Immune cells lacking SUV39H1

Abstract

To provide compositions and engineered immune cells for use in adoptive cell therapy of cancer, and to provide methods of producing the immune cells.SOLUTION: Provided is a composition comprising immune cells engineered to lack Suv39h1. Preferably, the engineered immune cells further comprise a genetically engineered antigen receptor that specifically binds to a target antigen. The present invention also provides a method for obtaining genetically engineered immune cells, comprising a step consisting of inhibiting Suv39h1 expression and / or activity in immune cells, and optionally further comprising a step consisting of introducing into the immune cells a genetically engineered antigen receptor that specifically binds to a target antigen.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the field of adoptive therapy. The present invention provides immune cells lacking Suv39h1 with enhanced survival, potential for reconstitution, and central memory phenotype after adoptive transfer.

Background Art

[0002] Many research institutions have shown that adoptive T cell therapy (ATCT) using T cells with recombinant T cell receptor (TCR) and chimeric antigen receptor (CAR) technologies is very promising in initial clinical trials for several malignancies (Kershaw MH, Westwood JA, Darcy PK. Nat Rev Cancer. 2013;13:525 - 541).

[0003] A newly emerging theme is that the effective engraftment and long - term persistence of therapeutic T cells correlate with positive treatment effects. From several pre - clinical trials, it has become clear that naive T cells and early - differentiated T cells possess increased long - term persistence capacity (Berger C et al., J Clin Invest. 2008;118:294 - 305; Hinrichs CS et al., Proc Natl Acad Sci 2009;106:17469 - 17474; Tanel A et al., Expert Rev Vaccines. 2009;8(3):299 - 312) and can induce a strong anti - tumor response (Gattinoni L et al., J Clin Investig. 2005;115:1616 - 1626; Lugli E et al., J Clin Invest. 2013;123:594 - 599). Furthermore, the increase in the persistence of adoptively transferred cells seems to depend on the acquisition of a central memory T cell (TCM) population (Powell DJ et al., Blood. 2005;105(1):241 - 50; Huang J, Khong HT et al., J Immunother. 2005;28:258 - 267).

[0004] Stable gene transfer is routinely achieved in the clinical setting by the use of gamma-retroviral vectors to introduce CARs (see, e.g., Guest RD et al., Cancer Immunol Immunother. 2014;63:133-145) and TCRs (see in particular Johnson LA et al., Blood. 2009;114(3):535-46) into polyclonal T cells, and no overtly harmful safety signals have been shown in patients receiving CAR T cell transplantation over a period of more than 10 years (Scholler J et al., Sci Transl Med.2012;4:132ra153).

[0005] For efficient transduction with retroviral or lentiviral vectors, primary T cells need to be actively proliferating (Stacchini A et al., Leuk Res.1999;23:127-136), which is generally achieved by mitogenic stimulation of resting primary T cells.

[0006] However, upon activation, T cells progress in an irreversible linear fashion towards an effector (TE) phenotype (Mahnke YD et al., Eur J Immunol.2013;43:2797-2809; Farber DL.Semin Immunol.2009;21:84-91). Thus, mitogenic activation for retroviral or lentiviral transduction drives T cell differentiation from naive to the TE phenotype. When combined with an ex-vivo culture protocol to expand the number of transduced T cells to the numbers required for clinical application (about 10 9 -10 11 ), T cells are driven towards a more differentiated phenotype, which is not optimal for systemic persistence.

[0007] Thus, adoptive T cell therapies, including CAR T cell-based therapies, have shown significant therapeutic success, particularly in the treatment of certain blood cancers over the past few years, but efficacy has been demonstrated in only a few blood cancer types and a small number of solid tumor types. It has been hypothesized that the low efficacy of the treatment may be due to restricted survival of T cells after adoptive transfer. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0008] Accordingly, there is still a need in the art for modified or engineered T cells that exhibit enhanced central memory phenotype and enhanced survival after adoptive transfer. In particular, there is still a need for the provision of immune cells, particularly T cells, that are useful in adoptive therapy and that support effective and wide-scale cancer treatment. MEANS FOR SOLVING THE PROBLEMS

[0009] The inventors have surprisingly discovered that T cells deficient in Suv39h1 result in enhanced central memory phenotype and enhanced survival after adoptive transfer. In particular, the inventors have shown that T cells deficient in Suv39h1 accumulate and are highly efficiently reprogrammed into long-lived central memory T cells that express both CD44 and CD62L. Accordingly, the present invention relates to modified or engineered immune cells, particularly modified T cells, in which Suv39h1 is inactivated.

[0010] Accordingly, the modified or engineered immune cells described above are of great interest for use in adoptive therapy. Accordingly, the present invention more particularly relates to engineered or modified immune cells deficient in Suv39h1, preferably further comprising a genetically engineered antigen receptor that specifically binds to a target antigen.

[0011] Typically, the engineered immune cells according to claim 1 are T cells or NK cells, particularly CD4+ or CD8+ T cells. Preferred cells are T NCells, TSC M , TC M or TE M may be selected from cells and combinations thereof.

[0012] Typically, the engineered immune cells are isolated from a subject. The subject is preferably suffering from cancer or at risk of developing cancer.

[0013] The target antigen to which the genetically engineered antigen receptor specifically binds is preferably expressed in cancer cells and / or is a universal tumor antigen.

[0014] The genetically engineered antigen receptor can be a chimeric antigen receptor (CAR) containing an extracellular antigen recognition domain that specifically binds to the target antigen. The genetically engineered antigen receptor can also be a T cell receptor (TCR).

[0015] The activity and / or expression of Suv39h1 in the engineered immune cells is preferably selectively inhibited or blocked. In one embodiment, the engineered immune cells express a Suv39h1 nucleic acid encoding a non-functional Suv39h1 protein.

[0016] The present invention also includes a step of inhibiting the expression and / or activity of Suv39h1 in immune cells; optionally, a step of introducing a genetically engineered antigen receptor that specifically binds to target cells into the immune cells, relating to a method for producing genetically engineered immune cells.

[0017] Inhibition of Suv39h1 activity and / or expression preferably includes contacting or bringing into contact the cells with at least one agent that inhibits the expression and / or activity of Suv39h1 and / or disrupts the Suv39h1 gene. The agent can be selected from small molecule inhibitors; antibody derivatives, aptamers, nucleic acid molecules that block transcription or translation, or gene editing agents.

[0018] The present invention also relates to the engineered immune cells described herein, or a composition comprising said engineered immune cells, for use in adoptive cell therapy, particularly adoptive cancer therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019]

Figure 1

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Modes for Carrying Out the Invention

[0020] Definitions As used herein, the term "antibody" is used in the broadest sense and includes intact antibodies, as well as functional (antigen-binding) antibody fragments including Fab fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (VH) regions capable of specifically binding to an antigen, single-chain antibody fragments including single-chain variable fragments (scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments, including polyclonal and monoclonal antibodies. The term includes genetically engineered and / or other modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecificities, such as bispecific antibodies, diabodies, tribodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise indicated, the term "antibody" should be understood to include its functional antibody fragments. The term also includes intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0021] "Antibody fragment" means a molecule other than an intact antibody that includes a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabody; linear antibody; variable heavy chain (VH) region, single-chain antibody molecule, e.g., scFv and single-domain VH single antibody; and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment, e.g., scFv, that includes a variable heavy chain region and / or a variable light chain region.

[0022] "Single-domain antibody" is an antibody fragment that includes all or a portion of the heavy-chain variable domain of an antibody, or all or a portion of the light-chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody.

[0023] As used herein, “suppression” of gene expression means that the expression of one or more gene products encoded by a target gene in a cell is eliminated or reduced as compared to the expression level of the gene product in the absence of suppression. Exemplary gene products include the mRNA and protein products encoded by that gene. Suppression is in some cases transient or reversible and in other cases permanent. Suppression is in some cases suppression of a functional or full-length protein or mRNA, despite the fact that truncated or non-functional products may be produced. In some embodiments herein, as opposed to expression, the activity or function of a gene is suppressed. Gene suppression is generally induced by artificial methods, i.e., by the addition or introduction of a compound, molecule, complex, or composition and / or by disruption of the nucleic acid of the gene or nucleic acids associated with the gene, e.g., at the DNA level. Exemplary methods for gene suppression include gene silencing, knockdown, knockout, and / or gene disruption techniques, e.g., gene editing. Examples include antisense techniques, e.g., RNAi, siRNA, shRNA, and / or ribozymes, which generally result in a transient reduction in expression, and gene editing techniques, which result in inactivation or disruption of a target gene, e.g., by inducing cleavage and / or homologous recombination.

[0024] As used herein, "disruption" of a gene means a change in the gene sequence at the DNA level. Examples include insertions, mutations, and deletions. Disruption typically results in the suppression and / or loss of expression of the normal or "wild-type" product encoded by the gene. Examples of such gene disruptions include insertions of genes or parts of genes, frameshift mutations and missense mutations, deletions, knock-ins, and knockouts, including deletion of the entire gene. Such disruptions occur in the coding region, for example, in one or more exons, such that, for example, due to the insertion of a stop codon, a full-length product, a functional product, or any product cannot be produced. Such disruptions can also occur by disruption of a promoter or enhancer or other region affecting transcriptional activation so as to block transcription of the gene. Gene disruption includes gene targeting, including targeted gene inactivation by homologous recombination.

[0025] The cells of the present invention The cells according to the present invention are typically eukaryotic cells such as mammalian cells (also referred to as animal cells in the present invention), for example human cells.

[0026] More specifically, the cells of the present invention are bone marrow cells or lymphoid cells derived from blood, bone marrow, lymph, or lymphoid organs (especially the thymus), and include cells of the immune system (i.e., immune cells), for example cells of innate or adaptive immunity, i.e., lymphocytes, typically T cells and / or NK cells.

[0027] According to the present invention, the cells are preferably lymphocytes including, in particular, T cells, B cells, and NK cells.

[0028] The cells according to the present invention may be immune cell progenitor cells, for example lymphocyte progenitor cells, more preferably T cell progenitor cells.

[0029] T cell progenitors typically express a set of consensus markers including CD44, CD117, CD135, and Sca-1. See also Petrie HT, Kincade PW. Many roads, one destination for T cell progenitors. The Journal of Experimental Medicine. 2005;202(1):11~13.

[0030] The cells are typically primary cells, such as cells isolated directly from a subject and / or cells isolated and frozen from a subject.

[0031] With respect to the subject to be treated, the cells of the present invention can be allogeneic and / or autologous.

[0032] In some embodiments, the cells are one or more subsets of T cells or other cell types, such as the entire T cell population, CD4+ cells, CD8+ cells, and partial progenitor populations thereof, such as those defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence ability, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation.

[0033] Among the subtypes and partial progenitor populations of T cells and / or CD4+ T cells and / or CD8+ T cells, T cells include naive T (T N ) cells, effector T cells (T EFF ), memory T cells and subtypes thereof, such as stem cell memory T (TSC M ), central memory T (TC M ), effector memory T (T EM) or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), naive T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal associated invariant T (MAIT) cells, natural and adaptive regulatory T (Treg) cells, helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. The cells according to the invention have stem / memory characteristics and higher reconstitution ability upon inhibition of Suv39h1 T EFF cells, and T N cells, TSC M TC M TE M cells, and combinations thereof are preferred.

[0034] In some embodiments, one or more of the T cell populations are enriched or depleted of cells that are positive for or express one or more specific markers, such as surface markers, or negative for or express them at relatively low levels. In some cases, such markers are absent or expressed at relatively low levels in a particular population of T cells (e.g., non-memory cells) but present or expressed at relatively high levels in a particular other population of T cells (e.g., memory cells). In one embodiment, the cells (e.g., CD8 + cells or T cells, e.g., CD3 +The cells are enriched (i.e., positively selected) for cells that are positive for CD117, CD135, CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L or express them at high surface levels, and / or depleted (e.g., negatively selected) for cells that are positive for CD45RA or express them at high surface levels of CD45RA. In some embodiments, the cells are enriched or depleted for cells that are positive for CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127) or express them at high surface levels. In some examples, the CD8+ T cells are enriched for CD45RO-positive (or CD45RA-negative) and CD62L-positive cells.

[0035] For example, according to the present invention, the cells may comprise a CD4+ T cell population and / or a CD8+ T cell subpopulation, e.g., a subpopulation enriched for central memory (T CM ) cells. Alternatively, the cells may be other types of lymphocytes including natural killer (NK) cells, MAIT cells, innate lymphoid cells (ILC), and B cells.

