SUV39H1-deficient immune cells

Abstract

The present invention relates to engineered immune cells deficient in Suv39h1. Preferably, the engineered immune cells further comprise a genetically engineered antigen receptor that specifically binds to a target antigen. The present invention also relates to a method for obtaining genetically engineered immune cells, comprising a step of inhibiting the expression and / or activity of Suv39h1 in immune cells; and optionally further comprising a step of introducing a genetically engineered antigen receptor that specifically binds to a target antigen into the immune cells. The present invention also encompasses the use of the engineered immune cells in adoptive therapy, particularly in the treatment of cancer.

Description

[0001] This application is a divisional application of an invention patent application with an application date of June 20, 2018, an application number of 201880053053.6, and an invention name of “SUV39H1-deficient immune cells”. Technical Field

[0002] The present invention relates to the field of adoptive therapy. The present invention provides Suv39h1-deficient immune cells with enhanced survival, reconstitution potential and central memory phenotype after adoptive transfer. Background Art

[0003] Adoptive T cell therapy (ATCT) using T cells equipped with recombinant T cell receptor (TCR) and chimeric antigen receptor (CAR) technology has shown very encouraging activity in early clinical testing against several malignancies at many institutions (Kershaw MH, Westwood JA, Darcy PK. Nat Rev Cancer. 2013; 13:525–541).

[0004] An emerging theme is that effective engraftment and long-term persistence of therapeutic T cells are associated with positive treatment outcomes. Several preclinical studies have shown that naive and early differentiated T cells have enhanced long-term persistence (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 effective anti-tumor responses (Gattinoni L et al., J Clin Investig. 2005; 115: 1616–1626; Lugli E et al. J Clin Invest. 2013; 123: 594–599). Furthermore, enhanced persistence of adoptively transferred cells appears to be dependent 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).

[0005] Stable gene transfer has been routinely achieved in the clinic, particularly by using gammaretroviral vectors to transduce polyclonal T cells with CAR (e.g., see Guest RD et al., Cancer Immunol Immunother. 2014; 63: 133–145) and TCR (see especially Johnson LA et al., Blood. 2009; 114(3): 535–46), where these engineered cells have shown no significant adverse safety profiles in patients transplanted with CAR T cells for more than 10 years (Scholler J et al., Sci Transl Med. 2012; 4: 132ra153).

[0006] For efficient transduction by retroviral vectors or lentiviruses, primary T cells need to be actively proliferating (Stacchini A et al., Leuk Res. 1999; 23: 127–136), which is usually achieved by mitogenic stimulation of quiescent primary T cells.

[0007] However, once activated, T cells will develop towards the effector (TE) phenotype in an irreversible linear manner (Mahnke YD et al., Eur J Immunol. 2013; 43: 2797–2809; Farber DL. Semin Immunol. 2009; 21: 84-91). Therefore, retroviral or lentiviral transduction of mitogenic activation promotes T cell differentiation from the initial type to the TE phenotype. Combined with the in vitro culture protocol, the number of transduced T cells can be expanded to the number required for clinical application (about 10 9 –10 11 ), T cells tend toward a more differentiated phenotype, which is not optimal for systemic retention.

[0008] Therefore, although adoptive T-cell therapy, including CAR T-cell-based therapy, has achieved remarkable therapeutic success in the treatment of certain hematological cancers over the past few years, it has only shown effectiveness in a few hematological cancer types and a few solid tumor types. It has been hypothesized that limited T-cell survival after adoptive transfer may lead to low therapeutic efficacy.

[0009] Therefore, there is still a need in the art for modified or engineered T cells that exhibit an enhanced central memory phenotype and enhanced survival after adoptive transfer. In particular, there is still a need to provide immune cells, particularly T cells, that can be used for adoptive therapy, which significantly supports effective and large-scale cancer treatment. Brief description of the invention

[0010] The inventors have now surprisingly found that Suv39h1-deficient T cells carry an enhanced central memory phenotype and enhanced survival after adoptive transfer. In particular, the inventors have shown that Suv39h1-deficient T cells accumulate and reprogram with increased efficiency into long-lived central memory T cells expressing both CD44 and CD62L. Thus, the present invention relates to modified or engineered immune cells, in particular modified T cells, in which Suv39h1 is inactivated.

[0011] Thus, the modified or engineered immune cells are of great interest for their use in adoptive therapy.Therefore, the present invention more specifically relates to Suv39h1-deficient engineered or modified immune cells, wherein the immune cells preferably further comprise a genetically engineered antigen receptor that specifically binds to a target antigen.

[0012] Typically, the engineered immune cells of claim 1 are T cells or NK cells, in particular CD4+ or CD8+ T cells. Preferred cells may be selected from T N Cells, TSC M , TC M or TE M Cells and their combinations.

[0013] Typically, the engineered immune cells are also isolated from a subject. Preferably, the subject has cancer, or is at risk of developing cancer.

[0014] The target antigen to which the genetically engineered antigen receptor specifically binds is preferably expressed on cancer cells and / or is a universal tumor antigen.

[0015] The genetically engineered antigen receptor can be a chimeric antigen receptor (CAR), which comprises an extracellular antigen recognition domain that specifically binds to a target antigen. The genetically engineered antigen receptor can also be a T cell receptor (TCR).

[0016] Preferably, the activity and / or expression of Suv39h1 in the engineered immune cell is selectively inhibited or blocked. In one embodiment, the engineered immune cell expresses a Suv39h1 nucleic acid encoding a non-functional Suv39h1 protein.

[0017] The present invention also relates to a method for producing genetically engineered immune cells, which comprises the steps of inhibiting the expression and / or activity of Suv39h1 in immune cells; and optionally introducing a genetically engineered antigen receptor that specifically binds to a target antigen into the immune cells.

[0018] Preferably, inhibiting the activity and / or expression of Suv39h1 comprises contacting or bringing the cell into contact with at least one agent that inhibits the expression and / or activity of Suv39h1 and / or destroys the Suv39h1 gene. The agent may be selected from a small molecule inhibitor; an antibody derivative; an aptamer; a nucleic acid molecule that blocks transcription or translation; or a gene editing agent.

[0019] The present invention also relates to the use of the engineered immune cells as described herein or a composition comprising the engineered immune cells in adoptive cell therapy, in particular adoptive therapy for cancer. DETAILED DESCRIPTION OF THE INVENTION

[0021] definition

[0022] The term "antibody" is used in the broadest sense herein and includes polyclonal and monoclonal antibodies, including complete antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, variable heavy chain (VH) regions capable of specific binding to antigens, single-chain antibody fragments (including single-chain variable fragments (scFv)) and single-domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified immunoglobulin forms, such as intracellular antibodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecific antibodies (e.g., bispecific antibodies), double antibodies, three antibodies and four antibodies, tandem di-scFv, tandem tri-scFv. Unless otherwise indicated, the term "antibody" should be understood to encompass its functional antibody fragments. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0023] "Antibody fragment" refers to a molecule other than an intact antibody that contains 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; diabodies; linear antibodies; variable heavy chain (VH) regions; single-chain antibody molecules (such as scFvs) and single-domain VH monobodies; and multispecific antibodies formed from antibody fragments. In a specific embodiment, the antibody is a single-chain antibody fragment comprising a variable heavy chain region and / or a variable light chain region, such as scFv.

[0024] A "single domain antibody" is an antibody fragment that comprises all or a portion of the heavy chain variable domain 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.

[0025] As used herein, "inhibition" of gene expression refers to the elimination or reduction of expression of one or more gene products encoded by the subject gene in the cell compared to the expression level of the gene product without inhibition. Exemplary gene products include mRNA and protein products encoded by the gene. In some cases, inhibition is transient or reversible, while in other cases, inhibition is permanent. Although truncated or non-functional products may be produced, functional or full-length proteins or mRNAs may be inhibited in some cases. In some embodiments herein, in contrast to expression, gene activity or function is inhibited. Gene inhibition is usually induced by artificial methods, i.e., by adding or introducing compounds, molecules, complexes or compositions, and / or by destroying the nucleic acid of the gene or nucleic acid associated with the gene (such as at the DNA level). Exemplary methods for gene inhibition include gene silencing, knockdown, knockout and / or gene disruption techniques, such as gene editing. Examples include antisense techniques, such as RNAi, siRNA, shRNA and / or ribozymes, which generally result in a transient reduction in expression; and gene editing techniques, which result in inactivation or destruction of targeted genes, such as by inducing breakage and / or homologous recombination.

