art, as described elsewhere herein. Standard procedures are used to cryopreserve the CAR-expressing T cells for storage and/or recovery for use in a human subject. In one embodiment, transduction, culturing and/or expansion of T cells in vitro is carried out in the absence of non-human animal derived products, such as fetal calf serum and fetal bovine serum.

[0111] For cloning polynucleotides, a vector can be introduced into a host cell (an isolated host cell) to allow replication of the vector itself and thereby amplify copies of the polynucleotide contained therein. Cloning vectors can contain sequence components that typically include, but are not limited to, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements can be appropriately selected by one skilled in the art. For example, the origin of replication can be selected to facilitate autonomous replication of the vector in the host cell.

[0112] In certain specific embodiments, the present invention provides isolated host cells comprising a vector provided herein. Host cells comprising a vector can be used to express or clone a polynucleotide contained in the vector. Suitable host cells can include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells, such as mammalian cells, especially human cells.

[0113] The vector can be introduced into the host cell using any suitable methods known in the art, including but not limited to DEAE-dextran mediated delivery, calcium phosphate precipitation method, cationic lipid mediated delivery, liposome mediated transfection, electroporation, microparticle bombardment, receptor mediated gene delivery, polylysine mediated delivery, histone, chitosan and peptides. Standard methods for transfecting and transforming cells to express a vector of interest are well known in the art. In a further embodiment, a mixture of different expression vectors can be used to genetically modify a donor population of immune effector cells, wherein each vector encodes a different CAR as disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, wherein a portion of the engineered cells express more than one different CAR.

[0114] In one embodiment, the present invention provides a method for storing genetically engineered cells expressing a CAR or TCR. It involves cryopreserving the immune cells so that the cells remain viable after thawing. A fraction of the CAR-expressing immune cells can be cryopreserved by methods known in the art to provide a continuous source of such cells for future treatment of patients affected by cancer. If necessary, the cryopreserved transformed immune cells can be thawed, grown, and expanded to produce more such cells.

[0115] In some embodiments, the cells are formulated by first harvesting them from the culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in an amount effective for treatment. A suitable infusion medium may be any isotonic medium formulation, normal saline, Normosol™ R (Abbott), or Plasma-Lyte™ A (Baxter), but 5% dextrose in water or lactated Ringer's may also be used. The infusion medium may be supplemented with human serum albumin.

d. Allogeneic CAR T cells (ALLOCAR T™)

[0116] The process for producing allogeneic CAR T cell therapies or AlloCARs™ involves collecting healthy, selected, screened, and tested T cells from healthy donors. The T cells are then engineered to express CARs that recognize specific cell surface proteins expressed on hematologic or solid tumors. The allogeneic T cells are gene edited to reduce the risk of graft-versus-host disease (GvHD) and prevent allogeneic rejection. The T cell receptor gene (e.g., TCRα, TCRβ) is knocked out to avoid GvHD. The CD52 gene can be knocked out to make the CAR T cell product resistant to anti-CD52 antibody treatment. Thus, anti-CD52 antibody treatment can be used to suppress the host immune system and allow engraftment of the CAR T cells to achieve their full therapeutic effect. The engineered T cells then undergo a purification step and are ultimately cryopreserved in vials for delivery to patients.

e. Autologous CAR T cells (AUTOCAR T™)

[0117] Autologous chimeric antigen receptor (CAR) T-cell therapy involves collecting a patient's own cells (e.g., white blood cells, including T cells) and genetically engineering the T cells to express CARs that recognize a target expressed on the cell surface of one or more specific cancer cells and kill the cancer cells. The engineered cells are then cryopreserved and subsequently administered to the patient.

5. Pharmaceutical compositions and therapy

[00207] In some embodiments, the cells are formulated by first harvesting them from the culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in an amount effective for treatment. A suitable infusion medium may be any isotonic medium formulation, normal saline, Normosol™ R (Abbott), or Plasma-Lyte™ A (Baxter), but 5% dextrose in water or lactated Ringer's may also be used. The infusion medium may be supplemented with human serum albumin.

[00208] In embodiments, the desired therapeutic amounts of cells in the composition are typically at least 2 cells (e.g., a subset of at least 1 CD8+ central or stem memory T cell and at least 1 CD4+ helper T cell; or two or more CD8+ central or stem memory T cells; or a subset of two or more CD4+ helper T cells) or more often greater than 10 ² cells and up to and including 10 ⁶ cells, up to and including 10 ⁷ , 10 ⁸ or 10 ⁹ cells, and may be greater than 10 ¹⁰ cells. The number of cells will depend on the desired route of administration for which the composition is intended and the type of cells included therein. The desired cell density is typically greater than 10 ⁶ cells/mL and typically greater than 10 ⁷ cells/mL, typically 10 ⁸ cells/mL or greater. A clinically significant amount of immune cells can be divided into multiple infusions that collectively equal or exceed 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ or 10 ¹² cells. In some aspects of the present invention, particularly since all of the infused cells will be redirected to a specific target antigen, a smaller number of cells in the range of about 10 ⁵ /kilogram or about 10 ⁶ /kilogram (10 ⁶ -10 ¹¹ per patient) can be administered. CAR therapies can be administered multiple times at doses in these ranges. The cells can be autologous, allogeneic or heterologous to the patient receiving the therapy.