[0036] The cells and compositions containing the cells for manipulation according to the present invention are isolated from a sample, particularly a biological sample, e.g., a biological sample obtained from or derived from a subject. Typically, the subject is in need of and / or receiving cell therapy (adoptive cell therapy). The subject is preferably a mammal, particularly a human. In one embodiment of the present invention, the subject has cancer.

[0037] Samples include tissues, liquids, and other samples taken directly from a subject, as well as samples obtained from one or more processing steps, such as separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing, and / or incubation. A biological sample may be a sample obtained directly from a biological source or a processed sample. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissues and organ samples, including processed samples derived therefrom. Samples from which cells are derived or samples in which cells are isolated are preferably blood or blood-derived samples, or apheresis products or leukoreduction products, or derived therefrom. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy material, tumors, leukemia, lymphoma, lymph nodes, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, and / or cells derived therefrom. Samples include samples derived from autologous and allogeneic sources in the context of cell therapy (typically adoptive cell therapy).

[0038] In some embodiments, the cells are derived from a cell line, such as a T cell line. The cells can also be obtained from a heterologous source, such as a mouse, rat, non-human primate, or pig. The cells are preferably human cells.

[0039] Cells lacking Suv39h1 As used herein, the term "Suv39h1" or "H3K9-histone methyltransferase Suv39h1" has its general meaning in the art and refers to the histone methyltransferase "suppressor of variegation 3-9 homolog 1 (Drosophila)" that specifically trimethylates the Lys-9 residue of histone H3 using monomethylated H3-Lys-9 as a substrate (see also Aagaard L, Laible G, Selenko P, Schmid M, Dorn R, Schotta G, Kuhfittig S, Wolf A, Lebersorger A, Singh PB, Reuter G, Jenuwein T (June 1999) "Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 encode centromere-associated proteins which complex with the heterochromatin component M3 1" EMBO J 1 8(7):1923-38). The histone methyltransferase is also known as MG44, KMT1A, SUV39H, SUV39H1, histone-lysine N-methyltransferase SUV39H1, H3-K9-HMTase 1, OTTHUMP00000024298, Su(var)3-9 homolog 1, lysine N-methyltransferase 1A, histone H3-K9 methyltransferase 1, position-effect variegation 3-9 homolog, histone-lysine N-methyltransferase or H3 lysine-9 specific 1. The human Suv39h1 methyltransferase is called O43463 in UNIPROT and is encoded by the gene Suv39h1 (gene ID: 6839 in NCBI) located on chromosome x. The term Suv39h1 according to the present invention also encompasses all orthologs of SUV39H1, such as SU(VAR)3-9.

[0040] As used herein, the expression "lacking Suv39h1" according to the present invention means inhibition or blockade of Suv39h1 activity (i.e., methylation of Lys-9 of histone H3 by H3K9-histone methyltransferase) in the cells according to the present invention.

[0041] The "inhibition of Suv39h1 activity" according to the present invention means a decrease in Suv39h1 activity of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the activity or level of the non-inhibited Suv39h1 protein. It is preferred that inhibition of Suv39h1 activity results in the substantially undetectable activity of Suv39h1 in the cell.

[0042] Inhibition of Suv39h1 activity can also be achieved by suppression of Suv39h1 gene expression or by disruption of the Suv39h1 gene. According to the present invention, the suppression reduces the expression of Suv39h1 in cells, particularly immune cells of the present invention, by at least 50, 60, 70, 80, 90, or 95% relative to the same cells produced by the method in the absence of suppression. Gene disruption can also result in reduced expression of the Suv39h1 protein or expression of a non-functional Suv39h1 protein.

[0043] The "non-functional" Suv39h1 protein as used herein means a protein whose activity has been reduced or whose detectable activity is absent as described above.

[0044] Accordingly, an inhibitor of Suv39h1 activity in the cells according to the present invention can be selected from any compound or agent that does not have the ability to inhibit methylation of Lys-9 of histone H3 by a natural or H3K9-histone methyltransferase or the ability to inhibit the expression of the H3K9-histone methyltransferase SUV39H1 gene.

[0045] Inhibition of Suv39h1 in immune cells according to the present invention can be permanent, irreversible or temporary or reversible. However, it is preferred that the Suv39h1 inhibition is permanent and irreversible. Inhibition of Suv39h1 in cells can be achieved before or after injection of the cells into the target patient, as described below.

[0046] Genetically engineered cells according to the invention In some embodiments, the cells comprise one or more nucleic acids introduced via genetic engineering encoding one or more antigen receptors.

[0047] Typically, the nucleic acids are heterologous (i.e., nucleic acids not normally found in, for example, the cells being engineered and / or the organism from which such cells are derived). In some embodiments, the nucleic acids are not naturally occurring, including chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

[0048] Among the antigen receptors according to the present invention are genetically engineered T cell receptors (TCRs) and their components, as well as functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs).

[0049] Chimeric antigen receptor (CAR) In some embodiments, the engineered antigen receptors comprise chimeric antigen receptors (CARs), including activating CARs or stimulatory CARs, co-stimulatory CARs (see WO 2014 / 055668), and / or inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Med., 5(215), December 2013).

[0050] A chimeric antigen receptor (CAR) (also known as a chimeric immune receptor, chimeric T cell receptor, artificial T cell receptor) is an engineered receptor that confers any specificity to immune effector cells (T cells). Typically, these receptors are used to transplant the specificity of monoclonal antibodies to T cells, and transcription of their coding sequences is facilitated by retroviral vectors.

[0051] In some embodiments, a CAR generally includes an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components via a linker and / or transmembrane domain(s). Such molecules typically mimic or resemble signals through natural antigen receptors, signals through such receptors in combination with co-stimulatory receptors, and / or signals through co-stimulatory receptors alone.

[0052] In some embodiments, a CAR is constructed to have specificity for a particular antigen (or marker or ligand), e.g., an antigen expressed on a particular cell type targeted by adoptive therapy such as a cancer marker. Thus, a CAR typically includes, in its extracellular portion, one or more antigen-binding molecules, e.g., one or more antigen-binding fragments, domains, or portions, or one or more antibody variable domains, and / or antibody molecules.

[0053] The portions used to bind to an antigen fall into one of three general categories: single-chain antibody fragments (scFvs) derived from antibodies, Fab’ selected from libraries, or native ligands involved in their cognate receptors (for first-generation CARs). Success stories in each of these categories have been reported, in particular, in Sadelain M, Brentjens R, Riviere I. The basic principles of chimeric antigen receptor (CAR) design. Cancer discovery. 2013;3(4):388 - 398 (see especially Table 1), which is incorporated herein. scFvs derived from murine immunoglobulins are commonly used because they can be readily derived from well-characterized monoclonal antibodies.

[0054] Typically, a CAR includes a single-chain antibody fragment (scFv) derived from one or more antigen-binding portions of an antibody molecule, e.g., the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).

[0055] In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds to a target cell or disease, such as an antigen like a cancer marker or cell surface antigen of tumor cells or cancer cells, e.g., any target antigen described herein or well-known in the art.

[0056] In some embodiments, the CAR comprises an antibody or antigen-binding fragment (e.g., scFv) that specifically recognizes an antigen expressed on the surface of a cell, such as an intact antigen.

[0057] In some embodiments, the CAR comprises a TCR-like antibody, e.g., an antibody or antigen-binding fragment (e.g., scFv), that specifically recognizes an intracellular antigen, such as a tumor-associated antigen, presented on the cell surface as an MHC-peptide complex. In some embodiments, the antibody or antigen-binding portion thereof that recognizes the MHC-peptide complex can be expressed in a cell as part of a recombinant receptor, e.g., an antigen receptor. Among the antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Generally, a CAR comprising an antibody or antigen-binding fragment that exhibits TCR-like specificity for a peptide-MHC complex can also be referred to as a TCR-like CAR.

[0058] In some embodiments, the antigen-specific binding or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains within the CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid the association of such a domain with the transmembrane domains of the same or different surface membrane proteins and to minimize the interaction with other members of the receptor complex.

[0059] In some embodiments, the transmembrane domain is derived from either a natural source or a synthetic source. When the source is natural, the domain can be derived from a membrane-bound protein or a transmembrane protein. Transmembrane regions include those derived from the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 (i.e., those containing at least their transmembrane region(s)). The transmembrane domain can also be synthetic.

[0060] In some embodiments, a short oligopeptide linker or a polypeptide linker, e.g., a linker of 2 - 10 amino acids in length, is present to form a connection between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0061] CARs generally contain at least one intracellular signaling component. First-generation CARs typically had the intracellular domain from the CD3ζ chain, a major transducer of signals from the endogenous TCR. Second-generation CARs typically further contain intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic terminus of the CAR, providing additional signals to T cells. In preclinical studies, second-generation CARs have been shown to improve the anti-tumor activity of T cells. More recently, third-generation CARs combine multiple signaling domains, e.g., CD3ζ - CD28 - 41BB or CD3ζ - CD28 - OX40, to enhance potency.

[0062] For example, a CAR can include an intracellular component of the TCR complex, such as a TCR CD3+ chain that mediates T cell activation and cytotoxicity, such as the CD3 zeta chain. Thus, in some embodiments, the antigen-binding molecule is linked to one or more cell signaling modules. In some embodiments, the cell signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. A CAR can also further include one or more additional molecules, such as a portion of the Fc receptor gamma, CD8, CD4, CD25, or CD16.

[0063] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the CAR activates at least one of the normal effector functions or responses of the corresponding non-engineered immune cell (typically a T cell). For example, the CAR can induce T cell functions, such as cytolytic activity or T helper activity, secretion of cytokines or other factors.

[0064] In some embodiments, the intracellular signaling domain(s) includes the cytoplasmic sequence of a T cell receptor (TCR), and in some embodiments also, in the native context, the cytoplasmic sequence of a co-receptor that acts in concert with such a receptor to initiate signaling after antigen receptor binding, and / or any derivative or variant of such a molecule, and / or any synthetic sequence having the same functional capacity.

[0065] T cell activation is, in some embodiments, described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act antigen-independently to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some embodiments, the CAR includes one or both of such signaling components.

[0066] In some embodiments, the CAR comprises a primary cytoplasmic signaling sequence that modulates primary activation of the TCR complex either in a stimulatory or inhibitory manner. A primary cytoplasmic signaling sequence that acts in a stimulatory manner may include a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signaling sequences that include an ITAM include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signaling molecule(s) of the CAR comprise a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3 zeta.

[0067] The CAR can also include the signaling domain and / or transmembrane portion of a co-stimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some embodiments, the same CAR includes both an activation component and a co-stimulatory component; alternatively, one activation domain is provided by the CAR, while the co-stimulatory component is provided by another CAR that recognizes a different antigen.

[0068] The CAR or other antigen receptor can also be an inhibitory CAR (e.g., iCAR) and includes intracellular components that attenuate or suppress a response, such as an immune response. Examples of such intracellular signaling components are those identified as immune checkpoint molecules, including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, and EP2 / 4 adenosine receptors including the A2AR. In some embodiments, the engineered cell comprises a signaling domain of such an inhibitory molecule, or an inhibitory CAR derived therefrom, that acts to attenuate its response. Such CARs are used, for example, to reduce the likelihood of off-target effects where the antigen recognized by the activation receptor, e.g., the CAR, is also expressed or may be expressed on the surface of normal cells.

[0069] TCR In some embodiments, the genetically engineered antigen receptor comprises a recombinant T cell receptor (TCR) and / or a TCR cloned from a native T cell.

[0070] The term "T cell receptor" or "TCR" refers to a molecule that includes variable alpha and beta chains (also known as TCRα and TCRβ, respectively) or variable gamma and delta chains (also known as TCRγ and TCRδ, respectively) and can specifically bind to an antigen peptide bound to an MHC receptor. In some embodiments, the TCR is of the αβ type. Typically, TCRs that exist in the αβ and γδ types are generally structurally similar, but the T cells expressing them may have different anatomical locations or functions. TCRs can be identified on the surface of cells or in a soluble form. Generally, TCRs are identified on the surface of T cells (or T lymphocytes), where they generally play a role in recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, the TCR may also include a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, for example, Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd edition, Current Biology Publications, p. 4:33, 1997). For example, in some aspects, each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Unless otherwise specified, the term "TCR" is to be understood to encompass its functional TCR fragments. This term also encompasses intact or full-length TCRs, including αβ or γδ type TCRs.