[0026] As used herein, the "destruction" of a gene refers to the change of the gene sequence at the DNA level. Examples include insertion, mutation and deletion. Typically, the destruction causes the inhibition of expression of the normal or "wild type" product of the gene encoding and / or does not express at all. Examples of such gene disruption are insertion, frameshift and missense mutation, deletion, knock-in and knock-out of a gene or a part of a gene, including the deletion of the entire gene. Such disruption may occur in the coding region, for example in one or more exons, resulting in the inability to produce a full-length product, a functional product or any product, such as by inserting a stop codon. Such disruption may also occur through the destruction of a promoter or enhancer or other regions that affect transcriptional activation, thereby preventing gene transcription. Gene disruption includes gene targeting, including targeted gene inactivation by homologous recombination.

[0027] Cells of the present invention

[0028] Typically, the cells according to the invention are eukaryotic cells, such as mammalian cells (also referred to herein as animal cells), for example human cells.

[0029] More particularly, the cells of the present invention are derived from blood, bone marrow, lymph or lymphoid organs (particularly thymus), and are cells of the immune system (i.e. immune cells), such as cells of innate or adaptive immunity. For example, myeloid or lymphocytes, including lymphocytes, are typically T cells and / or NK cells.

[0030] Preferably, according to the present invention, the cells are in particular lymphocytes, including T cells, B cells and NK cells.

[0031] The cell according to the invention may also be an immune cell progenitor cell, such as a lymphoid progenitor cell, more preferably a T cell progenitor cell.

[0032] Typically, T cell progenitors express a set of common 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.

[0033] Typically, the cells are primary cells, such as cells isolated directly from a subject and / or isolated from a subject and frozen.

[0034] The cells of the invention may be allogeneic and / or autologous to the subject to be treated.

[0035] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as complete T cell populations, CD4+ cells, CD8+ cells, and subsets thereof, such as those defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization and / or retention capacity, antigen specificity, type of antigen receptor, presence in a specific organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation.

[0036] T cells and / or subtypes and subsets of CD4+ and / or CD8+ T cells are naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, such as stem cell memory T (TSC M ), central memory T(TC M ), effect memory T(T EM ) or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring 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, α / βT cells and δ / γT cells. Preferably, the cells according to the present invention are T cells with stem / memory properties and higher reconstitution capacity due to the inhibition of Suv39h1.EFF cells, and T N Cells, TSC M TC M TE M Cells and their combinations.

[0037] In some embodiments, one or more T cell populations are enriched or depleted for cells that are positive for or express high levels of one or more specific markers (e.g., surface markers), or cells that are negative for or express relatively low levels of one or more markers. In some cases, these markers are those that are absent or expressed at relatively low levels on certain T cell populations (such as non-memory cells), but present or expressed at relatively high levels on certain other T cell populations (such as memory cells). In one embodiment, cells (such as, CD8 + cells or T cells, for example, CD3 + 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 that express high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127). In some examples, CD8+ T cells are enriched for cells that are positive for CD45RO (or negative for CD45RA) and positive for CD62L.

[0038] For example, according to the present invention, the cells may include a CD4+ T cell population and / or a CD8+ T cell subset, such as enriched central memory (T CM Alternatively, the cell may be a subset of other types of lymphocytes, including natural killer (NK) cells, MAIT cells, innate lymphoid cells (ILCs), and B cells.

[0039] According to the present invention, the cells and compositions containing cells for engineering are separated from samples, particularly biological samples, such as obtained from or derived from a subject. Typically, the subject needs cell therapy (adoptive cell therapy) and / or is a subject who will receive cell therapy. The subject is preferably a mammal, particularly a human. In one embodiment of the invention, the subject suffers from cancer.

[0040] Samples include tissues, fluids and other samples taken directly from a subject, as well as samples from one or more processing steps such as separation, centrifugation, genetic engineering (e.g., viral vector transduction), washing and / or incubation. Biological samples can be samples directly obtained from biological sources or processed samples. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat; tissue and organ samples, including processed samples derived therefrom. Preferably, the sample from which cells are derived or separated is a blood or blood-derived sample, or is derived from apheresis or leukocyte apheresis products. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemias, lymphomas, lymph nodes, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissues and / or cells derived therefrom. Under cell therapy (typically adoptive cell therapy), samples include samples of autologous and allogeneic origin.

[0041] In some embodiments, the cell is derived from a cell line, such as a T cell line. These cells can also be obtained from a xenogeneic source, such as a mouse, rat, non-human primate, or pig. Preferably, the cell is a human cell.

[0042] Suv39h1-deficient cells

[0043] 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 chromatin 3-9 homolog 1 (Drosophila)", which 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 (Jun 1999). "Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9encode 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 spot 3-9 homolog, histone lysine N-methyltransferase or H3 lysine 9 specific 1. Human Suv39h1 methyltransferase is called O43463 in UNIPROT and is encoded by the gene Suv39h1 (gene ID in NCBI: 6839) located on chromosome x. The term Suv39h1 according to the present invention also covers all orthologs of SUV39H1, such as SU(VAR)3-9.

[0044] As used herein, the expression "Suv39h1 deficient" according to the present invention means that Suv39h1 activity is inhibited or blocked in the cell according to the present invention (ie Lys-9 of histone H3 is methylated by H3K9-histone methyltransferase).

[0045] According to the present invention, "inhibiting Suv39h1 activity" means that the Suv39h1 activity is reduced by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more compared to the activity or level of the non-inhibited Suv39h1 protein. Preferably, inhibiting Suv39h1 activity results in the absence of substantially detectable Suv39h1 activity in the cell.

[0046] Inhibition of Suv39h1 activity can also be achieved by inhibiting Suv39h1 gene expression or by Suv39h1 gene disruption. According to the invention, the inhibition reduces the expression of Suv39h1 in cells, in particular in immune cells of the invention, by at least 50, 60, 70, 80, 90 or 95% relative to the same cells produced by the method in the absence of the inhibition. Gene disruption can also result in reduced expression of Suv39h1 protein or in expression of non-functional Suv39h1 protein.

[0047] A "non-functional" Suv39h1 protein refers to a protein having reduced activity or lacking detectable activity as described above.

[0048] Therefore, the inhibitor of Suv39h1 activity in the cell according to the present invention can be selected from any compound or agent that naturally or not has the ability to inhibit the methylation of Lys-9 of histone H3 by H3K9-histone methyltransferase or inhibits the expression of H3K9-histone methyltransferase SUV39H1 gene.

[0049] Inhibition of Suv39h1 in immune cells according to the invention may be permanent and irreversible or transient and reversible. However, preferably, Suv39h1 inhibition is permanent and irreversible. Inhibition of Suv39h1 in cells may be achieved before or after infusion of the cells into the target patient as described below.

[0050] Genetically engineered cells according to the invention

[0051] In some embodiments, the cells comprise one or more nucleic acids encoding one or more antigen receptors introduced by genetic engineering.

[0052] Typically, the nucleic acid is heterologous (i.e., for example, it is not normally present in the cell to be engineered and / or the organism from which such cell is derived). In some embodiments, the nucleic acid is not naturally occurring, including chimeric combinations of nucleic acids encoding various domains from a variety of different cell types.

[0053] Antigen receptors according to the present invention are genetically engineered T cell receptors (TCR) and components thereof, as well as functional non-TCR antigen receptors, such as chimeric antigen receptors (CAR).

[0054] Chimeric Antigen Receptor (CAR)

[0055] In some embodiments, the engineered antigen receptor comprises a chimeric antigen receptor (CAR), including an activating or stimulatory CAR, a co-stimulatory CAR (see WO2014 / 055668) and / or an inhibitory CAR (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December 2013)).

[0056] Chimeric antigen receptors (CARs) (also called chimeric immunoreceptors, chimeric T cell receptors, artificial T cell receptors) are engineered receptors that can transfer any specificity to immune effector cells (T cells). Typically, these receptors are used to transfer the specificity of monoclonal antibodies to T cells, facilitating the transfer of their coding sequences via retroviral vectors.

[0057] In some aspects, CAR generally comprises an extracellular antigen (or ligand) binding domain connected to one or more intracellular signaling components via a joint and / or a transmembrane domain. This molecule typically simulates or approximates the signal by a natural antigen receptor, the signal by this receptor combined with a costimulatory receptor and / or the signal by only a costimulatory receptor.