[00209] The CAR-expressing cell populations of the present invention can be administered either alone or as a pharmaceutical composition in combination with diluents and/or other components such as IL-2 or other cytokines or cell populations. The pharmaceutical compositions of the present invention can comprise a CAR or TCR-expressing cell population, e.g., the T cells described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can comprise buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose, or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide) and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[00210] Pharmaceutical compositions (solutions, suspensions, etc.) can include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides which can serve as a solvent or suspending medium, polyethylene glycols, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The preparation for parenteral administration may be placed in ampoules, disposable syringes or multiple-dose vials made of glass or plastic. The pharmaceutical composition for injection is preferably sterile.

6. Treatment methods

[00211] The present invention provides methods for treating or preventing a disease (e.g., cancer) in a patient, comprising administering to the patient in need thereof an effective amount of at least one CAR or immune cells comprising a CAR disclosed herein.

[00212] Methods of treating diseases or disorders including cancer are provided. In some embodiments, the present invention relates to generating a T cell-mediated immune response in a subject comprising administering to the subject an effective amount of engineered immune cells of the present application. In some embodiments, the T cell-mediated immune response is directed against a target cell or cells. In some embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR). In some embodiments, the target cell is a tumor cell. In some aspects, the present invention provides a method of treating or preventing a cancer, said method comprising administering to a subject in need thereof an effective amount of at least one isolated antigen-binding domain described herein. In some aspects, the present invention provides a method of treating or preventing a cancer, said method comprising administering to a subject in need thereof an effective amount of at least one immune cell, wherein the immune cell comprises at least one chimeric antigen receptor, T cell receptor and/or isolated antigen binding domain described herein. The CAR immune cells of the present invention can be used to treat cancers associated with aberrant expression of biomarkers. In some embodiments, the CAR immune cells of the present invention can be used to treat small cell lung cancer, melanoma, low grade gliomas, glioblastoma, medullary thyroid cancer, carcinoid tumors, dispersed neuroendocrine tumors of the pancreas, bladder and prostate, testicular cancer and lung adenocarcinoma with neuroendocrine features. In exemplary embodiments, CAR-containing immune cells, such as CAR T cells of the present invention, are used to treat small cell lung cancer.

[00213] Also provided are methods for reducing the size of a tumor in a subject, comprising administering to the subject an engineered cell of the present invention to the subject, wherein the cell comprises a chimeric antigen receptor comprising an antigen-binding domain and binds to an antigen on the tumor.

[00214] In some embodiments, the subject has a solid tumor or a hematologic malignancy, such as lymphoma or leukemia. In some embodiments, the engineered cell is delivered to the tumor bed. In some embodiments, the cancer is present in the bone marrow of the subject. In some embodiments, the engineered cells are autologous immune cells, such as autologous T cells. In some embodiments, the engineered cells are allogeneic immune cells, such as allogeneic T cells. In some embodiments, the engineered cells are heterologous immune cells, such as heterologous T cells. In some embodiments, the engineered cells of the present application are transfected or transduced in vivo. In other embodiments, the engineered cells are transfected or transduced ex vivo. As used herein, the term "cell in vitro" refers to any cell that is cultured ex vivo.

[00215] The terms "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dosage" of a therapeutic agent, such as engineered CAR T cells, mean any amount that, when used alone or in combination with another therapeutic agent, protects a subject from developing a disease or promotes regression of the disease, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevents worsening or disability due to the disease. The ability of a therapeutic agent to promote regression of the disease can be assessed using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[00216] The terms "patient" and "subject" are used interchangeably and include human subjects and non-human animal subjects, as well as subjects with formally diagnosed disorders, subjects without formally recognized disorders, subjects receiving medical care, subjects at risk of developing disorders, etc.

[00217] The terms "treat" and "treatment" include therapeutic treatments, prophylactic treatments, and uses in which the risk of a subject developing a disorder or other risk factor is reduced. Treatment does not require a complete cure of the disorder and includes embodiments in which the manifestation of symptoms or major risk factors are reduced. The term "prevent" does not require 100% elimination of the probability of an event. Rather, it means that the probability of occurrence of the event is reduced by the presence of the compound or by use of the method.

[00218] The desired therapeutic amounts of cells in the composition are typically at least 2 cells (e.g., a subset of at least 1 CD8+ central memory T cell and at least 1 CD4+ helper T cell), or more typically are greater than 10 ² cells and no more than 10 ⁶ , no more than 10 ⁸ or 10 ⁹ cells, inclusive, and may be greater than 10 ¹⁰ cells. The amount of cells will depend on the desired route of administration for which the composition is intended and the type of cells included therein. The desired cell density is typically greater than 10 ⁶ cells/mL and typically greater than 10 ⁷ cells/mL, typically 10 ⁸ cells/mL or greater. A clinically significant amount of immune cells can be divided into multiple infusions that collectively equal or exceed 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ or 10 ¹² cells. In some aspects of the present invention, particularly since all of the infused cells will be redirected to a specific target antigen, a smaller number of cells in the range of 10 ⁶ /kilogram (10 ⁶ -10 ¹¹ per patient) can be administered. CAR-based therapies can be administered multiple times at doses in these ranges. The cells can be autologous, allogeneic or heterologous to the patient undergoing therapy.