[0071] Accordingly, for the purposes of this specification, references to a TCR include any TCR or functional fragment thereof, such as the antigen-binding portion of a TCR that binds to a specific antigen peptide bound to an MHC molecule, i.e., an MHC-peptide complex. The "antigen-binding portion" or "antigen-binding fragment" of a TCR can be used interchangeably and refers to a molecule that contains a part of the structural domain of the TCR but binds to the antigen (e.g., MHC-peptide complex) to which the full TCR binds. In some cases, the antigen-binding portion includes the variable domains of the TCR, such as the variable alpha and variable beta chains of the TCR, that are sufficient to form a binding site for binding to a specific MHC-peptide complex. For example, generally, each chain contains a variable domain that typically contains three complementarity-determining regions.

[0072] In some embodiments, the variable domains of the TCR chains associate to form loops or complementarity-determining regions (CDRs) similar to immunoglobulins, which confer antigen recognition, determine peptide specificity by forming the binding site of the TCR molecule, and determine peptide specificity. Typically, similar to immunoglobulins, the CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., Pwc. Nat’l Acad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the major CDR involved in the recognition of processed antigens, although CDR1 of the alpha chain also interacts with the N-terminal portion of the antigen peptide, while CDR1 of the beta chain has been shown to interact with the C-terminal portion of the peptide. CDR2 is thought to recognize MHC molecules. In some embodiments, the variable region of the beta chain can include an additional hypervariable (HV4) region.

[0073] In some embodiments, the TCR chain includes a constant domain. For example, similar to immunoglobulins, the extracellular portion of the TCR chain (e.g., the α chain, β chain) can include two immunoglobulin domains, an N-terminal variable domain (e.g., Vα or Vβ; typically, amino acids 1-116 based on Kabat numbering, Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th edition) and one constant domain adjacent to the cell membrane (e.g., the α chain constant domain or Cα, typically amino acids 117-259 based on Kabat, the β chain constant domain or Cβ, typically amino acids 117-295 based on Kabat). For example, in some cases, the extracellular portion of the TCR formed by two chains includes two membrane-proximal constant domains and two membrane-distal variable domains containing CDRs. The constant domain of the TCR domain includes a short connecting sequence in which cysteine residues form disulfide bonds to link the two chains. In some embodiments, the TCR may further have additional cysteine residues in each of the α and β chains such that the TCR contains two disulfide bonds within the constant domain.

[0074] In some embodiments, the TCR chain can include a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain includes a cytoplasmic-side terminus. In some cases, this structure allows the TCR to bind to other molecules such as CD3. For example, a TCR that includes a constant domain and a transmembrane region can anchor the protein to the cell membrane and associate with the invariant subunits of the CD3 signaling apparatus or complex.

[0075] Generally, CD3 is a multi - protein complex that can possess three different chains (γ, δ, and ε) and the ζ chain in mammals. For example, in mammals, the complex can include a homodimer of the CD3γ chain, CD3δ chain, two CD3ε chains, and the CD3ζ chain. The CD3γ chain, CD3δ chain, and CD3ε chain are highly related cell - surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ chain, CD3δ chain, and CD3ε chain are negatively charged, which is characteristic of enabling these chains to associate with the positively charged T - cell receptor chains. The intracellular termini of the CD3γ chain, CD3δ chain, and CD3ε chain each contain one conserved motif known as an immunoreceptor tyrosine - based activation motif or ITAM, whereas each CD3ζ chain contains three conserved motifs. Generally, ITAM is involved in the signaling ability of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in signal propagation from the TCR to the cell. The CD3 - ε chain and ζ - chain together with the TCR form what is known as the T - cell receptor complex.

[0076] In some embodiments, the TCR may be a heterodimer of two chains α and β (or optionally γ and δ), or a single - chain TCR construct. In some embodiments, the TCR is, for example, a heterodimer containing two separate chains (α chain and β chain or γ chain and δ chain) linked by one or more disulfide bonds.

[0077] Exemplary antigen receptors that include a CAR and a recombinant TCR, and methods for engineering and introducing the receptors into cells include, for example, International Publication No. WO 2000 / 04257, International Publication No. WO 2013 / 126726, International Publication No. WO 2012 / 129514, International Publication No. WO 2014 / 031687, International Publication No. WO 2013 / 166321, International Publication No. WO 2013 / 071154, International Publication No. WO 2013 / 123061, U.S. Patent Application Publication No. 2002 / 0131960, Publication No. 2013 / 287748, Publication No. 2013 / 0149337, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application Publication No. 2537416, and / or those described in Sadelain et al., Cancer Discov. April 2013;3(4):388-398; Davila et al., (2013) PLoS ONE 8(4):e61338; Turtle et al., Curr. Opin. Immunol. October 2012;24(5):633-39; Wu et al., Cancer, March 2012, 18(2):160-75. In some embodiments, genetically engineered antigen receptors include the CAR described in U.S. Patent No. 7,446,190 and the CAR described in International Publication No. WO 2014 / 055668A1.

[0078] Antigen Among the antigens targeted by genetically engineered antigen receptors are those expressed in the context of a disease, condition, or cell type to be targeted via adoptive cell therapy. Diseases and conditions include proliferative, neoplastic, and malignant diseases and disorders, particularly cancer.

[0079] Cancer can be a solid cancer or a "liquid tumor," also known as a tumor of the hematopoietic and lymphoid tissues, including, in particular, leukemia and lymphoma, that affects, for example, the blood, bone marrow, and lymphatic system. Examples of liquid tumors include, for example, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL) (including various lymphomas such as mantle cell lymphoma, non-Hodgkin lymphoma (NHL), adenoma, squamous cell carcinoma, laryngeal cancer, gallbladder and bile duct cancer, and cancers of the retina such as retinoblastoma).

[0080] Solid cancers include, in particular, cancers that affect one of the organs selected from the group consisting of the colon, rectum, skin, endometrium, lung (including non-small cell lung cancer), uterus, bone (osteosarcoma, chondrosarcoma, Ewing sarcoma, fibrosarcoma, giant cell tumor, ameloblastoma, and chordoma), liver, kidney, esophagus, stomach, bladder, pancreas, neck, brain (e.g., meningioma, glioblastoma, low-grade astrocytoma, oligodendroglioma, pituitary tumor, schwannoma, and metastatic brain tumor), ovary, breast, head and neck region, testis, prostate, and thyroid.

[0081] The cancer according to the present invention is preferably a cancer that affects the above-mentioned blood, bone marrow, and lymphatic system. Typically, the cancer is multiple myeloma or is associated with multiple myeloma.

[0082] The disease according to the present invention also includes infectious diseases or conditions, such as, but not limited to, viral infections, retroviral infections, bacterial infections, and protozoal infections, immunodeficiency, cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus infections or conditions; autoimmune or inflammatory diseases or conditions, such as arthritis, such as rheumatoid arthritis (RA), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, asthma, and / or diseases or conditions associated with transplantation.

[0083] In some embodiments, the antigen is a polypeptide. In some embodiments, the antigen is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed in cells of a disease or condition, such as tumor cells or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed in normal cells and / or in engineered cells. In some such embodiments, the multi-targeting methods and / or gene disruption methods provided herein are used to improve specificity and / or efficacy.

[0084] In some embodiments, the antigen is a universal tumor antigen. The term "universal tumor antigen" generally refers to an immunogenic molecule, such as a protein, that is expressed at a higher level in tumor cells than in non-tumor cells and is expressed in tumors of various origins. In some embodiments, the universal tumor antigen is expressed in more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more human cancers. In some embodiments, the universal tumor antigen is expressed in at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or more different types of tumors. In some cases, the universal tumor antigen may be expressed in non-tumor cells, such as normal cells, but at a lower level than in tumor cells. In some cases, the universal tumor antigen is not expressed at all in non-tumor cells, for example, not expressed in normal cells. Exemplary universal tumor antigens include, for example, human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2 / neu, Wilms tumor gene 1 (WT1), livin, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, or cyclin (D1). Peptide epitopes of tumor antigens, including universal tumor antigens, are known in the art and, in some aspects, can be used to prepare MHC-restricted antigen receptors, such as TCRs or TCR-like CARs (see, for example, International Publication Nos. WO 2011009173 or WO 2012135854 of PCT applications, and US Patent Application Publication No. 20140065708).

[0085] In some embodiments, the antigen is expressed in multiple myeloma and is, for example, CD38, CD138, and / or CS-1. Other exemplary multiple myeloma antigens include CD56, TIM-3, CD33, CD123, and / or CD44. Antibodies or antigen-binding fragments directed against such antigens are known and are described, for example, in U.S. Patent Nos. 8,153,765, 8,603,477, 8,008,450; those described in U.S. Patent Application Publication No. 20120189622, and those described in International Publication Nos. 2006099875, 2009080829, or 2012092612 of PCT applications. In some embodiments, such antibodies or antigen-binding fragments thereof (e.g., scFv) can be used to generate a CAR.

[0086] In some embodiments, the antigen is expressed or upregulated on the surface of cancer cells or tumor cells, which may also be expressed in immune cells such as resting or activated T cells. For example, in some cases, the expression of hTERT, survivin, and other universal tumor antigens has been reported to be present in lymphocytes including activated T lymphocytes (see, for example, Weng et al., (1996) J Exp. Med., 183:2471-2479; Hathcock et al., (1998) J Immunol, 160:5702-5706; Liu et al., (1999) Proc. Natl Acad Sci. 96:5147-5152; Turksma et al., (2013) Journal of Translational Medicine, 11:152). Similarly, in some cases, CD38 and other tumor antigens are also expressed in immune cells such as T cells and can be upregulated, for example, in activated T cells. For example, in some embodiments, CD38 is a known T cell activation marker.

[0087] In some embodiments provided herein, immune cells, such as T cells, can be engineered to suppress or disrupt the gene encoding the antigen of the immune cell so that the expressed genetically engineered antigen receptor does not specifically bind to the antigen in the context of its expression in the immune cell itself. Thus, in some aspects, this can avoid off-target effects, such as binding of engineered immune cells to each other, which can, for example, reduce the efficacy of the engineered cells in adoptive cell therapy.

[0088] In some embodiments, for example in the case of an inhibitory CAR, the target is an off-target marker, for example an antigen that is not expressed in diseased cells or cells to be targeted, but is expressed in normal or non-diseased cells, which express a disease-specific target that is targeted by an activating or stimulating receptor in the same engineered cell. Exemplary such antigens are MHC molecules, such as MHC class I molecules, which, for example, are downregulated in relation to the treatment of a disease or condition, but continue to be expressed in non-target cells.

[0089] In some embodiments, the engineered immune cells can contain an antigen that targets one or more other antigens. In some embodiments, the one or more other antigens are tumor antigens or cancer markers. In some embodiments, other antigens targeted by the antigen receptor on the provided immune cells include, for example, orphan tyrosine kinase receptor ROR1, tEGFR, Her2, Ll-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EphA2, ErbB2, 3 or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, Ll-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gplOO, carcinoembryonic antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms tumor 1 (WT-1), cyclin, such as cyclin A1 (CCNA1), and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV, or other pathogens.

[0090] In some embodiments, the CAR binds to a pathogen-specific antigen. In some embodiments, the CAR is specific for viral antigens (e.g., HIV, HCV, HBV, etc.), bacterial antigens, and / or parasitic antigens.

[0091] In some embodiments, the cells of the invention are genetically engineered to express on the cell surface two or more genetically engineered receptors, each receptor recognizing a different antigen and typically each comprising a different intracellular signaling component. Such multi-targeting approaches are described, for example, in International Patent Application Publication No. WO 2014 / 055668 A1 (e.g., a combination of an activating CAR and a co-stimulatory CAR that targets two different antigens that are individually present on off-target cells, e.g., normal cells, but only together on the cells of the disease or condition to be treated), and Fedorov et al., Sci. Transl. Med., 5(215) (December 2013) (cells expressing an activating CAR and an inhibitory CAR, e.g., where the activating CAR binds to one antigen that is expressed on both normal cells or non-diseased cells and the cells of the disease or condition to be treated, and the inhibitory CAR binds to another antigen that is expressed only on normal cells or cells where treatment is not desired).