[0058] In some embodiments, CAR is constructed to have specificity for a specific antigen (or marker or ligand), such as an antigen expressed in a specific cell type to be targeted by adoptive therapy, such as a cancer marker. Therefore, CAR typically includes one or more antigen binding molecules, such as one or more antigen binding fragments, domains or parts, or one or more antibody variable domains, and / or antibody molecules in its extracellular part.

[0059] The parts used to bind antigens are generally divided into three categories: single-chain antibody fragments (scFv) derived from antibodies, Fabs selected from libraries, or natural ligands that bind to their cognate receptors (for first-generation CARs). Successful examples of each of these categories are particularly reported in Sadelain M, Brentjens R, and Riviere I. The basic principles of chimeric antigen receptor (CAR) design are described in Cancer discovery. 2013; 3 (4): 388-398 (see Table 1 in particular) and are included in this application. ScFvs derived from mouse immunoglobulins are commonly used because they are easily derived from well-characterized monoclonal antibodies.

[0060] Typically, a CAR includes one or more antigen-binding portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb).

[0061] In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds an antigen, such as a cancer marker or a cell surface antigen of a cell or disease to be targeted (such as a tumor cell or cancer cell), such as any target antigen described herein or known in the art.

[0062] In some embodiments, the CAR contains an antibody or antigen binding fragment (e.g., scFv) that specifically recognizes an antigen (such as an intact antigen) expressed on the surface of a cell.

[0063] In some embodiments, CAR contains TCR-like antibodies, such as antibodies or antigen-binding fragments (e.g., scFv), which specifically recognize intracellular antigens presented on the cell surface with MHC-peptide complexes, such as tumor-associated antigens. In some embodiments, antibodies or antigen-binding portions thereof that recognize MHC-peptide complexes can be expressed on cells as part of a recombinant receptor (such as an antigen receptor). There are functional non-TCR antigen receptors in the antigen receptor, such as chimeric antigen receptors (CAR). Typically, CARs containing antibodies or antigen-binding fragments that exhibit TCR-like specificity for peptide-MHC complexes can also be referred to as TCR-like CARs.

[0064] In some aspects, antigen-specific binding or recognition components are connected to one or more transmembrane and intracellular signaling domains. In some embodiments, CAR includes a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of these domains to the transmembrane domains of the same or different surface membrane proteins to minimize the interaction with other members of the receptor complex.

[0065] In some embodiments, the transmembrane domain is derived from a natural or synthetic source. If the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein. The transmembrane region includes those derived from (i.e., at least comprising its transmembrane region) T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154 α, β or ζ chain. The transmembrane domain can also be synthetic.

[0066] In some embodiments, a short oligo- or polypeptide linker is present, e.g., a linker of 2 to 10 amino acids in length, and forms a connection between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0067] CAR generally includes at least one or more intracellular signaling components. Typically, the first generation CAR has an intracellular domain of CD3ζ chain, which is the main signal transmitter from endogenous TCR. Typically, the second generation CAR also includes intracellular signaling domains from various costimulatory protein receptors (eg, CD28, 41BB, ICOS) to the cytoplasmic tail of CAR, To provide other signals to T cells. Preclinical studies have shown that the second generation improves the anti-tumor activity of T cells. Recently, the third generation CAR combines multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to enhance efficacy.

[0068] For example, CAR may include intracellular components of the TCR complex, such as TCR CD3+ chains that mediate T cell activation and cytotoxicity, such as CD3ζ chains. Therefore, in some aspects, the antigen binding molecule is connected 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. CAR may further include a portion of one or more other molecules (such as Fc receptor gamma, CD8, CD4, CD25, or CD16).

[0069] In some embodiments, once connected to CAR, the cytoplasmic domain or intracellular signaling domain of CAR activates at least one of the responses of normal effector functions or corresponding non-engineered immune cells (typically T cells). For example, CAR can induce T Cell function, such as cytolytic activity or T helper activity, cytokine or other factor secretion.

[0070] In some embodiments, the intracellular signaling domain includes the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects also includes co-receptors that naturally cooperate with such receptors to initiate signal transduction upon antigen receptor binding, and / or any derivatives or variants of such molecules and / or any synthetic sequences having the same functional capability.

[0071] In some aspects, T cell activation is described as being mediated by two types of cytoplasmic signaling sequences: cells (primary cytoplasmic signaling sequences) that initiate antigen-dependent primary activation by TCR; and cells that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some aspects, CAR includes one or two of this signaling component.

[0072] In some aspects, CAR includes a primary cytoplasmic signaling sequence that regulates the primary activation of the TCR complex in a stimulatory or inhibitory manner. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif, which is referred to as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs containing primary cytoplasmic signaling sequences include sequences derived from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signaling molecule in CAR contains a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3 ζ.

[0073] 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 aspects, the same CAR includes both activation components and co-stimulatory components. Alternatively, the activation domain is provided by one CAR, while co-stimulation is provided by another CAR that recognizes another antigen.

[0074] CAR or other antigen receptors may also be inhibitory CAR (e.g., iCAR), and include weakening or suppressing the intracellular components of responses (such as immune responses). Examples of such intracellular signaling components are those found on immune checkpoint molecules, including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptors, EP2 / 4 adenosine receptors (including A2AR). In some aspects, engineered cells include inhibitory CAR, which includes the signaling domain of this inhibitory molecule or the signaling domain derived from this inhibitory molecule, so that it plays a role in weakening its response. When the antigen identified by an activation receptor (e.g., CAR) is also expressed or can also be expressed on the surface of normal cells, this CAR is, for example, used to reduce the possibility of off-target effects.

[0075] TCR

[0076] In some embodiments, the genetically engineered antigen receptor includes a recombinant T cell receptor (TCR) and / or a TCR cloned from a naturally occurring T cell.

[0077] "T cell receptor" or "TCR" refers to a molecule containing variable α and β chains (respectively referred to as TCRRa and TCRp) or variable γ and δ chains (respectively referred to as TCRy and TCR5), and it is capable of specifically binding to an antigenic peptide bound to an MHC receptor. In some embodiments, the TCR is in the αβ form. Typically, TCRs in the αβ and γδ forms are generally structurally similar, but the T cells expressing them may have different anatomical locations or functions. The TCR can be found on the surface of a cell or in a soluble form. Typically, TCRs are found on the surface of T cells (or T lymphocytes) that are generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, the TCR may also contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, for example, Janeway et ah, Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p.4:33, 1997). For example, in some aspects, each chain of a TCR may have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with an invariant protein of the CD3 complex that participates in mediating signal transduction. Unless otherwise indicated, the term "TCR" should be understood to encompass functional TCR fragments thereof. The term also encompasses complete or full-length TCRs, including TCRs in αβ or γδ form.

[0078] Therefore, for the purposes of this article, reference to a TCR includes any TCR or functional fragment, such as an antigen binding portion of a TCR that binds to a specific antigenic peptide (i.e., an MHC-peptide complex) bound to an MHC molecule. An "antigen binding portion" or "antigen binding fragment" of a TCR that can be used interchangeably refers to a molecule that contains a portion of a domain of a TCR but binds to an antigen (e.g., an MHC-peptide complex) bound by a complete TCR. In some cases, the antigen binding portion contains a variable domain of a TCR, such as a variable α chain and a variable β chain of a TCR, which is sufficient to form a binding site that binds to a specific MHC-peptide complex, such as a binding site that typically contains three complementary determining regions per chain.

[0079] In some embodiments, the variable domains of the TCR chains associate to form a loop or complementary determining region (CDR) similar to an immunoglobulin, which confers antigen recognition and determines peptide specificity by forming the binding site of the TCR molecule. Typically, as with immunoglobulins, CDRs are separated by framework regions (FRs) (see, for example, Jores et al., Pwc. Nat'l Acad. Sci. USA 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, although it has been shown that the CDR1 of the α chain interacts with the N-terminal portion of the antigenic peptide, and the CDR1 of the β chain interacts with the C-terminal portion of the peptide, CDR3 is the main CDR responsible for recognizing processed antigens. CDR2 is believed to recognize MHC molecules. In some embodiments, the variable region of the β chain may contain other hypervariable (HV4) regions.