[00219] In some embodiments, the therapeutically effective amount of CAR T cells is about 1×10 ⁵ cells/kg, about 2×10 ⁵ cells/kg, about 3×10 ⁵ cells/kg, about 4×10 ⁵ cells/kg, about 5×10 ⁵ cells/kg, about 6×10 ⁵ cells/kg, about 7×10 ⁵ cells/kg, about 8×10 ⁵ cells/kg, about 9×10 ⁵ cells/kg, 2×10 ⁶ cells/kg, about 3×10 ⁶ cells/kg, about 4×10 ⁶ cells/kg, about 5×10 ⁶ cells/kg, about 6×10 ⁶ cells/kg, about 7×10 ⁶ cells/kg, about 8×10 ⁶ cells/kg, about 9×10 ⁶ cells/kg, approximately 1×10 ⁷ cells/kg, approximately 2×10 ⁷ cells/kg, approximately 3×10 ⁷ cells/kg, approximately 4×10 ⁷ cells/kg, approximately 5×10 ⁷ cells/kg, approximately 6×10 ⁷ cells/kg, approximately 7×10 ⁷ cells/kg, approximately 8×10 ⁷ cells/kg, or approximately 9×10 ⁷ cells/kg.

[00220] In some embodiments, target doses for CAR+/CAR-T+/TCR+ cells range from 1×10 ⁶ to 2×10 ⁸ cells/kg, such as 2×10 ⁶ cells/kg. It should be understood that doses above and below this range may be appropriate for certain subjects, and appropriate dose levels can be determined by a healthcare provider as needed. In addition, multiple doses of cells may be provided according to the present invention.

[00221] In some aspects, the present invention provides a pharmaceutical composition comprising at least one antigen-binding domain described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

[00222] The CAR-expressing cell populations of the present invention can be administered either alone or as a pharmaceutical composition in combination with diluents and/or other components such as IL-2 or other cytokines or cell populations. The pharmaceutical compositions of the present invention can comprise a CAR or TCR-expressing cell population, e.g., the T cells described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can comprise buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose, or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide) and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[00223] Pharmaceutical compositions (solutions, suspensions, etc.) can include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides which can serve as a solvent or suspending medium, polyethylene glycols, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The preparation for parenteral administration may be placed in ampoules, disposable syringes or multiple-dose vials made of glass or plastic. The pharmaceutical composition for injection is preferably sterile.

[00224] In some embodiments, when administered to a patient, engineered immune cells expressing any of the antigen-specific CARs described herein on their cell surface can reduce, kill, or lyse endogenous antigen-expressing cells of the patient. In one embodiment, the percentage of reduction or lysis of endogenous antigen-expressing cells or cells from a cell line expressing the antigen by the engineered immune cells expressing any of the antigen-specific CARs described herein is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or greater. In one embodiment, the percentage of reduction or lysis of endogenous antigen-expressing cells or cells from an antigen-expressing cell line by the engineered immune cells expressing antigen-specific CARs is from about 5% to about 95%, from about 10% to about 95%, from about 10% to about 90%, from about 10% to about 80%, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 20% to about 90%, from about 20% to about 80%, from about 20% to about 70%, from about 20% to about 60%, from about 20% to about 50%, from about 25% to about 75%, or from about 25% to about 60%. In one embodiment, the endogenous antigen expressing cells are endogenous antigen expressing cells of the bone marrow.

[00225] In one embodiment, the percentage of reduction or lysis of target cells, such as a cell line expressing an antigen, by engineered immune cells expressing an antigen-specific CAR of the present invention on their cell surface membrane can be measured using the assay disclosed herein.

[00226] The methods may further comprise administering one or more chemotherapeutic agents. In some specific embodiments, the chemotherapeutic agent is a lymphodepleting (preconditioning) chemotherapeutic agent. For example, methods for improving the physical condition of a patient in need of T cell therapy include administering to the patient specific effective doses of cyclophosphamide (from 200 mg/ ^m2 /day to 2000 mg/ ^m2 /day, from about 100 mg/ ^m2 /day to about 2000 mg/ ^m2 /day; e.g., about 100 mg/ ^m2 /day, about 200 mg/ ^m2 /day, about 300 mg/ ^m2 /day, about 400 mg/ ^m2 /day, about 500 mg/ ^m2 /day, about 600 mg/ ^m2 /day, about 700 mg/ ^m2 /day, about 800 mg/ ^m2 /day, about 900 mg/ ^m2 /day, about 1000 mg/ ^m2 /day, about 1500 mg/ ^m2 /day or approximately 2000 mg/ ^m2 /day) and certain doses of fludarabine (from 20 mg/ ^m2 /day to 900 mg/ ^m2 /day, from about 10 mg/ ^m2 /day to about 900 mg/ ^m2 /day; for example, about 10 mg/ ^m2 /day, about 20 mg/ ^m2 /day, about 30 mg/ ^m2 /day, about 40 mg/ ^m2 /day, about 40 mg/ ^m2 /day, about 50 mg/ ^m2 /day, about 60 mg/ ^m2 /day, about 70 mg/ ^m2 /day, about 80 mg/ ^m2 /day, about 90 mg/ ^m2 /day, about 100 mg/ ^m2 /day, about 500 mg/ ^m2 /day or approximately 900 mg/ ^m2 /day). A preferred dosing regimen involves treating a patient with approximately 300 mg/ ^m2 /day of cyclophosphamide and approximately 30 mg/ ^m2 /day of fludarabine daily for three days prior to administration of a therapeutically effective amount of engineered T cells to the patient.

[00227] In some embodiments, lymphodepletion further comprises administering an anti-CD52 antibody. In some embodiments, the anti-CD52 antibody is administered at a dose of about 13 mg/day IV.

[00228] In other embodiments, each of the antigen-binding domain, the transduced (or otherwise engineered) cells, and the chemotherapeutic agent is administered in an amount effective to treat a disease or condition in a subject.