[0092] In some situations, overexpression of a stimulatory factor (e.g., a lymphokine or cytokine) can be toxic to the subject. Thus, in some situations, the engineered cells include genetic segments that render the cells sensitive to negative selection in vivo, e.g., when administered in adoptive immunotherapy. For example, in some embodiments, the cells are engineered such that they can be eliminated as a result of a change in the in vivo state of the patient to whom they are administered. A negatively selectable phenotype can be obtained by insertion of a gene that confers sensitivity to a drug administered, e.g., a compound. Negatively selectable genes include the herpes simplex virus type I thymidine kinase (HSV-I TK) gene that confers ganciclovir sensitivity (Wigler et al., Cell 11:223, 1977); the cellular hypoxanthine phosphoribosyl transferase (HPRT) gene, the cellular adenine phosphoribosyl transferase (APRT) gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[0093] In other embodiments of the present invention, cells, such as T cells, are not engineered to express a recombinant receptor, but rather a natural antigen receptor specific for a desired antigen, such as tumor-infiltrating lymphocytes and / or T cells cultured in vitro or ex vivo during the incubation step(s), to promote the proliferation of cells having a specific antigen specificity. For example, in some embodiments, the cells are generated for adoptive cell therapy by isolating tumor-specific T cells, such as autologous tumor-infiltrating lymphocytes (TILs). Direct targeting of human tumors using autologous tumor-infiltrating lymphocytes can, in some cases, mediate tumor regression (see Rosenberg SA et al., (1988) N Engl J Med. 319:1676-1680). In some embodiments, the lymphocytes are extracted from the excised tumor. In some embodiments, such lymphocytes are expanded in vitro. In some embodiments, such lymphocytes are cultured with lymphokines (such as IL-2). In some embodiments, such lymphocytes mediate specific lysis of autologous tumor cells but not of allogeneic tumor cells or autologous normal cells.

[0094] Among additional nucleic acids for introduction, such as genes, there are those that improve the efficacy of therapy, for example, by promoting the survival and / or function of the transplanted cells; genes that provide genetic markers for selecting and / or evaluating cells, for example, for evaluating in vivo survival or localization; genes that improve safety, for example, as described in Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991), and Riddell et al., Human Gene Therapy 3:319 - 338 (1992), such as genes that render cells sensitive to negative selection in vivo; also refer to the publications of international applications PCT / US91 / 08442 and PCT / US94 / 05601 by Lupton et al., which describe the use of bifunctional selectable fusion genes derived from fusing a dominant positive selection marker with a negative selection marker. For example, refer to columns 14 - 17 of U.S. Patent No. 6,040,177 to Riddell et al.

[0095] Method for obtaining cells according to the present invention The present invention also relates to a method for producing modified or engineered immune cells, comprising the step of inhibiting the expression and / or activity of Suv39h1 in immune cells.

[0096] The method for obtaining cells according to the present invention preferably further comprises the step of introducing a genetically engineered antigen receptor that specifically binds to a target antigen into the immune cells.

[0097] Inhibition of the expression and / or activity of Suv39h1 and introduction of a genetically engineered antigen receptor that specifically binds to a target antigen in immune cells can be carried out simultaneously or consecutively in any order.

[0098] Inhibition of Suv39h1 According to the present invention, the engineered immune cells can be contacted with at least one agent that inhibits or blocks the expression and / or activity of Suv39h1.

[0099] The agent can be selected from the following small molecule inhibitors: typically, antibody derivatives such as intrabodies, nanobodies, affibodies that block or inhibit the expression or activity of Suv39h1; typically, aptamers that block or inhibit the expression or activity of Suv39h1; nucleic acid molecules that block transcription or translation, such as antisense molecules complementary to Suv39h1; RNA interference agents (e.g., small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), or piwiRNA (piRNA)); ribozymes, and combinations thereof.

[0100] Also, at least one agent may be an exogenous nucleic acid comprising a) an engineered non-natural Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) guide RNA that hybridizes to the Suv39h1 genomic nucleic acid sequence, and / or b) a nucleotide sequence encoding a CRISPR protein (typically, a type II Cas9 protein). Optionally, the cell is transgenic to express the Cas9 protein. Also, the agent can be a zinc finger protein (ZFN) or a TAL protein.

[0101] The term "organic small molecule" refers to a molecule of a size comparable to that of organic molecules commonly used in pharmaceuticals. This term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred organic small molecules have a size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0102] In certain embodiments, an inhibitor of the H3K9 histone methyltransferase SUV39H1 is chaetocin (CAS 28097-03-2) as described in Greiner D, Bonaldi T, Eskeland R, Roemer E, Imhof A. "Identification of a specific inhibitor of the histone methyltransferase SU(VAR)3-9", Nat Chem Biol. August 2005;l(3):143-5.; Weber, H.P. et al. "The molecular structure and absolute configuration of chaetocin", Acta Cryst, B28, 2945-2951 (1972); Udagawa, S. et al. "The production of chaetoglobosins, sterigmatocystin, O-methylsterigmatocystin, and chaetocin by Chaetomium spp. and related fungi", Can. J. Microbiol, 25, 170-177 (1979); and Gardiner, D.M. et al. "The epipolythiodioxopiperazine (ETP) class of fungal toxins: distribution, mode of action, functions and biosynthesis", Microbiol, 151, 1021-1032 (2005). For example, chaetocin is commercially available from Sigma Aldrich.

[0103] Inhibitors of SUV39H1 can also be ETP69 (Rac-(3S,6S,7S,8aS)-6-(benzo[d][1,3]dioxol-5-yl)-2,3,7-trimethyl-1,4-dioxohexahydro-6H-3,8a-epidithiopyrrolo[1,2-a]pyrazine-7-carbonitrile), a racemic analog of the epidithiodiketopiperazine alkaloid ketosin A (see International Publication No. 2014066435, also see Baumann M, Dieskau AP, Loertscher BM et al., Tricyclic Analogues of Epidithiodioxopiperazine Alkaloids with Promising In Vitro and In Vivo Antitumor Activity. Chemical science (Royal Society of Chemistry: 2010). 2015;6:4451-4457 and Snigdha S, Prieto GA, Petrosyan A et al., H3K9me3 Inhibition Improves Memory, Promotes Spine Formation, and Increases BDNF Levels in the Aged Hippocampus. The Journal of Neuroscience. 2016;36(12):3611-3622).

[0104] The inhibitory activity of the compound can be determined using various methods described in Greiner D. et al., Nat Chem Biol. August 2005;l(3):143-5 or Eskeland, R. et al., Biochemistry 43, 3740-3749 (2004).

[0105] Inhibition of Suv39h1 within cells can be achieved before or after injection into the target patient. In some embodiments, the inhibition defined above is performed in vivo after administering the cells to the subject. Typically, the Suv39h1 inhibitor defined herein can be included in a composition containing the cells. Also, Suv39h1 can be administered separately, simultaneously, or after administering the cell(s) to the subject.

[0106] Typically, inhibition of Suv39h1 according to the present invention can be achieved by incubating the cells according to the present invention with a composition containing at least one of the aforementioned pharmacological inhibitors. The inhibitor is included during the in vitro expansion of anti-tumor T cells, thereby altering their reconstitution, survival, and therapeutic effect after adoptive transfer.

[0107] Inhibition of Suv39h1 in cells according to the present invention can be achieved using intrabodies. Intrabodies are antibodies that bind intracellularly to their antigen after being produced in the same cell (for reviews, see, for example, Marshall AL, Dubel S, and Boldicke T, "Specific in vivo knockdown of protein function by intrabodies," MAbs. 2015;7(6):1010-35, also Van Impe K, Bethuyne J, Cool S, Impens F, Ruano-Gallego D, De Wever O, Vanloo B, Van Troys M, Lambein K, Boucherie C et al., "A nanobody targeting the F-actin capping protein CapG restrains breast cancer metastasis," Breast Cancer Res 2013;15:R116; Hyland S, Beerli RR, Barbas CF, Hynes NE, Wels W., "Generation and functional characterization of intracellular antibodies interacting with the kinase domain of human EGF receptor," Oncogene 2003;22:1557-67; Lobato MN, Rabbitts TH., "Intracellular antibodies and challenges facing their use as therapeutic agents," Trends Mol Med 2003;9:390-6, and Donini M, Morea V, Desiderio A, Pashkoulov D, Villani ME, Tramontano A, Benvenuto E., "Engineering stable cytoplasmic intrabodies with designed specificity," J Mol Biol. July 4, 2003;330(2):323-32).

[0108] An intrabody can be produced by cloning each cDNA from existing hybridoma clones, or more simply, a new scFv / Fab can be selected from in vitro display techniques such as phage display that results in genes necessary to encode antibodies from the start, enabling the pre-design of the fine specificity of the antibody in more detail. In addition, bacterial, yeast, mammalian cell surface display, and ribosome display can be used. However, the most commonly used in vitro display system for the selection of specific antibodies is phage display. In a procedure called panning (affinity selection), recombinant antibody phages are selected by incubation of the antigen with the antibody phage repertoire. This process is repeated several times, resulting in an enriched antibody repertoire containing specific antigen binders for almost all possible targets. To date, in vitro assembled recombinant human antibody libraries have already yielded thousands of novel recombinant antibody fragments. It should be noted that a prerequisite for specific protein knockdown by cytoplasmic intrabodies is that the antigen is neutralized / inactivated by antibody binding. Five different approaches have emerged for generating suitable antibodies: 1) in vivo selection of functional intrabodies in eukaryotes such as yeast and prokaryotes such as Escherichia coli (antigen-dependent and -independent); 2) generation of antibody fusion proteins to improve cytosolic stability; 3) use of special frameworks to improve cytosolic stability (e.g., by grafting CDRs in stable antibody frameworks or introducing synthetic CDRs); 4) use of single-domain antibodies to improve cytosolic stability; and 5) selection of disulfide bond-free stable intrabodies. These approaches are described in detail especially in Marschall, A.L. et al., mAbs 2015, as mentioned above.

[0109] The most commonly used format for intrabodies is the scFv, consisting of the heavy (H) and light (L) chain variable antibody domains (VH and VL) held together by a short flexible linker sequence (often (Gly4Ser)3) to avoid the need to separately express and assemble the two antibody chains of a full IgG or Fab molecule. Alternatively, a Fab format that further includes the C1 domain of the heavy chain and the constant region of the light chain has been used. In recent years, a new possible format for intrabodies, the scFab, has been described. The scFab format offers the prospect of easier subcloning of the Fab gene into intracellular expression vectors, but a future challenge is whether it offers any advantage over the well-established scFv format. In addition to scFv and Fab, bispecific formats have been used as intrabodies. A bispecific Tie-2×VEGFR-2 antibody targeted to the ER demonstrated an extended half-life compared to the monospecific antibody counterpart. A bispecific transmembrane intrabody was developed as a special format for simultaneously recognizing intracellular and extracellular epitopes of epidermal growth factor, combining the distinct features of the related monospecific antibodies, namely, inhibition of autophosphorylation and ligand binding.

[0110] Another intrabody format particularly suitable for cytoplasmic expression is the single-domain antibody (also called nanobody), which is derived from camelids or consists of one human VH domain or one human VL domain. Such single-domain antibodies often have advantageous properties, such as high stability; excellent solubility; ease of library cloning and selection; and high expression yields in E. coli and yeast.

[0111] Intrabodym genes can be expressed inside target cells after transfection with an expression plasmid or viral transduction with a recombinant virus. Typically, selection aims to achieve optimal intrabodym transfection and production levels. The success of transfection and subsequent intrabodym production can be analyzed by immunoblot detection of the produced antibody, but for the evaluation of the exact intrabodym / antigen interaction, co-immunoprecipitation of HEK293 cell extracts transiently co-transfected with the corresponding antigen and intrabodym expression plasmid can be used.

[0112] Inhibition of Suv39h1 in the cells according to the invention can also be achieved using aptamers that inhibit or block the expression or activity of Suv39h1. Aptamers are a class of molecules that represent an alternative to antibodies from the perspective of molecular recognition. Aptamers are oligonucleotide (DNA or RNA) or oligopeptide sequences that have the ability to recognize substantially any class of target molecules with high affinity and specificity.

[0113] Oligonucleotide aptamers can be isolated by the in vitro evolution method of a random sequence library (Systematic Evolution of Ligands by Exponential enrichment (SELEX)), as described by Tuerk C. and Gold L in 1990. The random sequence library can be obtained by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer of a unique sequence that is ultimately chemically modified. The possible modifications, uses, and advantages of this class of molecules are outlined by Jayasena S.D. in 1999.

[0114] Peptide aptamers consist of antibody variable regions that are sterically constrained and displayed by a platform protein such as Escherichia coli thioredoxin A, which is selected from a combinatorial library by the two-hybrid method (Colas P, Cohen B, Jessen T, Grishina I, McCoy J, Brent R. "Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2", Nature. April 11, 1996; 380(6574):548-550).