[0080] In some embodiments, the TCR chain contains a constant domain. For example, like immunoglobulins, the extracellular portion of a TCR chain (e.g., α-chain, β-chain) can contain two immunoglobulin domains at the N-terminus, a variable domain (e.g., Va or Vp; 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 ed.) and a constant domain adjacent to the cell membrane (e.g., α-chain constant domain or Ca, typically amino acids 117-259 based on Kabat, β-chain constant domain or Cp, typically amino acids 117-295 based on Kabat). For example, in some cases, the extracellular portion of a TCR formed by two chains contains two membrane proximal constant domains and two membrane distal variable domains containing CDRs. The constant domain of a TCR domain contains a short linker sequence in which cysteine ​​residues form a disulfide bond, thereby forming a connection between the two chains. In some embodiments, The TCR may have an additional cysteine ​​residue in each of the α and β chains such that the TCR contains two disulfide bonds in the constant domain.

[0081] In some embodiments, the TCR chain may contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, this structure allows TCR to associate with other molecules (such as CD3). For example, a TCR containing a constant domain with a transmembrane region can anchor the protein in the cell membrane and associate with the invariant subunit of the CD3 signaling device or complex.

[0082] Typically, CD3 is a multiprotein complex that can have three different chains (γ, δ and ε) in mammals and ζ chains. For example, in mammals, the complex may contain a homodimer of CD3y chain, CD35 chain, two CD3s chains and CD3ζ chain. CD3y, CD35 and CD3s chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of CD3y, CD35 and CD3s chains are negatively charged, which is a feature that allows these chains to associate with positively charged T cell receptor chains. The intracellular tails of CD3y, CD35 and CD3s chains each contain a single conserved motif, called an immunoreceptor tyrosine-based activation motif or ITAM, and each CD3ζ chain has three. Typically, ITAM participates in the signal transduction ability of the TCR complex. These auxiliary molecules have a negatively charged transmembrane region and play a role in propagating signals from TCR to cells. CD3 and ζ chains form the so-called T cell receptor complex together with TCR.

[0083] In some embodiments, the TCR can be a heterodimer of two chains α and β (or optionally γ and δ), or it can be a single-chain TCR construct. In some embodiments, the TCR is a heterodimer (α and β chains or γ and δ chains) containing two separate chains (such as connected by one or more disulfide bonds).

[0084] Exemplary antigen receptors, including CARs and recombinant TCRs, and methods of engineering and introducing the receptors into cells, include, for example, those described in International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061; U.S. Patent Application Publication Nos. US2002131960, US2013287748, US20130149337; 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 those described in European Patent Application No. EP2537416, and / or those described in Sadelain et al., Cancer Discov. 2013 April; 3(4):388-398; Davila et al. (2013) PLoS ONE 8(4):e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5):633-39; Wu et al., Cancer, 2012 March 18(2):160-75. In some aspects, the genetically engineered antigen receptors include CARs as described in U.S. Patent No.: 7,446,190, and those described in International Patent Application Publication No.: WO / 2014055668A1.

[0085] antigen

[0086] Among the antigens targeted by genetically engineered antigen receptors are antigens expressed in the context of diseases, disorders or cell types targeted by adoptive cell therapy. These diseases and disorders include proliferative, neoplastic and malignant diseases and disorders, more particularly cancer.

[0087] Cancer can be a solid cancer or a "liquid tumor", such as cancers affecting the blood, bone marrow and lymphatic system, also known as tumors of the hematopoietic system and lymphoid tissue, which particularly include leukemias and lymphomas. For example, liquid tumors include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic 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 cancer and bile duct cancer, retinal cancer (such as retinoblastoma)).

[0088] Solid cancers particularly include cancers affecting one of the organs selected from the group consisting of colon, rectum, skin, endometrium, lung (including non-small cell lung cancer), uterus, bone (such as osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, giant cell tumor, adamantoma and chordoma), liver, kidney, esophagus, stomach, bladder, pancreas, cervix, brain (such as meningioma, glioblastoma, low grade astrocytoma, oligodendroglioma, pituitary tumor, schwannoma and metastatic brain cancer), ovary, breast, head and neck, testicle, prostate and thyroid.

[0089] Preferably, the cancer according to the invention is a cancer affecting the blood, bone marrow and lymphatic system as described above. Typically, the cancer is or is associated with multiple myeloma.

[0090] Diseases according to the present invention also encompass infectious diseases or disorders, such as, but not limited to, viral, retroviral, bacterial and protozoan infections, immunodeficiency, cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus; autoimmune or inflammatory diseases or disorders, such as arthritis (e.g., rheumatoid arthritis (RA)), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease, multiple sclerosis, asthma and / or transplant-related diseases or disorders.

[0091] In some embodiments, the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells (e.g., tumors or pathogenic cells) of a disease or condition compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or expressed on engineered cells. In some such embodiments, the multi-target and / or gene disruption methods provided herein are used to improve specificity and / or efficacy.

[0092] In some embodiments, the antigen is a universal tumor antigen. The term "universal tumor antigen" refers to an immunogenic molecule, such as a protein, which is usually expressed at a higher level in tumor cells than in non-tumor cells, and is also expressed in tumors of different origins. In some embodiments, the expression of universal tumor antigens in human cancer exceeds 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or more. In some embodiments, universal tumor antigens are expressed in at least three, at least four, at least five, at least six, at least seven, at least eight or more different types of tumors. In some cases, universal tumor antigens can be expressed in non-tumor cells (such as normal cells), but their expression level is lower than the expression level in tumor cells. In some cases, universal tumor antigens are not expressed at all in non-tumor cells, such as not expressed in normal cells. For example, exemplary universal tumor antigens include 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 cell cycle protein (D1). Peptide epitopes of tumor antigens including universal tumor antigens are known in the art, and in some aspects, can be used to generate MHC restricted antigen receptors, such as TCR or TCR-like CAR (e.g., see published PCT application No. WO2011009173 or WO2012135854 and published U.S. application No. US20140065708).

[0093] In some aspects, the antigen is expressed on multiple myeloma, such as 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 for such antigens are known, for example, including U.S. Patent Nos. 8,153,765; 8,603477; 8,008,450; U.S. Publication Application No. US20120189622 and those described in the published international PCT application Nos. WO2006099875, WO2009080829 or WO201209261. In some embodiments, the antibody or its antigen-binding fragment (e.g., scFv) can be used to generate CAR.

[0094] In some embodiments, the antigen may be an antigen expressed or upregulated on a cancer cell or tumor cell, but may also be expressed in an immune cell such as a resting or activated T cell. For example, in some cases, it is reported that expression of hTERT, survivin and other universal tumor antigens is present in lymphocytes, including activated T lymphocytes (e.g., see 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 may also be expressed in immune cells such as T cells, such as upregulated in activated T cells. For example, in some aspects, CD38 is a known T cell activation marker.

[0095] In some embodiments provided herein, immune cells (such as T cells) can be engineered to inhibit or destroy genes encoding antigens in immune cells so that the expressed genetically engineered antigen receptors do not specifically bind to antigens when expressed on the immune cells themselves. Therefore, in some aspects, this can avoid off-target effects, such as the binding of engineered immune cells to themselves, which may reduce the efficacy of engineered immune cells, such as those associated with adoptive cell therapy.

[0096] In some embodiments, such as in the case of an inhibitory CAR, the target is an off-target marker, such as an antigen that is not expressed on a diseased cell or cell to be targeted but is expressed on a normal or non-diseased cell, which in the same engineered cell also expresses a disease-specific target targeted by an activating or stimulating receptor. An example of such an antigen is an MHC molecule, such as a class I MHC molecule, for example, which is relevant to the treatment of a disease or disorder in which these molecules are downregulated but remain expressed in non-targeted cells.

[0097] In some embodiments, the engineered immune cells may contain antigens that target 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 antigen receptors on provided immune cells may include orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3 or 4, FBP, fetal acetylcholine e receptor, GD2, GD3, HMW-MAA, IL-22R-α, IL-13R-α2, kdr, kappa light chain, Lewis Y, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gplOO, tumor-fetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrinB2, CD 123, CS-1, c-Met, GD-2 and MAGE A3, CE7, Wilms Tumor 1 (WT-1), cell cycle proteins (such as cyclin A1 (CCNA1)), and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens.

[0098] In some embodiments, CAR binds to pathogen-specific antigens. In some embodiments, CAR is specific for viral antigens (such as HIV, HCV, HBV, etc.), bacterial antigens, and / or parasite antigens.

[0099] In some embodiments, the cell of the present invention is genetically engineered to express two or more genetically engineered receptors on a cell, each receptor recognizes different antigens, and typically, each receptor includes different intracellular signaling components. For example, this multi-target strategy is described in International Patent Application Publication No. WO 2014055668 A1 (description of the combination of activation and costimulation CAR, for example, targeting is present alone in a target (such as a normal cell) Outside, but only present together in two different antigens on the cell of the disease to be treated or the condition) and Fedorov et al., Sci.Transl.Medicine, 5 (215) (December 2013) (description of cells expressing activation and inhibitory CAR, such as wherein activation CAR is combined with a kind of antigen expressed on normal or non-diseased cells and disease to be treated or the condition cell, and inhibitory CAR is combined with another antigen expressed only on normal cells or cells that do not need treatment).