[00229] In certain certain embodiments, compositions comprising CAR-expressing immune effector cells disclosed herein can be administered in combination with any number of chemotherapeutic agents, which can be administered in any order. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamin resin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, calichemycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; Antimetabolites such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; Purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; Androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; Adrenal cortex depressants such as aminoglutethimide, mitotane, trilostane; a folic acid supplement such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamole; nitracrine; pentostatin; fenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as paclitaxel (TAXOL®, Bristol-Myers Squibb) and doxetaxel (TAXOTERE®, Rhne-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylmycetes (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing. Also included within this definition are antihormonal agents that regulate or suppress the action of hormones on tumors such as antiestrogens, including, for example, tamoxifen, raloxifene, 4(5)-imidazoles, aromatase inhibitors, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing. Combinations of chemotherapeutic agents, including but not limited to CHOP, i.e., cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone, are also administered as needed.

[00230] In some embodiments, the chemotherapeutic agents are administered at the same time or one week after administration of the engineered cell, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered 1-4 weeks, or 1 week-1 month, 1 week-2 months, 1 week-3 months, 1 week-6 months, 1 week-9 months, or 1 week-12 months after administration of the engineered cell, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least 1 month before administration of the cell, polypeptide, or nucleic acid. In some embodiments, the methods further comprise administering two or more chemotherapeutic agents.

[00231] A number of additional therapeutic agents may be used in combination with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab.

[00232] Additional therapeutic agents suitable for use in the combination of the present invention include, but are not limited to, ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (KADCYLA®, imatinib (Gleevec®), cetuximab (Erbitux®, panitumumab) (Vectibix®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, mazitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, sabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, mTOR inhibitors such as everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, CDK inhibitors such as the CDK inhibitor (palbociclib).

[00233] In some embodiments, a composition comprising immune cells comprising a CAR can be administered in a therapeutic regimen for preventing cytokine release syndrome (CRS) or neurotoxicity. A therapeutic regimen for preventing cytokine release syndrome (CRS) or neurotoxicity can include administration of lenzilumab, tocilizumab, atrial natriuretic peptide (ANP), anakinra, iNOS inhibitors (e.g., L-NIL or 1400W). In additional embodiments, a composition comprising immune cells comprising a CAR can be administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDs) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolate. Illustrative NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates. Illustrative analgesics include acetaminophen, oxycodone, tramadol, and propoxyphene hydrochloride. Illustrative glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Illustrative biologic response modifiers include molecules directed against cell surface markers (eg, CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (eg, etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®), chemokine inhibitors, and adhesion molecule inhibitors. Biologic response modifiers include monoclonal antibodies as well as recombinant forms of the molecules. Illustrative DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), and minocycline.

[00234] In some specific embodiments, the compositions described herein are administered in combination with a cytokine. Examples of cytokines include lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian suppressor; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factors; integrin; thrombopoietin (TPO); Nerve growth factors (NGFs) such as NGF-beta; Platelet-derived growth factor; Transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; Insulin-like growth factors I and II; Erythropoietin (EPO); Osteoinductive factors; Interferons such as interferon-alpha, -beta and -gamma; Colony stimulating factors (CSFs) such as macrophage CSF (M-CSF); Granulocyte and macrophage CSF (GM-CSF) and Granulocyte CSF (G-CSF); Interleukins (ILs) such as IL-1, IL-1-alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-21; tumor necrosis factor such as KaKTNF-alpha or TNF-beta; and other polypeptide factors including LIF and KIT ligand (KL). As used herein, the term "cytokine" includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native sequence cytokines.

7. Methods of sorting and exhaustion

[00235] In some embodiments, methods are provided for sorting a population of immune cells in vitro, wherein a subset of the population of immune cells comprises engineered immune cells expressing antigen-specific CARs comprising epitopes specific for monoclonal antibodies (e.g., exemplary mimotope sequences). The method comprises contacting the population of immune cells with a monoclonal antibody specific for the epitopes and selecting immune cells that bind to the monoclonal antibody to obtain a population of cells enriched in engineered immune cells expressing the antigen-specific CAR.

[00236] In some embodiments, said monoclonal antibody specific for said epitope is optionally conjugated to a fluorophore. In this embodiment, the step of selecting cells that bind to the monoclonal antibody can be performed by fluorescence-activated cell sorting (FACS).

[00237] In some embodiments, said monoclonal antibody specific for said epitope is optionally conjugated to a magnetic particle. In this embodiment, the step of selecting cells that bind to the monoclonal antibody can be performed by magnetically activated cell sorting (MACS).

[00238] In some embodiments, the mAb used in the method of sorting immune cells expressing a CAR is selected from alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10 and/or ustekinumab. In some embodiments, said mAb is rituximab. In another embodiment, said mAb is QBEND-10. In another embodiment, the mAb binds to TCRα or TCRβ.

[00239] In some embodiments, the population of CAR-expressing immune cells obtained using the in vitro method of sorting CAR-expressing immune cells described above comprises at least 70%, 75%, 80%, 85%, 90%, 95% CAR-expressing immune cells. In some embodiments, the population of CAR-expressing immune cells obtained using the in vitro method of sorting CAR-expressing immune cells comprises at least 85% CAR-expressing immune cells.

[00240] In some embodiments, the population of CAR-expressing immune cells obtained using the in vitro method of sorting CAR-expressing immune cells described above exhibits increased in vitro cytotoxic activity compared to the initial (unsorted) cell population. In some embodiments, said in vitro cytotoxic activity is increased by 10%, 20%, 30%, or 50%. In some embodiments, the immune cells are T cells.

[00241] In some embodiments, the mAb is pre-bound to a support or surface. Non-limiting examples of a solid support may include a bead, an agarose bead, a magnetic bead, a plastic well plate, a glass well plate, a ceramic well plate, a column, or a cell culture bag.