[0115] Inhibition of Suv39h1 in cells according to the present invention can also be achieved using affibody molecules. Affibodies are small proteins engineered to bind with high affinity to a number of target proteins or peptides and, as they mimic monoclonal antibodies, are members of the family of antibody mimics (for a review, see Lofblom J, Feldwisch J, Tolmachev V, Carlsson J, Stahl S, Frejd FY. Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett. June 18, 2010; 584(12):2670 - 80). Affibody molecules are based on engineered variants (Z - domains) of the B - domain within the immunoglobulin - binding region of staphylococcal protein A and, in theory, bind specifically to a given target. Affibody molecule libraries are generally constructed by combinatorial randomization of amino acids 13 in helices 1 and 2 that originally constitute the Fc - binding surface of the Z - domain. The library is typically displayed on phage and subsequently biopanned against the desired target. When increasing primary affinity, affinity maturation can generally be achieved by either helix shuffling or sequence alignment combined with directed combinatorial mutagenesis, which results in improved binders. Newly identified molecules with altered binding surfaces generally maintain the original helical structure as well as high stability, although specific exceptions with interesting properties have been reported. Due to their small size and rapid folding properties, affibody molecules can be produced by chemical peptide synthesis.

[0116] In other embodiments of the invention, inhibition of Suv39h1 activity can be achieved by gene repression / suppression by RNA interference (RNAi), such as gene knockdown using small interfering RNA (siRNA), small hairpin RNA (shRNA), or ribozymes. siRNA technology includes those based on RNAi that utilize double-stranded RNA molecules having sequences homologous to the nucleotide sequence of mRNA transcribed from a gene, and sequences complementary to the nucleotide sequence. siRNA can generally be siRNA comprising a plurality of RNA molecules that are homologous / complementary to one region of the mRNA transcribed from a gene or to a different region.

[0117] Antisense oligonucleotides, including antisense RNA molecules and antisense DNA molecules, act to directly block the translation of the H3K9-histone methyltransferase Suv39h1, thus preventing protein translation or increasing mRNA degradation, and thus reducing the level of the H3K9-histone methyltransferase SUV39H1, and thus its activity in cells. For example, antisense oligonucleotides complementary to specific regions of the mRNA transcript sequence encoding the H3K9-histone methyltransferase SUV39H1 of at least about 15 bases can be synthesized, for example, by conventional phosphodiester techniques and administered, for example, by intravenous injection or infusion. Methods for using antisense techniques to specifically inhibit the gene expression of genes whose sequences are known are well known in the art (see, for example, U.S. Patent Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

[0118] As used herein, "RNA interfering agent" is defined as any agent that interferes with or inhibits the expression of a target biomarker gene by RNA interference (RNAi). Such RNA interfering agents include, but are not limited to, nucleic acid molecules comprising RNA molecules homologous to the target gene of the present invention (e.g., Suv39h1), or fragments thereof, small interfering RNAs (siRNAs), and small molecules that interfere with or inhibit the expression of a target nucleic acid by RNA interference (RNAi).

[0119] Small interfering RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. By contacting a subject or cell with a small double-stranded RNA (dsRNA) or a vector or construct that causes the production of a small double-stranded RNA such that the expression of the H3K9-histone methyltransferase SUV39H1 gene is specifically inhibited, the expression of the H3K9-histone methyltransferase SUV39H1 gene can be reduced (i.e., RNA interference or RNAi). Methods for selecting appropriate dsRNAs or dsRNA-encoding vectors are well known in the art for genes whose sequences are known (see, e.g., Tuschl, T. et al. (1999); Elbashir, S.M. et al. (2001); Hannon, G.J. (2002); McManus, M.T. et al. (2002); Brummelkamp, T.R. et al. (2002); U.S. Patent Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01 / 36646, WO 99 / 32619, and WO 01 / 68836). The phosphodiester bonds of the siRNAs of the present invention are advantageously protected, in whole or in part. This protection is generally carried out by chemical routes using methods known in the art. The phosphodiester bonds can be protected, for example, by thiol or amine functional groups or by phenyl groups. The 5' and / or 3' ends of the siRNAs of the present invention are also advantageously protected using, for example, the above-described techniques for protecting phosphodiester bonds. The siRNA sequence advantageously comprises at least 12 contiguous dinucleotides or derivatives thereof.

[0120] As used herein, the term "siRNA derivative" with respect to the present nucleic acid sequence refers to a nucleic acid having a percentage of identity of at least 90%, preferably at least 95%, for example, at least 98%, more preferably at least 98% with erythropoietin or a fragment thereof.

[0121] As used herein, the expression "percentage of identity" between two nucleic acid sequences means the percentage of identical nucleic acids between the two sequences to be compared, obtained by the best alignment of said sequences, and this percentage is purely statistical and the differences between these two sequences are randomly distributed across the nucleic acid sequences. As used herein, "best alignment" or "optimal alignment" means an alignment that gives the highest percentage of identity determined (see below). Sequence comparison between two nucleic acid sequences is usually achieved by comparing these sequences previously aligned according to the best alignment; this comparison is achieved in segments of the comparison to identify and compare local regions of similarity. The best sequence alignment for performing the comparison can be achieved, in addition to by hand, by using the general homology algorithm developed by SMITH and WATERMAN (Ad. App. Math. Vol. 2, p. 482, 1981), by using the local homology algorithm developed by NEDDLEMAN and WUNSCH (J. Mol. Biol, Vol. 48, p. 443, 1970), by using the similarity method developed by PEARSON and LIPMAN (Proc. Natl. Acd. Sci. USA, Vol. 85, p. 2444, 1988), by using computer software using such algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr. Madison, WI USA), by using the MUSCLE multiple alignment algorithm (Edgar, Robert C, Nucleic Acids Research, Vol. 32, p. 1792, 2004). It is preferred that BLAST software can be used to obtain the best local alignment.The percentage of identity between two nucleic acid sequences is determined by comparing these two optimally aligned sequences, and in order to obtain the optimal alignment between these two sequences, the nucleic acid sequence can include additions or deletions relative to the reference sequence. The percentage of identity is calculated by determining the number of identical positions between these two sequences, dividing this number by the total number of positions being compared, and multiplying the resulting value by 100 to obtain the percentage of identity between these two sequences.

[0122] shRNA (small hairpin RNA) can also function as an inhibitor of expression for use in the present invention. shRNA typically consists of a short (e.g., 19 - 25 nucleotides) antisense strand, followed by a 5 - 9 nucleotide loop, and a similar sense strand. Alternatively, the sense strand may precede the nucleotide loop structure and the antisense strand may follow.

[0123] Ribozymes can also function as inhibitors of expression for use in the present invention. A ribozyme is an enzymatic RNA molecule that can catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence - specific hybridization of the ribozyme molecule to a complementary target RNA, followed by nucleotide strand cleavage. Engineered hairpin or hammerhead - type motif ribozyme molecules that specifically and efficiently catalyze the nucleotide strand cleavage of the H3K9 - histone methyltransferase SUV39H1 mRNA sequence are thus useful within the scope of the present invention. The specific ribozyme cleavage site within any potential RNA target is first identified by scanning the target molecule for ribozyme cleavage sites that typically include the following sequences: GUA, GUU, and GUC. Once identified, a short RNA sequence between approximately 15 - 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features such as secondary structures that may render the oligonucleotide sequence unsuitable.

[0124] Both antisense oligonucleotides and ribozymes useful as inhibitors of expression can be prepared by known methods. Such methods include techniques for chemical synthesis, for example, by solid-phase phosphoramadite chemical synthesis. Alternatively, antisense RNA molecules can be produced by in vitro or in vivo transcription of DNA sequences encoding the RNA molecules. Such DNA sequences can be incorporated into a variety of vectors that incorporate suitable RNA polymerase promoters, such as the T7 or SP6 polymerase promoter. Various modifications to the oligonucleotides of the present invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of ribonucleotide or deoxyribonucleotide flanking sequences to the 5' and / or 3' ends of the molecule, or the use of phosphorothioates or 2'-O-methyls instead of phosphodiester linkages within the oligonucleotide backbone.

[0125] The antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the present invention can be delivered in vivo either alone or in association with a vector. In its broadest sense, a "vector" is any vehicle that can facilitate the transfer of an antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid into a cell, preferably a cell that expresses H3K9-histone methyltransferase SUV39H1. Preferably, the vector transports the nucleic acid to the cell with reduced degradation as compared to the degree of degradation that would occur in the absence of the vector. Generally, vectors useful in the present invention include, but are not limited to, plasmids, phagemids, viruses, and other vehicles derived from viral or bacterial sources that have been engineered by the insertion or incorporation of an antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence. Viral vectors are the preferred type of vector and include, but are not limited to, nucleic acid sequences derived from the following viruses: retroviruses such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus and Rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type virus; polyomavirus; Epstein-Barr virus; papillomavirus; herpes virus; vaccinia virus; poliovirus; and RA virus such as retrovirus. Other vectors known in the art but not named can be readily used.

[0126] The preferred viral vectors are based on non-cytolytic eukaryotic viruses in which non-essential genes have been replaced by the gene of interest. Non-cytolytic viruses include retroviruses (e.g., lentiviruses), and their life cycle involves reverse transcription from genomic viral RNA to DNA and subsequent proviral integration into the host cell DNA. Retroviruses are approved for human gene therapy trials. The most useful retroviruses are replication-defective (i.e., they can direct the synthesis of the desired protein but cannot produce infectious particles). Such genetically modified retroviral expression vectors have general utility for high-efficiency gene transduction in vivo. Standard protocols for producing replication-defective retroviruses (including steps of uptake of exogenous genetic material into a plasmid, transfection of a packaging cell line with the plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture medium, and infection of target cells with the viral particles) are provided in Kriegler, 1990 and Murry, 1991).

[0127] Viruses preferred for certain applications are adenoviruses and adeno-associated (AAV) viruses, double-stranded DNA viruses that have already been approved for human use in gene therapy. In fact, 12 different AAV serotypes (AAV1 - 12), each with different tissue tropisms, are known (Wu, Z Mol Ther 2006;14:316 - 27). Recombinant AAV is derived from the dependent parvovirus AAV2 (Choi, VW J Virol 2005;79:6801 - 07). Adeno-associated viruses types 1 - 12 can be engineered to be replication-defective and can infect a wide range of cell types and species (Wu, Z Mol Ther 2006;14:316 - 27). This further has advantages such as heat and lipid solvent stability; high transduction frequencies in diverse lineages of cells including hematopoietic cells; and the lack of superinfection inhibition, thus allowing multiple lineages of transduction. According to reports, adeno-associated viruses integrate into human cell DNA in a site-specific manner, thereby minimizing the potential for insertional mutagenesis and variability of transgene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infection has been followed in tissue culture for more than 100 passages in the absence of selection pressure, suggesting that adeno-associated virus genome integration is a relatively stable event. Adeno-associated viruses can also function in an episomal manner.

[0128] Other vectors include plasmid vectors. Plasmid vectors are widely described in the art and are well known to those skilled in the art. See, for example, Sambrook et al., 1989. In recent years, plasmid vectors have been used as DNA vaccines for delivering antigen - encoding genes to cells in vivo. Plasmid vectors are particularly advantageous for this use because they do not have many of the same safety concerns as viral vectors. However, such plasmids having a promoter compatible with the host cell can express peptides from genes operably encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC / CMV, SV40, and pBlueScript. Other plasmids are well known to those skilled in the art. Moreover, plasmids can be custom - designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids can be delivered by various parenteral, mucosal, and topical routes. For example, DNA plasmids can be injected intramuscularly, intradermally, subcutaneously, or by other routes. This can also be administered by intranasal spray or droplets, rectal suppositories, and orally. This can also be administered to epithelial or mucosal surfaces using a gene gun. Plasmids can be given in aqueous solution, dried onto the surface of gold particles, or associated with another DNA delivery system including, but not limited to, liposomes, dendrimers, cochleate delivery vehicles, and microencapsulation.

[0129] The antisense oligonucleotides, siRNA, shRNA, or ribozyme nucleic acid sequences according to the present invention are generally placed under the control of a heterologous regulatory region, for example, a heterologous promoter. The promoter can be specific for Müller glial cells, microglial cells, endothelial cells, pericytes, and astrocytes. For example, specific expression in Müller glial cells can be obtained by the promoter of a suitable glutamine synthetase gene. The promoter can be, by way of example, a viral promoter such as the CMV promoter, or any synthetic promoter.

[0130] Inhibition or disruption of the Suv39h1 gene Inhibition of Suv39h1 in the cells according to the present invention can also be achieved by suppressing or disrupting the Suv39h1 gene, for example, by deletion, such as deletion of the whole gene, exon or region, and / or substitution with an exogenous sequence, and / or mutation, such as a frameshift mutation or missense mutation within the gene, typically within the exon of the gene. In some embodiments, a premature stop codon is incorporated into the gene by disruption so that the Suv39h1 protein is not expressed or is non-functional. The disruption is generally carried out at the DNA level. The disruption is generally permanent, irreversible or not transient.