[0100] In some cases, the overexpression of stimulatory factors (such as lymphokines or cytokines) may be toxic to the subject. Therefore, in some cases (such as when applied in adoptive immunotherapy), engineered cells include gene fragments that make cells easy to be negatively selected in vivo. For example, in some aspects, cells are engineered so that they can be eliminated due to changes in the patient's in vivo conditions of the patient who applies them. Negative selection phenotypes can be caused by inserting genes that confer sensitivity to the administered reagent (such as compounds). Negative selection genes include herpes simplex virus type I thymidine kinase (HSV-1TK) genes (Wigler et al., Cell II: 223, 1977) that confer ganciclovir sensitivity; cell hypoxanthine phosphoribosyl transferase (HPRT) gene; cell adenine phosphoribosyl transferase (APRT) gene; bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89: 33 (1992)).

[0101] In other embodiments of the present invention, cells (e.g., T cells) are not engineered to express recombinant receptors, but include naturally occurring antigen receptors specific for the desired antigen, such as tumor infiltrating lymphocytes and / or T cells cultured in vitro or ex vivo in an incubation step, to promote the expansion of cells with specific antigen specificity. For example, in some embodiments, cells for adoptive cell therapy are produced by separating tumor-specific T cells (e.g., autologous tumor infiltrating lymphocytes (TIL)). In some cases, direct targeting of human tumors using autologous tumor infiltrating lymphocytes can mediate tumor regression (see Rosenberg SA, et al. (1988) N Engl J Med. 319: 1676-1680). In some embodiments, lymphocytes are extracted from resected tumors. In some embodiments, such lymphocytes are amplified in vitro. In some embodiments, such lymphocytes are cultured together with lymphokines (e.g., IL-2). In some embodiments, such lymphocytes mediate specific lysis of autologous tumor cells, but do not mediate specific lysis of allogeneic tumors or autologous normal cells.

[0102] In other nucleic acids, for example, genes for introduction are those that improve therapeutic efficacy, such as by promoting the viability and / or function of transferred cells; provide gene markers to select and / or evaluate cells, such as genes that evaluate survival or localization in vivo; for example, as described in Lupton SD et al., Mol. and Cell Biol., 11: 6 (1991); and Riddell et al., Human Gene Therapy 3: 319-338 (1992), genes that improve safety by making cells susceptible to negative selection in vivo; also see the disclosures of PCT / US91 / 08442 and PCT / US94 / 05601 of Lupton et al., which describe the use of bifunctional selective fusion genes derived from fusion of dominant positive selective markers with negative selective markers. For example, see Riddell et al., U.S. Patent No. 6,040,177, columns 14-17.

[0103] Method for obtaining cells according to the invention

[0104] The present invention also relates to a method for producing a modified or engineered immune cell, which comprises the step of inhibiting the expression and / or activity of Suv39h1 in the immune cell.

[0105] Preferably, the method for obtaining the cell according to the present invention further comprises the step of introducing a genetically engineered antigen receptor that specifically binds to the target antigen into the immune cell.

[0106] The inhibition of the expression and / or activity of Suv39h1 and the introduction of a genetically engineered antigen receptor that specifically binds to a target antigen in an immune cell can be performed simultaneously or sequentially in any order.

[0107] Inhibition of Suv39h1

[0108] According to the present invention, the engineered immune cells may be contacted with at least one agent that inhibits or blocks the expression and / or activity of Suv39h1.

[0109] The agent may be selected from small molecule inhibitors. Antibody derivatives that typically block or inhibit the expression or activity of Suv39h1, such as intracellular antibodies, nanobodies or affinity antibodies; aptamers that typically 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 interfering agents (such as small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA) or piwiRNA (piRNA); ribozymes or combinations thereof.

[0110] The at least one agent may also be an exogenous nucleic acid comprising a) an engineered non-naturally occurring 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 wherein the cell is genetically modified to express the Cas9 protein. The agent may also be a zinc finger protein (ZFN) or a TAL protein.

[0111] The term "small organic molecule" refers to a molecule of a size comparable to that of an organic molecule commonly used in medicine. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size from up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0112] In a specific embodiment, the inhibitor of H3K9-histone methyltransferase SUV39H1 is chaetocin (CAS 28097-03-2), such as 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. 2005 Aug; 1(3): 143-5.; Weber, HP, 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, DM, et al, “The epipolythiodioxopiperazine (ETP) class of fungal toxins: distribution, mode of action, functions and biosynthesis”, Microbiol, 151, 1021-1032 (2005). For example, chaetocin can be commercially obtained from Sigma Aldrich.

[0113] The inhibitor 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), racemic analogs of epidithiodiketopiperazine alkaloid lipoprotein A (see WO2014066435, also see Baumann M, Dieskau AP, Loertscher BM, et al. Tricyclic Analogues of Epidithiodioxopiperazine Alkaloids with Promising In Vitro and In VivoAntitumor Activity. Chemical science (Royal Society of Chemistry: 2010). 2015; 6:4451-4457, and Snigdha S, Prieto GA, Petrosyan A,et al.H3K9me3 InhibitionImproves Memory,Promotes Spine Formation,and Increases BDNF Levels in theAged Hippocampus.The Journal of Neuroscience.2016;36(12):3611-3622).

[0114] The inhibitory activity of a compound can be determined using various methods, such as described by Greiner D. et al. Nat Chem Biol. 2005 Aug; 1(3): 143-5 or Eskeland, R. et al. Biochemistry 43, 3740-3749 (2004).

[0115] Inhibition of Suv39h1 in cells can be achieved before or after injection in a target patient. In some embodiments, inhibition as previously defined is performed in vivo after administering cells to a subject. Typically, a Suv39h1 inhibitor as defined herein can be included in a composition containing cells. Suv39h1 can also be administered separately before, simultaneously with, or after administering cells to a subject.

[0116] Typically, inhibition of Suv39h1 of the present invention can be achieved by incubating the cells of the present invention with a composition comprising at least one pharmacological inhibitor as described above, which is included during the expansion of anti-tumor T cells in vitro, thereby altering their reconstitution, survival and therapeutic efficacy after adoptive transfer.

[0117] Inhibition of Suv39h1 in cells according to the present invention can be achieved by intracellular antibodies. Intracellular antibodies are antibodies that are produced in the same cell and bind to its antigen in the cell (for example, see the following reviews, Marschall AL, Dübel Sand T“Specific in vivo knockdown of protein function byintrabodies”,MAbs. 2015;7(6):1010-35.but see also Van Impe K,Bethuyne J,CoolS,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 CapGrestrains breast cancer metastasis”.Breast Cancer Res 2013; 15:R116;Hyland S,Beerli RR,Barbas CF,Hynes NE,Wels W..“Generation and functionalcharacterization of intracellular antibodies interacting with the kinasedomain of human EGF receptor.Oncogene 2003;22:1557-67”;Lobato MN,Rabbitts TH.“Intracellular antibodies and challenges facing their use as therapeuticagents”.Trends Mol Med 2003;9:390-6,and Donini M,Morea V,Desiderio A,Pashkoulov D,Villani ME,Tramontano A,Benvenuto E.“Engineering stablecytoplasmic intrabodies with designed specificity”.J Mol Biol.2003Jul 4;330(2):323-32.)。

[0118] Intracellular antibodies can be generated by cloning corresponding cDNA from existing hybridoma clones, or more conveniently, new scFvs / Fab can be selected from in vitro display technology (such as phage display), which provides the necessary genes for encoding antibodies from the outset, and allows more detailed pre-design of antibody fine specificity. In addition, bacteria-, yeast-, mammalian cell surface display and ribosome display can be adopted. However, the most commonly used in vitro display system for selecting specific antibodies is phage display. In a process called panning (affinity selection), recombinant antibody phage is selected by incubating the antibody phage library with the antigen. Repeat this process several times to obtain an enriched antibody library containing a specific antigen binding agent for almost any possible target. So far, the recombinant human antibody library assembled in vitro has produced thousands of novel recombinant antibody fragments. It should be noted that the premise of knocking down specific proteins by cytoplasmic intracellular antibodies is to neutralize / inactivate the antigen by antibody binding. Five different approaches to producing suitable antibodies have emerged: 1) in vivo selection of functional intrabodies (antigen-dependent and independent) in eukaryotic organisms (such as yeast) and prokaryotes (such as E. coli); 2) production of antibody fusion proteins to improve cytoplasmic stability; 3) use of specific frameworks to improve cytoplasmic stability (e.g., by grafting CDRs or introducing synthetic CDRs in a stable antibody framework); 4) use of single domain antibodies to improve cytoplasmic stability; and 5) selection of stable intrabodies that do not contain disulfide bonds. Those approaches are particularly described in detail in Marschall, AL et al., mAbs 2015, as described above.