[00242] CAR-expressing immune cells to be administered to a recipient may be enriched in vitro from a starting population. Methods for expanding starting populations may include selecting cells that express an antigen, such as the CD34 antigen, using combinations of density gradient centrifugation, immunomagnetic bead purification, affinity chromatography, and fluorescence-activated cell sorting.

[00243] Flow cytometry can be used to quantify specific cell types in a cell population. In general, flow cytometry is a method for quantifying components or structural features of cells, primarily by optical means. Since different cell types can be distinguished by quantifying structural features, flow cytometry and cell sorting can be used to count and sort cells of different phenotypes in a mixture.

[00244] Flow cytometry analysis involves two main steps: 1) labeling selected cell types with one or more labeled markers and 2) determining the number of labeled cells relative to the total number of cells in the population. In some embodiments, the method of labeling cell types involves binding labeled antibodies to markers expressed by a particular cell type. The antibodies can either be directly labeled with a fluorescent compound or indirectly labeled using, for example, a second fluorescently labeled antibody that recognizes the first antibody.

[00245] In some embodiments, the method used to sort CAR-expressing T cells is magnetic activated cell sorting (MACS). Magnetic activated cell sorting (MACS) is a method for separating different populations of cells based on their surface antigens (CD molecules) using superparamagnetic nanoparticles and columns. MACS can be used to obtain a pure population of cells. Cells in a single-cell suspension can be magnetically labeled using microbeads. The sample is applied to a column consisting of ferromagnetic spheres coated with a cell-friendly coating that allows for rapid and gentle separation of the cells. Unlabeled cells pass through and magnetically labeled cells remain within the column. The pass-through can be collected as an unlabeled cell fraction. After a wash step, the column is removed from the separator and the magnetically labeled cells are eluted from the column.

[00246] A detailed protocol for purification of a specific cell population, such as T cells, can be found in Basu S et al. (2010). (Basu S, Campbell HM, Dittel BN, Ray A. Purification of specific cell population by fluorescence activated cell sorting (FACS). J Vis Exp. (41): 1546).

[00247] In some aspects, the present invention provides a method for depleting antigen-specific CAR-expressing immune cells by in vivo depletion. In vivo depletion may involve administering a treatment (e.g., a molecule that binds an epitope on a CAR) to a mammal to stop the proliferation of CAR-expressing immune cells by suppression or elimination.

[00248] In one aspect, the present invention provides a method for in vivo depletion of an engineered immune cell expressing a CAR comprising a mAb-specific epitope, comprising contacting said engineered immune cell or said CAR-expressing immune cell with at least one epitope-specific mAb. In another aspect, the present invention provides a method for in vivo depletion of a CAR-expressing immune cell that comprises a chimeric scFv (e.g., formed by inserting a mAb-specific epitope) by contacting said engineered immune cell with epitope-specific antibodies. In some embodiments, the immune cells are T cells and/or the antibodies are monoclonal.

[00249] According to one embodiment, the in vivo depletion of the engineered immune cells is performed on engineered immune cells that have been pre-sorted using the in vitro method of the present invention. In this case, the same infused mAb can be used. In some embodiments, the mAb-specific antigen is a CD20 antigen and the epitope-specific mAb is rituximab. In some embodiments, the present invention provides a method for in vivo depletion of an engineered immune cell expressing a CAR comprising an epitope specific for the mAb (a CAR-expressing immune cell) in a patient, comprising contacting said CAR-expressing immune cell with at least one epitope-specific mAb.

[00250] In some embodiments, the step of contacting said engineered immune cell or said CAR-expressing immune cell with at least one epitope-specific mAb comprises infusing the epitope-specific mAb (e.g., rituximab) into the patient. In some embodiments, the amount of the epitope-specific mAb administered to the patient is sufficient to kill at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the CAR-expressing immune cells in the patient.

[00251] In some embodiments, the step of contacting said engineered immune cell or said CAR-expressing immune cell with at least one epitope-specific mAb comprises infusing the patient with 375 mg/ ^m2 of rituximab one or more times. In some embodiments, the mAb (e.g., rituximab) is administered once per week.

[00252] In some embodiments, when immune cells expressing a CAR comprising a mAb-specific epitope (CAR-expressing immune cells) are depleted in a complement dependent cytotoxicity (CDC) assay using the mAb epitope-specific epitope, the number of viable CAR-expressing immune cells is reduced. In some embodiments, the number of viable CAR-expressing immune cells is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the mAb-specific epitope is a CD20 epitope or mimotope, and/or the mAb epitope-specific epitope is rituximab.

[00253] In certain specific embodiments, in vivo depletion of CAR-engineered immune cells is accomplished by infusion of bispecific antibodies. By definition, a bispecific monoclonal antibody (BsAb) is an artificial protein that is comprised of fragments of two different monoclonal antibodies and, therefore, binds to two different types of antigens. These BsAbs and their use in immunotherapy have been reviewed in Muller D and Kontermann RE (2010) Bispecific Antibodies for Cancer Immunotherapy, BioDrugs 24 (2): 89-98.

[00254] According to another specific embodiment, the infused bispecific mAb is capable of binding both a mAb-specific epitope carried on engineered immune cells expressing the chimeric scFv and a surface antigen on an effector and cytotoxic cell (e.g., immune cells such as lymphocytes, macrophages, dendritic cells, natural killer cells (NK cells), cytotoxic T lymphocytes (CTLs)). Thus, the depletion of engineered immune cells caused by the BsAb can occur due to antibody-dependent cellular cytotoxicity (ADCC). (Deo Y M, Sundarapandiyan K, Keler T, Wallace PK, and Graziano RF, (2000), Journal of Immunology, 165 (10): 5954-5961]).