[0131] In some embodiments, gene disruption or suppression is achieved using a gene editing agent, such as a DNA targeting molecule that specifically binds or hybridizes to a gene, such as a DNA binding protein or DNA binding nucleic acid, or a complex, compound, or composition containing the same. In some embodiments, the DNA targeting molecule comprises a DNA binding domain, such as a zinc finger protein (ZFP) DNA binding domain, a transcription activator-like protein (TAL) or TAL effector (TALE) DNA binding domain, a CRISPR DNA binding domain, or a DNA binding domain from a meganuclease.

[0132] The binding domains of the zinc finger, TALE, and CRISPR systems can be "engineered" to bind to a predetermined nucleotide sequence.

[0133] In some embodiments, the DNA targeting molecule, complex, or combination comprises a DNA binding molecule and one or more additional domains, such as an effector domain that promotes gene suppression or disruption. For example, in some embodiments, gene disruption is carried out by a fusion protein comprising a DNA binding protein and a heterologous regulatory domain or a functional fragment thereof.

[0134] Typically, the additional domain is a nuclease domain. Thus, in some embodiments, gene disruption is facilitated by gene editing or genome editing using a fusion protein composed of an engineered protein, such as a nuclease and a nuclease-containing complex, or a sequence-specific DNA binding domain fused or complexed with a non-specific DNA cleavage molecule such as a nuclease.

[0135] These target chimeric nucleases or nuclease-containing complexes effect precise genetic modifications by inducing a target double-strand or single-strand break and stimulating cellular DNA repair mechanisms, including error-prone non-homologous end joining (NHEJ) and homologous recombination repair (HDR). In some embodiments, the nuclease is an endonuclease, such as a zinc finger nuclease (ZFN), TALE nuclease (TALEN), RNA-guided endonuclease (RGEN), such as a CRISPR-associated (Cas) protein, or a meganuclease. Such systems are well known in the art (see, e.g., U.S. Patent No. 8,697,359; Sander and Joung (2014) Nat. Biotech. 32:347-355; Hale et al., (2009) Cell 139:945-956; Karginov and Hannon (2010) Mol. Cell 37:7; U.S. Patent Application Publication Nos. 2014 / 0087426 and 2012 / 0178169; Boch et al., (2011) Nat. Biotech. 29:135-136; Boch et al., (2009) Science 326:1509-1512; Moscou and Bogdanove (2009) Science 326:1501; Weber et al., (2011) PLoS One 6:e19722; Li et al., (2011) Nucl. Acids Res. 39:6315-6325; Zhang et al., (2011) Nat. Biotech. 29:149-153; Miller et al., (2011) Nat. Biotech. 29:143-148; Lin et al., (2014) Nucl. Acids Res. 42:e47). Such genetic strategies can use a constitutive expression system or an inducible expression system according to methods well known in the art.

[0136] ZFP and ZFN; TAL, TALE, TALEN In some embodiments, the DNA targeting molecule comprises a DNA binding protein, such as one or more zinc finger proteins (ZFPs) or transcription activator-like proteins (TALs), fused to an effector protein, such as an endonuclease. Examples include ZFNs, TALEs, and TALENs. See Lloyd et al., Frontiers in Immunology, 4(221), 1-7 (2013).

[0137] In some embodiments, the DNA targeting molecule comprises one or more zinc finger proteins (ZFPs) or domains thereof that bind DNA in a sequence-specific manner. A ZFP or domain thereof is a protein or an intradomain within a larger protein that binds DNA in a sequence-specific manner via one or more zinc finger regions of the amino acid sequence within a binding domain whose structure is stabilized by coordination of zinc ions. Generally, the sequence specificity of a ZFP can be modified by making amino acid substitutions at four helix positions (-1, 2, 3, and 6) on the zinc finger recognition helix. Thus, in some embodiments, the ZFP or ZFP-containing molecule is non-natural and is engineered, for example, to bind to a selected target site. See, for example, Beerli et al., (2002) Nature Biotechnol. 20:135-141; Pabo et al., (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al., (2001) Nature Biotechnol. 19:656-660; Segal et al., (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al., (2000) Curr. Opin. Struct. Biol. 10:411-416.

[0138] In some embodiments, the DNA targeting molecule is, or comprises, a zinc finger DNA binding domain fused to a DNA cleavage domain so as to form a zinc finger nuclease (ZFN). In some embodiments, the fusion protein comprises a cleavage domain (or cleavage half-domain) from at least one type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. In some embodiments, the cleavage domain is derived from the type IIS restriction endonuclease Fok I. See, for example, U.S. Patent Nos. 5,356,802; 5,436,150 and 5,487,994; and Li et al., (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al., (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al., (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al., (1994b) J. Biol. Chem. 269:31,978-31,982.

[0139] In one aspect, the ZFNs efficiently generate double-strand breaks (DSBs), for example, at a predetermined site in the coding region of a target gene (i.e., Suv39h1). Typical target gene regions include exons, regions encoding the N-terminal region, the first exon, the second exon, and promoter or enhancer regions. In some embodiments, transient expression of the ZFNs promotes highly efficient and permanent disruption of the target gene in the engineered cells. In particular, in some embodiments, delivery of the ZFNs results in permanent disruption of the gene with an efficiency exceeding 50%. Many gene-specific engineered zinc fingers are commercially available. For example, Sangamo Biosciences (Richmond, CA, USA), in collaboration with Sigma-Aldrich (St. Louis, MO, USA), has developed a platform for zinc finger construction (CompoZr) that allows researchers to avoid all zinc finger construction and validation, and provides zinc fingers specifically targeted to thousands of proteins. Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405. In some embodiments, commercially available zinc fingers are used or custom designed. (See, for example, Sigma-Aldrich catalog numbers CSTZFND, CSTZFN, CTI1-1KT, and PZD0020).

[0140] In some embodiments, the DNA targeting molecule comprises a natural or engineered (non-natural) transcription activator-like protein (TAL) DNA binding domain, such as a transcription activator-like effector (TALE) protein. See, e.g., U.S. Patent Application No. 20110301073. In some embodiments, the molecule is a DNA-binding endonuclease, such as a TALE-nuclease (TALEN). In some aspects, a TALEN is a fusion protein comprising a DNA binding domain derived from a TALE and a nuclease catalytic domain that cleaves a nucleic acid target sequence. In some embodiments, the TALE DNA binding domain is engineered to bind to a target sequence within a gene encoding a target antigen and / or an immunosuppressive molecule. For example, in some aspects, the TALE DNA binding domain can target CD38 and / or an adenosine receptor, such as A2AR.

[0141] In some embodiments, a TALEN recognizes and cleaves a target sequence within a gene. In some aspects, cleavage of the DNA results in a double-strand break. In some aspects, the cleavage stimulates the rate of homologous recombination or non-homologous end joining (NHEJ). Generally, NHEJ is an imperfect repair process that often results in a change in the DNA sequence at the cleavage site. In some aspects, the repair mechanism involves the rejoining of what remains at the two DNA ends either through direct religation (Critchlow and Jackson, Trends Biochem Sci. 1998 Oct;23(10):394-8) or through so-called microhomology-mediated end joining. In some embodiments, repair via NHEJ results in small insertions or deletions that can be used to disrupt and thereby suppress a gene. In some embodiments, the modification can be a substitution, deletion, or addition of at least one nucleotide. In some aspects, cells in which a cleavage-induced mutagenesis event, i.e., a mutagenesis event subsequent to an NHEJ event, has occurred can be identified and / or selected by methods well known in the art.

[0142] TALE repeats can be engineered to specifically target the Suv39h1 gene (Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405). A library of TALENs targeting 18,740 human protein-coding genes has been constructed (Kim et al., Nature Biotechnology. 31, 251-258 (2013)). Custom-designed TALE arrays are commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). In particular, TALENs targeting CD38 are commercially available (see Gencopoeia, catalog numbers HTN222870-1, HTN222870-2, and HTN222870-3, available from www.genecopoeia.com / product / search / detail.php?prt=26&cid=&key=HTN222870 on the World Wide Web). Exemplary molecules are described, for example, in U.S. Patent Application Nos. 2014 / 0120622 and 2013 / 0315884.

[0143] In some embodiments, the TALEN is introduced as a transgene encoded by one or more plasmid vectors. In some aspects, the plasmid vector can include a selectable marker that provides for the identification and / or selection of cells that have received the vector.

[0144] RGEN (CRISPR / Cas system) Gene suppression can be carried out using one or more DNA-binding nucleic acids, for example, through disruption via an RNA-guided endonuclease (RGEN), or other forms of suppression by another RNA-guided effector molecule. For example, in some embodiments, gene suppression can be carried out using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated proteins. See Sander and Joung, Nature Biotechnology, 32(4):347-355.

[0145] Generally, a "CRISPR system" comprehensively refers to transcripts and other elements that are involved in the expression of CRISPR-associated ("Cas") genes or induce their activity, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or the active partial tracrRNA), tracr-mate sequences (the "direct repeats" and the partially processed direct repeats by tracrRNA in the context of the endogenous CRISPR system), guide sequences (also called "spacers" in the context of the endogenous CRISPR system), and / or other sequences and transcripts derived from the CRISPR locus.

[0146] Typically, a CRISPR / Cas nuclease or CRISPR / Cas nuclease system includes a non-coding RNA molecule (guide RNA) that binds sequence-specifically to DNA, and a CRISPR protein having nuclease activity (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a type I, type II, or type III CRISPR system, e.g., a Cas nuclease. The CRISPR protein is preferably a cas enzyme, e.g., 9. Cas enzymes are well-known in the art. For example, the amino acid sequence of the Streptococcus pyogenes (S. pyogenes) Cas9 protein can be identified in the SwissProt database under accession number Q99ZW2. In some embodiments, the Cas nuclease and gRNA are introduced into cells. In some embodiments, the CRISPR system induces DSBs at the target site and subsequent breaks, as discussed herein. In other embodiments, a Cas9 variant, considered a "nickase," can be used to introduce a single-strand nick at the target site. Pairs of nickases are also used, e.g., to improve specificity, each directed by a pair of different gRNA target sequences. In yet another embodiment, catalytically inactive Cas9 can be fused to a heterologous effector domain, e.g., a transcriptional repressor, to affect gene expression.

[0147] Generally, the CRISPR system is characterized by elements that promote the formation of the CRISPR complex at the site of the target sequence. Typically, in the context of the formation of the CRISPR complex, a "target sequence" generally means a sequence that is designed such that the guide sequence has complementarity, where the formation of the CRISPR complex is promoted by hybridization between the target sequence and the guide sequence. Full complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote the formation of the CRISPR complex. The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. Generally, a sequence or template that can be used for recombination into a target locus containing the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In some embodiments, an exogenous template polynucleotide can be referred to as an editing template. In some embodiments, the recombination is homologous recombination.

[0148] In some embodiments, one or more vectors that drive the expression of one or more elements of the CRISPR system are introduced into a cell such that the expression of the elements of the CRISPR system directs the formation of the CRISPR complex at one or more target sites. For example, the Cas enzyme, the guide sequence linked to the tracr-mate sequence, and the tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined within a single vector, and one or more additional vectors can provide any components of the CRISPR system not included in the first vector. In some embodiments, the CRISPR system elements combined within a single vector can be arranged in any suitable orientation. In some embodiments, the CRISPR enzyme, the guide sequence, the tracr mate sequence, and the tracr sequence are operably linked to the same promoter and expressed. In some embodiments, the vector comprises a regulatory element operably linked to an enzyme coding sequence that encodes a CRISPR enzyme such as a Cas protein.

[0149] In some embodiments, a CRISPR enzyme combined with (optionally complexed with) a guide sequence is delivered to a cell. Typically, the CRISPR / Cas9 technology can be used to knockdown the gene expression of Suv39h1 in the engineered cells. For example, a Cas9 nuclease and a guide RNA specific to the Suv39h1 gene can be introduced into the cell using, for example, a lentiviral delivery vector or any of a number of known delivery methods or vehicles for introducing into cells, for example, any of a number of known methods or vehicles for delivering Cas9 molecules and guide RNAs (see also below).

[0150] Delivery of nucleic acids encoding gene disrupting molecules and complexes In some embodiments, a nucleic acid encoding a DNA targeting molecule, complex, or combination is administered or introduced into a cell. Typically, viral and non-viral gene delivery methods can be used to introduce nucleic acids encoding components of the CRISPR, ZFP, ZFN, TALE, and / or TALEN systems into cells in culture.

[0151] In some embodiments, a polypeptide is synthesized in situ in a cell as a result of introduction into the cell of a polynucleotide encoding the polypeptide. In some aspects, the polypeptide can be produced extracellularly and then introduced therein.