[0119] The most commonly used form of intracellular antibodies is scFv, which consists of H chain and L chain variable antibody domains (VH and VL) held together by a short and flexible linker sequence (usually (Gly4Ser)3) to avoid the need to express and assemble 2 antibody chains of complete IgG or Fab molecules separately. Alternatively, the Fab format has been used, which additionally contains the C1 domain of the heavy chain and the constant region of the light chain. Recently, a new possible form of intracellular antibodies, scFab, has been described. The scFab format is expected to make it easier to subclone the available Fab genes into intracellular expression vectors, but it remains to be seen whether it provides any advantages over the well-established scFv format. In addition to scFv and Fab, bispecific formats have been used as intracellular antibodies. Compared with monospecific antibody counterparts, the bispecific Tie-2x VEGFR-2 antibody targeting ER has been shown to have an extended half-life. A bispecific transmembrane intrabody was developed as a specific format to simultaneously recognize intracellular and extracellular epitopes of epidermal growth factor, combining the unique features of the related monospecific antibodies, namely inhibition of autophosphorylation and ligand binding.

[0120] Another intracellular antibody format particularly suitable for cytoplasmic expression is a single domain antibody (also called nanobody) derived from camelids or composed of one human VH domain or human VL domain. These single domain antibodies generally have favorable properties, such as high stability; good solubility; easy library cloning and selection; high expression yield in E. coli and yeast.

[0121] Following transfection with an expression plasmid or viral transduction with a recombinant virus, the intrabody gene can be expressed in the target cells. Typically, selection is intended to provide optimal intrabody transfection and production levels. Successful transfection and subsequent intrabody production can be analyzed by immunoblotting to detect the produced antibodies, however, to assess correct intrabody / antigen interactions, co-immunoprecipitation from HEK 293 cell extracts transiently co-transfected with the corresponding antigen and intrabody expression plasmids can be used.

[0122] Inhibition of Suv39h1 in cells according to the invention can also be achieved with aptamers that inhibit or block the expression or activity of Suv39h1. Aptamers are a class of molecules that represent alternatives to antibodies in terms of molecular recognition. Aptamers are oligonucleotides (DNA or RNA) or oligopeptide sequences that have the ability to recognize almost any type of target molecule with high affinity and specificity.

[0123] Oligonucleotide aptamers can be separated by systematic evolution of ligands by exponential enrichment (SELEX) of random sequence libraries, as described in Tuerk C. and Gold L., 1990. The random sequence library can be obtained by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer of unique sequence, which is ultimately chemically modified. The possible modifications, uses and advantages of this type of molecule have been reviewed in Jayasena SD, 1999.

[0124] Peptide aptamers consist of conformationally constrained antibody variable regions displayed by platform proteins (such as Escherichia coli thioredoxin A), which are selected from combinatorial libraries by a two-hybrid approach (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. 1996 Apr 11; 380(6574): 548-50).

[0125] Inhibition of Suv39h1 in cells according to the invention can also be achieved by affinity antibody molecules. Affibodies are small proteins engineered to mimic monoclonal antibodies in binding to a large number of target proteins or peptides with high affinity and are therefore members of the antibody mimetic family (see review J,Feldwisch J,Tolmachev V,Carlsson J, S, Frejd FY. Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett. 2010 Jun 18; 584 (12): 2670-80). Affibody molecules are based on engineered variants of the B domain (Z domain) in the immunoglobulin binding region of Staphylococcal protein A, and can theoretically bind specifically to any given target. Affibody molecule libraries are usually constructed by randomizing the combination of 13 amino acid positions in helices 1 and 2 that contain the original Fc binding surface of the Z domain. Typically, the library is displayed on phages and then bio-panned for the desired target. If the primary affinity increases, affinity maturation usually leads to improved binders and can be achieved by helical shuffling or sequence alignment combined with directed combinatorial mutagenesis. Recently identified molecules with altered binding surfaces generally retain the original helical structure and high stability, although unique exceptions with interesting properties have been reported. Due to their small size and rapid folding properties, affinity antibody molecules can be produced by chemical peptide synthesis.

[0126] In other embodiments of the invention, inhibition of Suv39h1 activity can be achieved by gene suppression / inhibition using RNA interference (RNAi) such as short interfering RNA (siRNA), short hairpin RNA (shRNA) or ribozyme knockdown. siRNA technology includes RNAi-based technologies 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 is typically homologous / complementary to one region of the mRNA transcribed from the gene, or may be an siRNA comprising multiple RNA molecules homologous / complementary to different regions.

[0127] Antisense oligonucleotides including antisense RNA molecules and antisense DNA molecules will directly block the translation of H3K9-histone methyltransferase Suv39h1, thereby preventing protein translation or increasing mRNA degradation, thereby reducing the level of H3K9-histone methyltransferase SUV39H1 and its activity in cells. For example, antisense oligonucleotides complementary to a unique region of the mRNA transcript sequence encoding H3K9-histone methyltransferase SUV39H1 can be synthesized, for example, by conventional phosphodiester technology, and administered by, for example, intravenous injection or infusion. Methods for using antisense technology to specifically inhibit gene expression of genes with known sequences are well known in the art (e.g., see 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).

[0128] "RNA interference agent" as used herein is defined as any agent that interferes with or inhibits the expression of a target biomarker gene by RNA interference (RNAi). Such RNA interference agents include, but are not limited to, nucleic acid molecules including RNA molecules homologous to the target gene of the present invention (e.g., Suv39h1), or fragments thereof, short interfering RNA (siRNA), and small molecules that interfere with or inhibit the expression of a target nucleic acid by RNA interference (RNAi).

[0129] Small inhibitory RNA (siRNA) can also be used as an expression inhibitor for the present invention. H3K9-histone methyltransferase SUV39H1 gene expression can be reduced by contacting a subject or cell with a small double-stranded RNA (dsRNA) or a vector or construct that causes the production of small double-stranded RNA, thereby specifically inhibiting H3K9-histone methyltransferase SUV39H1 gene expression (i.e., RNA interference or RNAi). For genes whose sequences are known, methods for selecting suitable dsRNA or dsRNA encoding vectors are well known in the art (e.g., see Tuschl, T. et al. (1999); Elbashir, SM et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, ​​TR. 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 WO01 / 68836). Advantageously, all or part of the phosphodiester bonds of the siRNA of the present invention are protected. Such protection is generally achieved by chemical means using methods known in the art. For example, the phosphodiester bonds can be protected by thiol or amine functional groups or by phenyl groups. Advantageously, the 5'- and / or 3'-end of the siRNA of the invention is also protected, for example using the above-mentioned techniques for protecting phosphodiester bonds. The siRNA sequence advantageously comprises at least twelve consecutive dinucleotides or derivatives thereof.

[0130] As used herein, the term "siRNA derivative" with respect to the nucleic acid sequence of the invention refers to a nucleic acid having an identity percentage of at least 90% with erythropoietin or a fragment thereof, preferably at least 95%, such as at least 98%, more preferably at least 98%.

[0131] As used herein, the expression "percent identity" between two nucleic acid sequences refers to the percentage of identical nucleic acids obtained with the best alignment of the sequences between the two sequences to be compared, which percentage is purely statistical and the differences between the two sequences are randomly distributed over the nucleic acid sequences. As used herein, "best alignment" or "optimized alignment" refers to the alignment with the highest percentage identity (see below) determined. Sequence comparison between two nucleic acid sequences is usually achieved by comparing these sequences that have been previously aligned according to the best alignment; the comparison is achieved on comparison segments in order to identify and compare similar local areas. In addition to manual operation, optimal sequence alignment can also be achieved by using the global 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 that adopts these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA in Wisconsin Genetics software Package, Genetics Computer Group, 575 Science Dr., Madison, WI USA), and by using the MUSCLE multiple alignment algorithm (Edgar, Robert C, Nucleic Acids Research, vol. 32, p: 1792, 2004). In order to obtain the best local comparison, preferably use BLAST software.Measure the identity percentage between two nucleotide sequences by comparing these two sequences of optimizing comparison, nucleotide sequence can comprise addition or disappearance with respect to reference sequence to obtain the optimization comparison between these two sequences.Through following calculation identity percentage: measure the number of identical positions between these two sequences, then this number is divided by the total number of comparison positions, then the result obtained is multiplied by 100 to obtain the identity percentage between these two sequences.