[00255] In some embodiments, the cytotoxic drug binds to epitope-specific mAbs that can be used to deplete CAR-expressing immune cells. By combining the targeting capabilities of monoclonal antibodies with the ability of cytotoxic drugs to kill cancer cells, an antibody-drug conjugate (ADC) can clearly differentiate between healthy and diseased tissue compared to the drug alone. Marketing approvals have been received for several ADCs; The technology for their production, especially on linkers, is described in ((Payne, G. (2003) Cancer Cell 3:207-212; Trail et al (2003) Cancer Immunol. Immunother. 52:328-337; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; US Patent No. 4975278).

[00256] In some embodiments, the epitope-specific mAb to be infused is pre-conjugated with a molecule capable of stimulating complement-dependent cytotoxicity (CDC). Thus, the complement system enhances or supplements the ability of antibodies to eliminate pathogens from the body. Upon stimulation, an activation cascade is initiated as a massive amplification of the response and activation of the membrane attack complex, which kills cells. Various molecules can be used for conjugation with mAbs, such as glycans (Courtois, A, Gac-Breton, S., Berthou, C, Guezennec, J., Bordron, A. and Boisset, C. (2012), Complement dependent cytotoxicity activity of therapeutic antibody fragments can be acquired by immunogenic glycan coupling, Electronic Journal of Biotechnology ISSN: 0717-3458; http://www.ejbiotechnology.info DOI: 10.2225/voll5-issue5).

8. Sets and products

[00257] The present invention provides kits comprising any of the cultured immune cells or engineered immune cells described herein and pharmaceutical compositions thereof. In some exemplary embodiments, a kit of the present invention comprises allogeneic CAR T cells for administration to a subject.

[00258] The present application also provides articles of manufacture comprising any of the therapeutic compositions or kits described herein. Examples of articles of manufacture include vials (e.g., sealed vials).

[00259] The present invention further provides kits comprising one or more containers containing a potassium solution at a desired concentration, and optionally one or more containers containing a solution containing one or more cytokines, such as IL-7 or IL-15.

EXAMPLES

[00260] As shown in the following examples, combinations of cytokine and metabolic modulators, conditions and concentrations of T cell expansion media can result in increased efficacy of allogeneic cell therapy products. For example, desired phenotypes of allogeneic CAR T cells can be achieved by using cell proliferation promoters such as IL-7, IL-15 and increased extracellular potassium.

1. Illustrative protocol for generating CAR T cells

[00261] As described herein, CAR T cells can be produced by various methods known in the art. An exemplary standard method is described herein.

[00262] To generate CAR T cells, PBMC can be purified from buffy coat samples from healthy volunteers using Ficoll density gradient medium (Ficoll Paque PLUS/GE Healthcare Life Sciences). T cells can be purified from PBMC using a commercially available T cell isolation kit (Miltenyi Biotec, catalog #130-096-535). Alternatively, primary human T cells can be directly purified from Leuko Paks (StemCell Technologies).

[00263] To produce CAR-encoding lentivirus, HEK-293T cells can be seeded at 0.4 million cells/ml in 2 ml DMEM (Gibco) supplemented with 10% FBS (Hyclone or JR Scientific) per well of a 6-well plate on day 0. On day 1, lentivirus can be produced by mixing together the lentiviral packaging vectors 1.5 μg psPAX2, 0.5 μg pMD2G, and 0.5 μg of the appropriate CAR transfer vector in 250 μl Opti-MEM (Gibco) per well of a 6-well plate (the “DNA mix”). 10 μl Lipofectamine 2000 (Invitrogen) in 250 μl Opti-MEM can be incubated at room temperature for 5 minutes and then added to the DNA mix. The mixture can be incubated at room temperature for 20 minutes and a total volume of 500 µl can be slowly added to the sides of the wells containing HEK-293T.

[00264] Purified T cells can be activated in a suitable medium. On day 2, the medium from each well of a 6-well plate can be replaced with 2 ml per well of T cell transduction medium, i.e., X-Vivo-15 supplemented with 10% FBS. On day 3, T cells can be resuspended at 0.5 million cells per ml in 1 ml of T cell transduction medium per well of a Grex-24 plate (Wilson Wolf, Cat #80192M). Lentiviral-containing supernatants from HEK293T cells can be collected and passed through a 0.45 micron filter (EMD Millipore) to remove cellular debris and then added to the T cells along with 100 IU/ml of human IL-2.

[00265] On day 5, 4.5 ml of T cell expansion medium (e.g., containing IL-7 + IL-15 and with or without extracellular potassium) can be added to each well of a Grex-24 plate. On day 9 and day 13, transduction efficiency can be determined by determining the percentage of T cells that recognize the desired antigen (e.g., recombinant antigen) using flow cytometry. Cells are expanded in larger flasks or G-Rex vessels (Wilson Wolf) as needed using the T cell expansion media described herein.

[00266] On day 14, the antigen-specific CAR T cells can be cryopreserved, such as by freezing the cells in a medium such as CryoStor® CS5, CryoStor® CS10, or CryoStor® CS2 (BioLife Solutions). The percentage of cells stained with the recombinant antigen can be normalized to clones immediately prior to cryopreservation.