[0152] Methods for introducing polynucleotide constructs into animal cells are known and include, by way of non-limiting example, stable transformation methods in which the polynucleotide construct is integrated into the genome of the cell, transient transformation methods in which the polynucleotide construct is not integrated into the genome of the cell, and virus-mediated methods.

[0153] In some embodiments, the polynucleotide can be introduced into cells by, for example, recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, etc. Transient transformation methods include microinjection, electroporation, or particle guns. The nucleic acid is administered in the form of an expression vector. The expression vector is preferably a retroviral expression vector, an adenoviral expression vector, a DNA plasmid expression vector, or an AAV expression vector.

[0154] Non-viral delivery methods of nucleic acids include lipofection, nucleofection, microinjection, biolistic, virosome, liposome, immunoliposome, polycation or lipid:nucleic acid conjugate, naked DNA, artificial virion, and drug-enhanced DNA uptake. Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic lipids and neutral lipids suitable for effective receptor recognition lipofection of polynucleotides include those of Felgner, International Publication Nos. 91 / 17424; 91 / 16024. Delivery can be delivery to cells (e.g., in vitro or ex vivo administration) or delivery to a target tissue (e.g., in vivo administration).

[0155] RNA or DNA viral systems include retroviruses, lentiviruses, adenoviruses, adeno-associated, and herpes simplex virus vectors for gene transfer.

[0156] For reviews of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., Current Topics in Microbiology and Immunology, Doerfler and Bohm (eds.) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0157] Reporter genes, including, but not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP), can encode gene products that function as markers for measuring changes or modifications in the expression of gene products by introducing them into cells.

[0158] Cell preparation Cell isolation involves one or more preparation and / or cell separation steps of non-affinity systems by techniques well known in the art. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents to, for example, remove unwanted components, concentrate desired components, and lyse or remove cells sensitive to specific reagents. In some examples, cells are separated based on one or more characteristics, such as density, adhesion characteristics, size, sensitivity, and / or resistance to specific components.

[0159] In some embodiments, the cell preparation includes steps for freezing the cells, for example for cryopreservation, either before or after isolation, incubation, and / or manipulation. In some aspects, any of a variety of known cryopreservation solutions and parameters can be used.

[0160] Typically, cells are incubated before or in connection with gene manipulation and / or Suv39h1 inhibition.

[0161] The incubation step can include culturing, incubation, stimulation, activation, amplification, and / or proliferation.

[0162] Inhibition of Suv39h1 according to the present invention can also be achieved in vivo after injection of the cells into the target patient. Typically, inhibition of suv39h1 is carried out using a pharmacological inhibitor as described above.

[0163] Inhibition of Suv39h1 by the foregoing method can also be carried out during the steps of stimulation, activation, and / or expansion. For example, PBMCs, or purified T cells, or purified NK cells, or purified lymphoid progenitor cells are expanded in vitro in the presence of a pharmacological inhibitor of Suv39h1 prior to adoptive transfer into a patient. In some embodiments, the composition or cells are incubated in the presence of a stimulating condition or a stimulant. Such conditions include those designed to mimic antigen exposure, to induce proliferation, expansion, activation, and / or survival of cells within a population, and / or for genetic manipulation, for example, to stimulate cells for the introduction of a genetically engineered antigen receptor.

[0164] The incubation conditions can include one or more of a specific medium, temperature, oxygen content, carbon dioxide content, time, drugs such as nutrients, amino acids, antibiotics, ions, and / or stimulants such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and other drugs designed to activate cells.

[0165] In some embodiments, the stimulating condition or drug includes one or more drugs, such as ligands, capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the drug turns on or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such drugs can include antibodies, such as antibodies specific for TCR components and / or costimulatory receptors, such as anti-CD3, anti-CD28, and / or one or more cytokines bound to a solid-phase carrier such as beads. Optionally, the expansion method can further include the step of adding anti-CD3 and / or anti-CD28 antibodies to the medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulant includes IL-2 and / or IL-15, for example, an IL-2 concentration of at least about 10 units / mL.

[0166] In some embodiments, the incubation is performed according to techniques such as those described in U.S. Patent No. 6,040,177 to Riddell et al., Klebanoff et al., J Immunother. 2012;35(9):651 - 660, Terakura et al., Blood. 2012;1:72 - 82, and / or Wang et al., J Immunother. 2012, 35(9):689 - 701.

[0167] In some embodiments, T cells are expanded by adding feeder cells, such as non - dividing peripheral blood mononuclear cells (PBMCs), to the starting culture composition (e.g., such that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells per T lymphocyte in the initial population to be expanded); and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some embodiments, the non - dividing feeder cells can include gamma - irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000 - 3600 rad, and cell division is blocked. In some embodiments, the feeder cells are added to the medium before adding the population of T cells.

[0168] In some embodiments, the stimulation conditions include a temperature suitable for the growth of human T lymphocytes, such as at least about 25°C, generally at least about 30°C, generally 37°C or about 37°C. Optionally, the incubation may further include adding non - dividing Epstein - Barr virus (EBV) - transformed lymphoblastoid cells (LCLs) as feeder cells. The LCLs can be irradiated with gamma rays in the range of about 6000 - 10,000 rad. The LCL feeder cells in some embodiments are provided in any suitable amount, such as at a ratio of at least about 10:1 of LCL feeder cells to initial T lymphocytes.

[0169] In embodiments, antigen-specific T cells, e.g., antigen-specific CD4+ and / or CD8+ T cells, are obtained by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, antigen-specific T cell lines or clones against cytomegalovirus antigens can be produced by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen.

[0170] In some embodiments, the method includes assessing the expression of one or more markers on the surface of the engineered or engineering cells. In one embodiment, the method includes assessing the surface expression of one or more target antigens (e.g., antigens recognized by a genetically engineered antigen receptor) that are required to be targeted by adoptive cell therapy, e.g., by an affinity-based detection method, e.g., by flow cytometry.

[0171] Vectors and methods for cell genetic engineering In some embodiments, genetic engineering includes the introduction of nucleic acids encoding other components for introduction into the genetically engineered component or cell, e.g., components encoding gene disruption proteins or nucleic acids.

[0172] Generally, engineering CAR into immune cells (e.g., T cells) requires culturing the cells so that transduction and expansion are possible. Various methods can be utilized for transduction, but stable gene transfer is required to sustain the expression of CAR in the engineered cells that expand and continue to survive by cloning.

[0173] In some embodiments, gene transfer is achieved by first stimulating cell growth, e.g., T cell growth, proliferation, and / or activation, followed by transduction of the activated cells and expansion of the culture to a number sufficient for clinical application.

[0174] Various methods for introducing a genetically engineered component, such as an antigen receptor, such as a CAR, are well known and can be used with the provided methods and compositions. Exemplary methods include methods for the transfer of nucleic acids encoding a receptor, including those via viruses, such as retroviruses or lentiviruses, transduction, transposons, and electroporation.

[0175] In some embodiments, the recombinant nucleic acid is introduced into cells using a vector derived from a recombinant infectious viral particle, such as simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV). In some embodiments, the recombinant nucleic acid is introduced into T cells using a recombinant lentiviral vector or a retroviral vector, such as a gammaretroviral vector (see, e.g., Koste et al. (2014) Gene Therapy April 3, 2014; Carlens et al. (2000) Exp Hematol 28(10):1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. November 2011; 29(11):550-557).

[0176] In some embodiments, retroviral vectors, such as those derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus-forming virus (SFFV), or adeno-associated virus (AAV), have long terminal repeat sequences (LTRs). Most retroviral vectors are derived from murine retroviruses. In some aspects, retroviruses include those derived from any avian or mammalian cell source. Retroviruses are typically amphotropic, meaning that the retrovirus can infect host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol, and / or env sequences. Numerous exemplary retroviral systems have been described (e.g., U.S. Patent Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman, (1989) BioTechniques 7:980-990; Miller, A.D., (1990) Human Gene Therapy 1:5-14; Scarpa et al., (1991) Virology 180:849-852; Burns et al., (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

[0177] Lentiviral transduction methods are also known. Exemplary methods are described in Wang et al., (2012) J. Immunother. 35(9):689-701; Cooper et al., (2003) Blood. 101:1637-1644; Verhoeyen et al., (2009) Methods Mol Biol. 506:97-114; and Cavalieri et al., (2003) Blood. 102(2):497-505.

[0178] In some embodiments, the recombinant nucleic acid is introduced into T cells via electroporation (see, e.g., Chicaybam et al., (2013) PLoS ONE 8(3):e60298, and Van Tedeloo et al., (2000) Gene Therapy 7(16):1431-1437). In some embodiments, the recombinant nucleic acid is introduced into T cells via transposition (see, e.g., Manuri et al., (2010) Hum Gene Ther 21(4):427-437; Sharma et al., (2013) Molec Ther Nucl Acids 2, e74, and Huang et al., (2009) Methods Mol Biol 506:115-126). Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection (described, e.g., in Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol. 7:2031-2034 (1987)).

[0179] Other approaches and vectors for introducing a genetically engineered nucleic acid encoding a genetically engineered product are those described, for example, in International Patent Application Publication No. WO 2014 / 055668 and U.S. Patent No. 7,446,190.

[0180] Compositions of the invention The present invention also includes compositions containing the cells described herein and / or cells produced by the provided methods. Typically, the compositions are pharmaceutical compositions and formulations for administration, such as adoptive cell therapy.

[0181] The pharmaceutical compositions of the present invention generally include at least one engineered immune cell of the present invention and a pharmaceutically acceptable carrier.

[0182] As used herein, the term "pharmaceutically acceptable carrier" includes physiological saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration.

[0183] The supplementary active compound can be further incorporated into the composition. In some embodiments, the selection of the carrier of the pharmaceutical composition is partially determined by a particular genetically engineered CAR or TCR, vector, or cell expressing the CAR or TCR, as well as the particular method used to administer the vector or host cell expressing the CAR. Thus, there are various suitable formulations. For example, the pharmaceutical composition can contain a preservative. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or its mixture is typically present in an amount of about 0.0001% to about 2% by weight of the total composition.

[0184] The pharmaceutical composition is formulated to be compatible with its intended route of administration.

[0185] Therapeutic methods The present invention also relates to the cells defined above in their use in adoptive therapy (particularly adoptive T cell therapy), typically in the treatment of cancer in a subject in need thereof.

[0186] As used herein, "treatment" or "treating" is defined as the application or administration to a patient in need thereof of a cell according to the present invention or a composition containing such cells for the purpose of curing, healing, alleviating, relieving, altering, correcting, remitting, improving, or affecting a disease such as cancer or any symptom of a disease (e.g., cancer). In particular, the terms "treating" or "treatment" mean reducing or alleviating at least one adverse clinical symptom associated with a disease such as cancer, e.g., pain, swelling, low blood cell count, etc.

[0187] Regarding cancer treatment, the terms "treating" or "treatment" also mean slowing or reversing the progression of the growth of neoplastic, uncontrolled cells, i.e., shrinking existing tumors and / or stopping tumor growth. The terms "treating" or "treatment" also mean inducing apoptosis in cancer or tumor cells in a subject.

[0188] The subject (i.e., patient) of the present invention is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be of any suitable age, including infant, juvenile, young, adult, and elderly subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent. In some examples, the patient or subject is a validated animal model for diseases, adoptive cell therapy, and / or for evaluating toxic outcomes such as cytokine release syndrome (CRS). In some embodiments of the present invention, the subject has cancer, is at risk of having cancer, or is in remission from cancer.

[0189] Cancer is a solid cancer or a "liquid tumor", e.g., a cancer affecting the blood, bone marrow, or lymphatic system, and is also known as a tumor of hematopoietic and lymphoid tissues, particularly including leukemia and lymphoma. Examples of liquid tumors include, for example, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL) (including various lymphomas such as mantle cell lymphoma, non-Hodgkin lymphoma (NHL), adenoma, squamous cell carcinoma, laryngeal cancer, gallbladder and bile duct cancer, and cancers of the retina such as retinoblastoma).

[0190] Solid cancer particularly includes cancer that affects one of the organs selected from the group consisting of colon, rectum, skin, endometrium, lung (including non-small cell lung cancer), uterus, bone (osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, giant cell tumor, adamantinoma, chordoma, etc.), liver, kidney, esophagus, stomach, bladder, pancreas, cervix, brain (meningioma, glioblastoma, low-grade astrocytoma, oligodendrocytoma, pituitary tumor, schwannoma, metastatic brain cancer, etc.), ovary, breast, head and neck region, testis, prostate, and thyroid.

[0191] The cancer according to the present invention is preferably a cancer that affects the blood, bone marrow, and lymphatic system as described above. Typically, the cancer is multiple myeloma or is associated with multiple myeloma.