[0132] ShRNA (short hairpin RNA) can also be used as an expression inhibitor for the present invention. Typically, shRNA consists of a short antisense strand (e.g., 19-25 nucleotides), followed by a 5-9 nucleotide loop and a similar sense strand. Alternatively, the sense strand can be before the nucleotide loop structure, and the antisense strand can be after the nucleotide loop structure.

[0133] Ribozymes can also be used as expression inhibitors for the present invention. Ribozymes are enzymatic RNA molecules that can catalyze specific cleavage of RNA. The mechanism of action of ribozymes involves sequence-specific hybridization of ribozyme molecules with complementary target RNA, followed by endonucleolytic cleavage. Therefore, engineered hairpin or hammerhead motif ribozyme molecules that specifically and effectively catalyze endonucleolytic cleavage of H3K9-histone methyltransferase SUV39H1 mRNA sequences are useful within the scope of the present invention. First, specific ribozyme cleavage sites in any potential RNA target are preliminarily identified by scanning the ribozyme cleavage sites of the target molecule, which typically include the following sequences: GUA, GUU and GUC. Once identified, the predicted structural features of short RNA sequences between about 15 to 20 ribonucleotides corresponding to the target gene region containing the cleavage site can be evaluated, such as secondary structures, which would make the oligonucleotide sequence unsuitable.

[0134] Antisense oligonucleotides and ribozymes that can be used as expression inhibitors can be prepared by known methods. These include techniques for chemical synthesis, such as by solid phase phosphoramidite chemical synthesis. Alternatively, antisense RNA molecules can be produced by transcribing the DNA sequence encoding the RNA molecule in vitro or in vivo. This DNA sequence can be incorporated into a variety of vectors, which have incorporated suitable RNA polymerase promoters (such as T7 or SP6 polymerase promoters). As a means to increase intracellular stability and half-life, various modifications of the oligonucleotide of the present invention can be introduced. Possible modifications include but are not limited to adding ribonucleotides or deoxyribonucleotides at the 5' and / or 3' ends of the molecule, or using phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkage in the oligonucleotide backbone.

[0135] The antisense oligonucleotides, siRNA, shRNA and ribozymes of the present invention can be delivered in vivo alone or in combination with a vector. In the broadest sense, a "vector" is a medium that can promote the transfer of antisense oligonucleotides, siRNA, shRNA or ribozyme nucleic acids to cells, preferably to cells expressing H3K9-histone methyltransferase SUV39H1. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the degree of degradation that would result in the absence of the vector. Generally, vectors that can be used in the present invention include, but are not limited to, plasmids, phagemids, viruses, other vectors derived from viral or bacterial sources, which have been controlled by the insertion or incorporation of antisense oligonucleotides, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are preferred vector types, including but not limited to nucleic acid sequences 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; polyoma virus; Epstein-Barr virus; papillomavirus; herpes virus; vaccinia virus; poliovirus; and RA virus (such as retrovirus). Other vectors not named but known in the art can be readily adopted.

[0136] Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced by target genes. Non-cytopathic viruses include retroviruses (e.g., lentiviruses), whose life cycle involves reverse transcription of genomic viral RNA into DNA, followed by integration of the provirus into host cell DNA. Retroviruses have been approved for human gene therapy trials. The most useful are those replication-defective retroviruses (i.e., capable of directing the synthesis of desired proteins, but unable to produce infectious particles). Such genetically altered retroviral expression vectors have general uses for efficient transduction of genes in vivo. Kriegler, 1990 and in Murry, 1991 provide standard protocols for producing replication-defective retroviruses (including incorporating exogenous genetic material into plasmids, transfecting packaging cells lined with plasmids, producing recombinant retroviruses by packaging cell lines, collecting viral particles from tissue culture media, and infecting target cells with viral particles).

[0137] For some applications, the preferred viruses are adenovirus and adeno-associated virus (AAV), which are double-stranded DNA viruses approved for human gene therapy. In fact, 12 different AAV serotypes (AAV1-12) are known, each with a different tissue tropism (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 virus 1-12 types can be engineered to be replication-deficient and can infect a variety of cell types and species (Wu, Z Mol Ther 2006; 14: 316-27). It also has advantages such as thermal and lipid solvent stability; high transduction frequency of multiple lineage cells including hematopoietic cells; and no excessive infection inhibition, so multiple series of transductions can be performed. It has been reported that adeno-associated viruses can integrate into human cell DNA in a site-specific manner, minimizing the possibility of insertional mutagenesis and the variability of inserted gene expression that characterizes retroviral infection. In addition, wild-type adeno-associated virus infection has been passaged more than 100 times in tissue culture without selective pressure, indicating that adeno-associated virus genomic integration is a relatively stable event. Adeno-associated viruses can also function in an extrachromosomal manner.

[0138] Other vectors include plasmid vectors. Plasmid vectors have been widely described in the art and are well known to those skilled in the art. For example, see Sambrook et al., 1989. In recent years, plasmid vectors have been used as DNA vaccines for delivering antigen encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety issues as many viral vectors. However, these plasmids with promoters compatible with host cells can express peptides from genes operably encoded in the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC / CMV, SV40 and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. In addition, restriction enzymes and ligation reactions can be used to customize the design of plasmids to remove and add specific DNA fragments. Plasmids can be delivered by various parenteral, mucosal and local routes. For example, DNA plasmids can be injected intramuscularly, intradermally, subcutaneously or by other routes. It can also be administered by intranasal sprays or drops, rectal suppositories and oral administration. It can also be applied to the epidermis or mucosal surface using a gene gun. The plasmid can be provided in aqueous solution, dried onto gold particles, or combined with another DNA delivery system including, but not limited to, liposomes, dendrimers, cochlear delivery vehicles, and microencapsulation.

[0139] Antisense oligonucleotides, siRNA, shRNA or ribozyme nucleic acid sequences according to the present invention are usually under the control of a heterologous regulatory region (e.g., a heterologous promoter). The promoter can be specific to Muller glial cells, microglia, endothelial cells, pericytes and astrocytes. For example, specific expression in Muller glial cells can be obtained by the promoter of the glutamine synthetase gene. Such as, the promoter can also be a viral promoter, such as a CMV promoter or any synthetic promoter.

[0140] Genetic repression or disruption of Suv39h1

[0141] Inhibition of Suv39h1 in cells according to the invention can also be achieved by inhibiting or destroying the Suv39h1 gene, such as by deletion, e.g., deletion of the entire gene, exons or regions, and / or replacement by exogenous sequences, and / or by mutations within the gene (typically, within the exons of the gene), such as frameshift or missense mutations. In some embodiments, the destruction results in premature stop codons being incorporated into the gene, so that the Suv39h1 protein is not expressed or is non-functional. The destruction is usually performed at the DNA level. The destruction is usually permanent, irreversible or non-temporary.

[0142] In some embodiments, gene disruption or inhibition is achieved using a gene editing agent, such as a DNA-targeting molecule that specifically binds or hybridizes to the gene, such as a DNA-binding protein or a DNA-binding nucleic acid, or a complex, compound, or composition containing them. 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 a TAL effector (TALE) DNA-binding domain, a clustered regularly interspaced short palindromic repeat (CRISPR) DNA-binding domain, or a DNA-binding domain from a meganuclease.

[0143] Zinc finger, TALE, and CRISPR system binding domains can be “engineered” to bind to predetermined nucleotide sequences.

[0144] 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, to facilitate inhibition or disruption of a gene. For example, in some embodiments, gene disruption is performed by a fusion protein comprising a DNA binding protein and its heterologous regulatory domain or a functional fragment thereof.

[0145] Typically, the additional domain is a nuclease domain. Thus, in some embodiments, engineered proteins such as nucleases and complexes or fusion proteins containing nucleases are used to promote gene disruption by gene or genome editing, and the engineered proteins consist of sequence-specific DNA binding domains fused or compounded with non-specific DNA cleavage molecules such as nucleases.