2. Supplementation IL-7+15

[00267] The data in the following examples demonstrate that, for example, the use of a first stimulant and a second stimulant, such as IL-7 (5000 IU/mL) and IL-15 (50 IU/mL), optionally with increased extracellular potassium (25 mM), can provide the desired phenotype of allogeneic CAR T cells compared to classical IL-2-based processes.

[00268] The present inventors first tested the addition of IL-7 and IL-15 to a culture medium in which the potassium content was maintained at the initial level of 4 mM. As shown in Fig. 1A, when compared to an IL-2-based process (“IL-2 IL-2” → 100 IU/ml IL-2 everywhere, fresh IL-2 was added on day 5), an IL-7 + IL-15-based process (either “IL-2 IL-7+15” → 100 IU/ml IL-2 until day 2, 5000 IU/ml IL-7 and 50 IU/ml IL-15 were added on day 5, or “IL-7+15 IL-7+15” → 5000 IU/ml IL-7 and 50 IU/ml IL-15 everywhere, with fresh IL-7 and IL-15 added on day 5) resulted in an increase in TSCM (CD62L+CD45RO”) among CD19-specific T cells with CAR in the final product. As shown in Fig. 1B, IL-7+15 culture conditions also enhance the ability of CD19-specific CAR T cells to release cytokines upon exposure to target cells.

3. Studies with potassium addition and titration

[00269] To further enhance the efficacy of the allogeneic CAR T cells of the present invention, the inventors of the present invention explored combining the benefits of adding IL-7+15 at the same concentrations as in the previous experiments with extracellular nutrients known to modulate T cell metabolism. Extracellular potassium has been shown to provide a suppressive mechanism by which the tumor microenvironment (TME) suppresses effector T cells in vivo. On the other hand, increased extracellular potassium during in vitro expansion of adoptive cell therapies (ACTs) results in the retention of T cells with a younger, less differentiated phenotype.

[00270] As shown in Fig. 2A, elevated extracellular potassium conditions (40 mM, open bars) resulted in increased TSCM cell counts of allogeneic CD19 CAR T cells in both the IL-2- and IL-7+15-containing methods compared to normal (4 mM) potassium concentrations (filled bars). However, Fig. 2B unexpectedly shows that elevated extracellular potassium conditions (40 mM, open bars) negatively impacted the expansion ability of allogeneic CAR T cells in the IL-2- and IL-7+15-containing methods compared to normal (4 mM) potassium concentrations (4 mM, filled bars).

[00271] To further enhance the benefits of high extracellular potassium levels during allogeneic CAR T cell production, the present inventors adjusted the amount of extracellular potassium that is particularly effective for the process of allogeneic CAR T cell production using IL-7+15. Extracellular potassium concentrations of 25 mM and 10 mM did not adversely affect the growth of human T cells during allogeneic CAR T cell production (Figure 3). In addition, the maximum level of TSCM cell preservation was achieved at 25 mM extracellular potassium (Figure 4).

4. Efficacy studies using combined IL-7+15 and potassium supplementation

[00272] The serial killing assay involves repeatedly exposing CAR T cells to their target, causing the CAR T cells to proliferate and, in some cases, differentiate and become exhausted. This assay has been used to examine the activity of allogeneic CAR T cells expanded under various conditions.

[00273] Having identified effective concentrations of extracellular potassium for producing allogeneic CAR T cells, the inventors then proceeded to evaluate whether the combination of IL-7+15 at a concentration of 25 mM potassium improved the potency of the allogeneic CAR T cells of the invention. The combination of the IL-7+15 method supplemented with 25 mM extracellular potassium resulted in maximal potency of CAR T cells compared to making similar products in IL-2 with normal (4 mM) potassium concentrations (Figure 5). Furthermore, the combination of the IL-7+15 method with 25 mM extracellular potassium significantly improved potency compared to the IL-2 method with 25 mM potassium and even the further improved IL-7+15 method with a baseline potassium level of 4 mM.

[00274] In conclusion, the present inventors have identified a highly effective combination of cytokines (IL-7 + IL-15) and metabolic modulators (extracellular potassium) that can maximize the efficacy of allogeneic CAR T cells.

[00275] Although the disclosed ideas have been described with reference to various applications, methods, kits and compositions, it should be understood that various changes and modifications can be made without departing from the teachings of this document and the claims presented below. The above examples are provided to better illustrate the disclosed ideas and are not intended to limit the scope of the ideas presented herein. Although the present ideas have been described in the definitions of these illustrative embodiments, one skilled in the art will readily appreciate that numerous variations and modifications of these illustrative embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the present teachings.

Claims (31)