[0192] In some embodiments, the subject has or is at risk of having an infectious disease or condition, such as, but not limited to, viral infection, retroviral infection, bacterial infection, and protozoal infection, immunodeficiency, cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus. In some embodiments, the disease or condition is an autoimmune or inflammatory disease or condition, such as arthritis, such as rheumatoid arthritis (RA), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, asthma, and / or a disease or condition associated with transplantation.

[0193] The present invention also includes a method of treatment, particularly adoptive cell therapy, preferably adoptive T cell therapy, which comprises administering the aforementioned composition to a subject in need thereof.

[0194] In some embodiments, the cells or compositions are administered to a subject, such as a subject having or at risk of having any of the cancers or diseases described above. In some aspects, the method treats, e.g., ameliorates, one or more symptoms of a disease or condition associated with cancer, e.g., restores, by reducing the tumor burden of the cancer that expresses an antigen recognized by the engineered cells.

[0195] Methods of administering cells for adoptive cell therapy are known and can be used in connection with the provided methods and compositions. For example, adoptive T cell therapy is described, e.g., in U.S. Patent Application No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85. See, e.g., Themeli et al., (2013) Nat Biotechnol. 31(10):928-933; Tsukahara et al., (2013) Biochem Biophys Res Commun 438(1):84-9; Davila et al., (2013) PLoS ONE 8(4):e61338.

[0196] In some embodiments, the cell therapy, such as adoptive cell therapy, such as adoptive T cell therapy, is performed by autologous transplantation, in which case the cells are isolated and / or prepared from the subject receiving the cell transplant or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject in need of treatment, e.g., a patient, and the cells are administered to the same subject after isolation and processing.

[0197] In some embodiments, cell therapy, such as adoptive cell therapy, such as adoptive T cell therapy, is performed by allogeneic transplantation, in which case the cells are isolated and / or prepared from a subject other than the subject to receive the cell therapy, or from the subject who will ultimately receive the cell therapy, such as a first subject. In such embodiments, the cells are then administered to a different subject of the same species, such as a second subject. In some embodiments, the first subject and the second subject are genetically identical. In some embodiments, the first subject and the second subject are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0198] The administration of at least one cell according to the present invention to a subject in need thereof can be combined with one or more additional therapeutic agents or combined with another therapeutic intervention, either simultaneously or sequentially in any order. In some situations, the cells are co-administered in a timely manner close enough to another therapy such that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cell population is administered prior to one or more additional therapeutic agents. In some embodiments, the cell population is administered after one or more additional therapeutic agents.

[0199] For cancer treatment, combined cancer treatments include, but are not limited to, chemotherapeutic agents, hormonal agents, anti-angiogenic agents, radiolabeled compounds, immunotherapy, surgery, cryotherapy, and / or radiation therapy.

[0200] Immunotherapies include, but are not limited to, immune checkpoint modulators (i.e., inhibitors and / or agonists), monoclonal antibodies, cancer vaccines.

[0201] The administration of cells in adoptive T cell therapy according to the present invention is preferably combined with the administration of an immune checkpoint modulator. The immune checkpoint modulator most preferably comprises an anti-PD-1 and / or anti-PDL-1 inhibitor.

[0202] The present invention also relates to the use of a composition comprising the engineered immune cells described herein for the manufacture of a medicament for treating cancer, an infectious disease or condition, an autoimmune disease or condition, or an inflammatory disease or condition in a subject.

Examples

[0203] 〔Results〕 Materials and methods: Syngeneic and Suv39h1-KO mice were intravenously infected with 5×10 3 CFU and challenged 40 days later with 2×10 6 CFU of recombinant Listeria monocytogenes (LM-OVA, derived from the wild-type 140403s strain) expressing OVA. Bacteria were grown in TSB medium (BD Bioscience) until the early logarithmic phase, and their growth was evaluated with an OD 600 photometer. Naive, dump - CD44 high K b -OVA + CD8 + T cells and related subsets were FACS sorted. For each subset analyzed, the inventors recovered 3 or 4 biological replicates. RNA was extracted using the Rneasy Micro Kit (QUIGEN) according to the manufacturer's protocol. Column DNAse treatment was included (QUIGEN). For each condition, cDNA was synthesized from the RNA using standard Affymetrix protocols. Labeled DNA was hybridized to Affymetrix Mouse Gene 2.1 ST and processed on an Affymetrix GeneTitan device.

[0204] Microarray data analysis: Microarray data was processed in R (version 3.0.0) using packages from Bioconductor. Raw data CEL files were used, and quality control analysis was performed using the ArrayQualityMetrics package. The raw data was preprocessed using the RMA method available in the oligo package. Probes without annotation were excluded from the analysis. Moderated t-tests were performed using the limma package, and p-values were adjusted using multiple testing with the Benjamini Hochberg method. Finally, the inventors considered that if the adjusted p-value was less than 5%, it was statistically significant.

[0205] Gene set enrichment analysis 。 Gene Set Enrichment Analysis (GSEA) was performed using genes with immunological characteristics (C7) and C2 (selected) gene sets from the Molecular Signatures DataBase (MSigDB database v5.1, http: / / software.broadinstitute.org / qsea / msiqdb / index.isp ). GMT files were downloaded along with gene symbol information. The GCT file consisted of a total of 41345 probes and was imported into GSEA. GSEA was run with default parameters except for the number of permutations (n = 10000). BubbleGUM analysis was performed as described above.

[0206] Analysis of tumor growth in Suv39h1-deficient CD8+ OT-1 cells was performed as follows: Total spleens and lymph nodes from male Suv39h1-KO CD45.2 OT-1 (specific for the OVA257-264 peptide SIINFEKL in the context of H2-Kb MHC class I) and syngeneic WT mice were cultured in complete medium (RPMI 1640 supplemented with 10% FBS, penicillin-streptomycin, and L-glutamine) for 3 days and then further activated with 100 UI / ml IL-2 and 1 μg / ml OVA257-264 peptide for another 3 days.

[0207] For tumor inoculation, 1×10 6Individual EL4-OVA lymphoma cells were subcutaneously injected into the right flank of CD45.1 / C57BL / 6 male mice. Seven days later, the mice were intravenously injected with 2×10 6 individuals of IL-2 / OVA-treated Suv39h1-KO CD45.2 OT-1 or syngeneic WT cells.

[0208] Mice were intraperitoneally injected with anti-PD1 (Bio X Cell, RMP-14) at a dose of 7.5 mg / Kg body weight per dose twice a week for 2 weeks. Tumor growth was measured manually using calipers.

[0209] Results : 1) Our results show that in cell populations obtained from Suv39h1-KO mice, early progenitor cells with a "stem cell-like" phenotype accumulate and are highly efficiently reprogrammed into long-lived central memory T cells that express both CD44 and CD62L, compared to cell populations obtained from wild-type mice. As shown in Figure 1, the proportion and number of central memory T cells (expressing both CD44 and CD62L) are both increased in Suv39h1-deficient mice.

[0210] 2) Transcriptome analysis was used to speculate on the mechanism of this unique phenotype: Wild-type and Suv39h1-deficient mice were immunized with Listeria monocytogenes expressing OVA, and OVA-specific T cells were isolated using fluorescence-activated cell sorting on day 7 after immunization.

[0211] After analyzing the cells by Affymetrix, the inventors confirmed that effector T cells from Suv39h1 express mRNA encoding high levels of stem cell-related proteins (CD8+ T stem cell-like memory characteristics (86 genes): Abcb1a Irf8 Rest Abcb1b Jarid2 Rif1 Alpl Kat6a Rnf138 Antxr2 Klf4 Rras Arl4c Ldha Sall4 Atr Ldhb Satb1 Baalc Ldhc Setbp1 Basp1 Ldhd Setdb1 Bcl6 Lect1 Skil Bub1 Lpin1 Smarcad1 Ccr7 Ly6a Sox2 Ccr9 Ly6e Spon1 Cd27 Map3k8 Stat3 Cxcr6 Mapk12 Tbx3 Dock9 Mcm3ap Tcf3 Dusp9 Mcoln2 Tcl1 Eomes Myc Tdgf1 Esrrb Nanog Tert Evl Ncor2 Tigit Fas Nr0b1 Tnfaip2 Fgf2 Nr1d2 Tnfrsf1b Fut4 P2ry14 Traf1 Gzmk Pax6 Traf4 Hand1 Pcgf2 Trib2 Hesx1 Plekha5 Txnip Ier3 Podxl Zfp42 Il2rb Pou5f1 Zfx Il7r Pou6f1 Zic3 Irf4 Prkce).

[0212] The inventors concluded that OVA-specific T cells from Suv39h1-deficient mice stimulated after Listeria monocytogenes infection express a "stem cell"-like mRNA signature.

[0213] 3) Suv39h1-deficient CD8+ T cells show increased survival and self-renewal in vivo.

[0214] Congenic CD45.2 Kb-OVA pentamer+CD8+ T cells isolated from WT and Suv39h1 KO LM-OVA-infected mice were transferred into naive recipient CD45.1 mice. Forty days after adoptive transfer, naive recipient mice were challenged with LM-OVA, and 4 days later, Kb-OVA pentamer+CD8+ T cells were analyzed by FACS.

[0215] As shown in Figure 3, the results indicate that Suv39h1-deficient cells express increased levels of "stem cell-like" characteristics. Suv39h1-deficient cells also survive well and are effectively repositioned in wild-type mice after adoptive transfer.

[0216] 4) Suv39h1-deficient CD8+ OT-1 cells control tumor growth in vivo Figures 4B and 4C show that mice transplanted with Suv39h1-KO OT-1 cells and treated with anti-PD1 monoclonal antibody controlled tumor growth better than mice that received the same treatment except using WT OT-1 cells. Notably, by 30 days after tumor injection, 4 out of 5 WT OT-1 transplanted mice showed tumor growth, while only 1 out of 5 mice injected with Suv39h1-KO OT-1 cells showed tumor growth.

[0217] Conclusion Lack of Suv39h1 activity in CD8+ T cells is associated with a good anti-tumor effect in a therapeutic approach based on adoptive T cell transfer.

Claims

Claim 1 A composition for use in adoptive cell therapy for cancer, comprising engineered immune cells, wherein the immune cells are NK cells or NK cell precursors engineered to be deficient in Suv39h1. Claim 2 The composition according to claim 1, wherein the immune cells further comprise a genetically engineered antigen receptor that specifically binds to a target antigen. Claim 3 The composition according to claim 2, wherein the target antigen is expressed in cancer cells and / or is a universal tumor antigen. Claim 4 The composition according to claim 2 or 3, wherein the genetically engineered antigen receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR) comprising one or more antigen-binding molecules in the extracellular portion. Claim 5 The composition according to any one of claims 2 to 4, wherein the immune cells comprise at least two genetically engineered receptors, each receptor recognizing a different antigen. Claim 6 The composition according to any one of claims 1 to 5, wherein the immune cells are autologous. Claim 7 The composition according to any one of claims 1 to 5, wherein the immune cells are allogeneic. Claim 8 The composition according to any one of claims 1 to 7, wherein the activity and / or expression of Suv39h1 in the engineered immune cells is selectively inhibited or blocked. Claim 9 The composition according to any one of claims 1 to 8, wherein the composition is administered in combination with an immune checkpoint molecule. Claim 10 The composition according to claim 9, wherein the immune checkpoint molecule is selected from a PD-1 inhibitor and a PDL-1 inhibitor. Claim 11 Engineered immune cells, wherein the immune cells are NK cells or NK cell precursors engineered to be deficient in Suv39h1 and further comprise a genetically engineered antigen receptor that specifically binds to a target antigen. Claim 12 The engineered immune cells according to claim 11, wherein the target antigen is expressed in cancer cells and / or is a universal tumor antigen. Claim 13 The engineered immune cells according to claim 11 or 12, wherein the genetically engineered antigen receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR) comprising one or more antigen-binding molecules in the extracellular portion. Claim 14 The engineered immune cell according to any one of claims 11 to 13, wherein the immune cell comprises at least two genetically engineered receptors, and each receptor recognizes a different antigen.

15. The engineered immune cell according to any one of claims 11 to 14, wherein the immune cell is autologous.

16. The engineered immune cell according to any one of claims 11 to 14, wherein the immune cell is allogeneic.

17. The engineered immune cell according to any one of claims 11 to 16, wherein the activity and / or expression of Suv39h1 in the engineered immune cell is selectively inhibited or blocked.

18. A method for producing a genetically engineered immune cell according to any one of claims 11 to 17, comprising the steps of inhibiting the expression and / or activity of Suv39h1 in the immune cell, and introducing a genetically engineered antigen receptor that specifically binds to a target antigen into the immune cell.

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