1. A cell culture medium for expanding T cells, comprising: a first stimulator and a second stimulator of cell proliferation, each independently selected from the group consisting of IL-7 and IL-15, and an extracellular modulator of cellular metabolism, which is extracellular potassium, at a concentration of no less than about 10 mM, wherein the first stimulator and the second stimulator are present at a concentration ratio of from about 250:1 to about 10:1 IU/mL. 2. A cell growth medium according to claim 1, wherein the first cell proliferation stimulator is IL-7. 3. A cell growth medium according to claim 1 or 2, wherein the second cell proliferation stimulator is IL-15. 4. A medium for growing cells according to any one of claims 1-3, wherein the first stimulant and the second stimulant are present in a concentration ratio of from about 250:1 to about 10:1, from about 200:1 to about 10:1, from about 150:1 to about 10:1, from about 100:1 to about 10:1, from about 500:1 to about 50:1, from about 250:1 to about 50:1, from about 200:1 to about 50:1, from about 150:1 to about 50:1, from about 100:1 to about 50:1, from about 500:1 to about 75:1, from about 250:1 to about 75:1, from about 200:1 to about 75:1, from about 150:1 to about 75:1, from about 100:1 to about 75:1, from about 10:1 to about 4:1, from about 8:1 to about 4:1, or from about 7:1 to about 6:1, and wherein the concentration is in IU/mL. 5. The cell growth medium of any one of claims 1-3, wherein the first stimulant and the second stimulant are present at a concentration ratio of about 150:1, about 140:1, about 130:1, about 120:1, about 110:1, about 100:1, about 90:1, about 80:1, or about 70:1, and wherein the concentration is in IU/mL. 6. The cell growth medium of any one of claims 1-5, wherein the first stimulator is IL-7 present at a concentration of about 100 IU/ml to about 5000 IU/ml; and the second stimulator is IL-15 present at a concentration of about 1 IU/ml to about 100 IU/ml. 7. The cell growth medium of claim 6, wherein IL-7 is present at a concentration of from about 300 IU/ml to about 5000 IU/ml, and IL-15 is present at a concentration of from about 25 IU/ml to about 50 IU/ml. 8. The cell growth medium of claim 7, wherein IL-7 is present at a concentration of approximately 5000 IU/ml and IL-15 is present at a concentration of approximately 50 IU/ml. 9. The cell growth medium of any one of claims 1-8, wherein the extracellular potassium is present at a concentration of from about 10 mM to about 25 mM, from about 20 mM to about 35 mM, or from about 20 mM to about 30 mM. 10. The cell growth medium of claim 9, wherein the extracellular potassium is present at a concentration of about 20 mM or about 25 mM. 11. A cell growth medium according to any one of claims 1-10, wherein the extracellular potassium is present in the form of KCl. 12. A method for producing a population of T cells in vitro, comprising culturing a starting population of T cells with a cell growth medium containing a first stimulator and a second stimulator of cell proliferation, each independently selected from the group consisting of IL-7 and IL-15, and an extracellular modulator of cellular metabolism, which is extracellular potassium, at a concentration of at least 10 mM, wherein the first stimulator and the second stimulator are present at a concentration ratio of from about 250:1 to about 10:1, and wherein the concentration is in IU/mL. 13. The method according to claim 12, wherein the cell growth medium is a cell growth medium according to any one of claims 1-11. 14. The method according to claim 12 or 13, wherein the obtained population of T cells is enriched with T _CM and/or T _SCM cells. 15. The method of any one of claims 12-14, wherein the resulting T cell population comprises at least about 30%, 35%, 40%, or 45% T _SCM cells. 16. The method of claim 15, wherein the resulting population of T cells comprises at least about 40% T _SCM cells. 17. The method of any one of claims 12-16, wherein the resulting T cell population is enriched in from about 100 times to about 1000 times more T _CM and/or T _SCM cells than the number of T _CM and/or T _SCM cells in the original T cell population, as measured over a period of about 7-16 days. 18. The method of claim 17, wherein the resulting T cell population is enriched in from about 100 times to about 1000 times more T _CM and/or T _SCM cells than the number of T _CM and/or T _SCM cells in the original T cell population, as measured over a period of about 10-14 days. 19. The method of claim 18, wherein the resulting T cell population is enriched in at least about 100 times or about 200 times more T _CM and/or T _SCM cells than the number of T _CM and/or T _SCM cells in the original T cell population. 20. The method according to any one of claims 12-19, wherein the initial population of T cells is a population of engineered T cells. 21. The method of claim 20, wherein the initial population of T cells is a population of T cells expressing one or more chimeric antigen receptors (CAR T cells). 22. The method according to any one of paragraphs 12-21, wherein the T cells are allogeneic T cells. 23. The method according to any one of claims 12-21, wherein the T cells are autologous T cells. 24. The method of claim 21, wherein the CAR T cells comprise at least about 30%, 35%, 40%, or 45% T _SCM cells. 25. The method of claim 24, wherein the CAR T cells comprise at least about 40% T _SCM cells. 26. A cell culture medium for expanding T cells, comprising: a first stimulator and a second stimulator of cell proliferation, each independently selected from the group consisting of IL-7 and IL-15; and optionally an extracellular modulator of cellular metabolism, which is extracellular potassium, at a concentration of no less than about 10 mM, wherein the first stimulator and the second stimulator are present at a concentration ratio of from about 250:1 to about 10:1, and wherein the concentration is in IU/mL. 27. The cell growth medium of claim 26, wherein the first stimulator is IL-7 present at a concentration of about 100 IU/ml to about 5000 IU/ml; and the second stimulator is IL-15 present at a concentration of about 1 IU/ml to about 100 IU/ml. 28. A cell culture medium according to any one of claims 1-11 or 26, 27, wherein the T cell is a CD4+ or CD8+ T cell. 29. A method for producing a population of T cells in vitro, comprising culturing a starting population of T cells with a cell growth medium comprising: a first stimulator and a second stimulator of cell proliferation, each independently selected from the group consisting of IL-7 and IL-15; and optionally an extracellular modulator of cellular metabolism, which is extracellular potassium, at a concentration of at least 10 mM, wherein the first stimulator and the second stimulator are present at a concentration ratio of from about 250:1 to 10:1, and wherein the concentration is in IU/ml. 30. The method of claim 28, wherein the first stimulant is IL-7 present at a concentration of about 100 IU/mL to about 5000 IU/mL; and the second stimulant is IL-15 present at a concentration of about 1 IU/mL to about 100 IU/mL. 31. The method according to any one of claims 12-25 or 29-30, wherein the T cell population is CD4+ or CD8+ T cells.