[0146] These targeted chimeric nucleases or complexes containing nucleases perform precise gene modification by inducing targeted double-strand breaks or single-strand breaks, stimulating cellular DNA repair mechanisms, including error-prone non-homologous end joining (NHEJ) and homology-directed repair (HDR). In some embodiments, the nuclease is an endonuclease, such as a zinc finger nuclease (ZFN), a TALE nuclease (TALEN), an RNA-guided endonuclease (RGEN), such as a CRISPR-associated (Cas) protein or a meganuclease. Such systems are well known in the art (e.g., see U.S. Pat. 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; US Pat. Publ. 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:el9722; Li et al. (2011) Nucl. Acids Res. 39:6315-6325; Zhang et al. (201 1) Nat. Biotech. 29:149-153; Miller et al. (2011) Nat. Biotech. 29: 143-148; Lin et al. (2014) Nucl. Acids Res. 42:e47). This genetic strategy can use a constitutive expression system or an inducible expression system according to methods well known in the art.

[0147] ZFP and ZFN; TAL, TALE and TALEN

[0148] In some embodiments, the DNA-targeting molecule includes 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).

[0149] In some embodiments, the DNA-targeting molecule comprises one or more zinc finger proteins (ZFPs) or domains thereof that bind to DNA in a sequence-specific manner. ZFPs or their domains are domains within a protein or a larger protein that bind to DNA in a sequence-specific manner by binding to one or more zinc finger regions of an amino acid sequence within the domain, and the structure of the domain is stabilized by zinc ion coordination. Typically, the sequence specificity of ZFPs can be altered by amino acid substitutions at four helical positions (-1, 2, 3, and 6) on the zinc finger recognition helix. Therefore, in some embodiments, ZFPs or molecules containing ZFPs are non-naturally occurring, for example, engineered 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. al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10: 411-416.

[0150] In some embodiments, the DNA-targeting molecule is or comprises a zinc finger DNA binding domain fused to a DNA cleavage domain 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 that may or may not be engineered. In some embodiments, the cleavage domain is from the type IIS restriction endonuclease FokI. For example, see U.S. Pat. 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.

[0151] In some aspects, ZFNs effectively generate double-strand breaks (DSBs), for example, at predetermined sites in the coding region of a target gene (i.e., Suv39h1). Typical targeted gene regions include exons, regions encoding N-terminal regions, first exons, second exons, and promoter or enhancer regions. In some embodiments, transient expression of ZFNs promotes efficient and permanent destruction of target genes in engineered cells. In particular, in some embodiments, delivery of ZFNs results in permanent destruction of genes with an efficiency exceeding 50%. Many gene-specific engineered zinc fingers are commercially available. For example, Sangamo Biosciences (Richmond, CA, USA) has collaborated with Sigma-Aldrich (St. Louis, MO, USA) to develop a zinc finger construction platform (CompoZr) that allows researchers to completely bypass the construction and verification of zinc fingers 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 commercially available zinc fingers (eg, see Sigma-Aldrich catalog numbers CSTZFND, CSTZFN, CTI1-1KT, and PZD0020).

[0152] In some embodiments, the DNA-targeting molecule comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as in a transcription activator-like protein effector (TALE) protein. For example, see U.S. Patent Publication No. 20110301073. In some embodiments, the molecule is a DNA binding endonuclease, such as a TALE nuclease (TALEN). In some aspects, TALEN is a fusion protein comprising a DNA binding domain derived from TALE and a nuclease catalytic domain to cut 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 adenosine receptors, such as A2AR.

[0153] In some embodiments, TALEN recognizes and cuts the target sequence in the gene. In some aspects, the cutting of DNA causes double-strand breaks. In some aspects, the rate of fracture stimulation homologous recombination or non-homologous end joining (NHEJ). Generally, NHEJ is an imperfect repair process, which usually causes the DNA sequence of the cutting site to change. In some aspects, the repair mechanism involves reconnecting the residues of the two DNA ends by directly reconnecting (Critchlow and Jackson, Trends Biochem Sci.1998Oct; 23 (10): 394-8) or by so-called microhomology-mediated end connection. In some embodiments, the repair via NHEJ causes a small insertion or deletion, and can be used to destroy and thus inhibit genes. In some embodiments, modification can be a substitution, deletion or addition of at least one nucleotide. In some aspects, it is possible to identify and / or select cells in which a mutagenic event (i.e., a mutagenic event continuous with an NHEJ event) induced by cleavage occurs by methods well known in the art.

[0154] TALE repeats can be assembled to specifically target the Suv39h1 gene (Gaj et al., Trends in Biotechnology, 2013, 31 (7), 397-405). A TALEN library for 18,740 human protein-coding genes has been established (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). Specifically, TALENs targeting CD38 are commercially available (see Gencopoeia, catalog numbers HTN222870-1, HTN222870-2, and HTN222870-3, available on the World Wide Web at www.genecopoeia.com / product / search / detail.php?prt=26&cid=&key=HTN222870). Exemplary molecules are described, for example, in U.S. Patent Publication Nos. US 2014 / 0120622 and 2013 / 0315884.

[0155] In some embodiments, TALEN is introduced as a transgene encoded by one or more plasmid vectors. In some aspects, the plasmid vector can contain a selection marker that provides for identification and / or selection of cells that accept the vector.

[0156] RGEN (CRISPR / Cas system)

[0157] One or more DNA binding nucleic acids can be used for gene suppression, such as destruction by RNA-guided endonucleases (RGENs), or other forms of suppression by another RNA-guided effector molecule. For example, in some embodiments, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins can be used for gene suppression. See Sander and Joung, Nature Biotechnology, 32(4): 347-355.

[0158] In general, "CRISPR system" refers broadly to transcripts and other elements involved in the expression of CRISPR-associated ("Cas") genes or directing their activity, including encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr pairing sequences (covering "direct repeats" and tracrRNA-processed partial direct repeats in endogenous CRISPR systems), guide sequences (also called "spacers" in endogenous CRISPR systems), and / or other sequences and transcripts from the CRISPR locus.

[0159] Typically, CRISPR / Cas nucleases or CRISPR / Cas nuclease systems include a non-coding RNA molecule (guide) RNA that specifically binds to DNA and a CRISPR protein (e.g., two nuclease domains) having a nuclease function. One or more elements of the CRISPR system may be derived from a type I, type II, or type III CRISPR system, such as a Cas nuclease. Preferably, the CRISPR protein is a cas enzyme, such as Cas9. Cas enzymes are well known in the art; for example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the SwissProt database with accession number Q99ZW2. In some embodiments, Cas nucleases and gRNA are introduced into cells. In some embodiments, the CRISPR system induces DSBs at the target site, which are then destroyed as described herein. In other embodiments, Cas9 variants considered to be "cutting enzymes" can be used to cut single strands at the target site. For example, paired cutting enzymes can also be used to improve specificity, each enzyme being guided by a pair of different gRNA targeting sequences. In still other embodiments, a catalytically inactive Cas9 can be fused to a heterologous effector domain, such as a transcriptional repressor, to affect gene expression.

[0160] In general, the CRISPR system is characterized by elements that promote the formation of CRISPR complexes at the target sequence site. Typically, in the case of forming a CRISPR complex, a "target sequence" generally refers to a sequence designed to have a complementary guide sequence, wherein the hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. If there is enough complementarity to cause hybridization and promote the formation of the CRISPR complex, it is not necessarily necessary to be fully complementary. The target sequence can include any polynucleotide, such as a DNA or RNA polynucleotide. In general, a sequence or template that can be used to reorganize into a target locus containing a target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In some aspects, an exogenous template polynucleotide may be referred to as an editing template. In some aspects, recombination is homologous recombination.

[0161] In some embodiments, one or more vectors driving the expression of one or more elements of the CRISPR system are introduced into cells so that the expression of the elements of the CRISPR system guides the formation of the CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence connected to a tracr-pairing sequence, and a tracr sequence that can be operably connected to a separate regulatory element on a separate vector. Alternatively, two or more elements expressed by the same or different regulatory elements can be combined in a single vector, wherein one or more additional vectors provide any component of the CRISPR system not included in the first vector. In some embodiments, the CRISPR system elements combined in a single vector can be arranged in any suitable direction. In some embodiments, the CRISPR enzyme, the guide sequence, the tracr mate sequence, and the tracr sequence are operably connected to the same promoter and expressed by the same promoter. In some embodiments, the vector comprises a regulatory element operably connected to an enzyme coding sequence (such as a Cas protein) encoding a CRISPR enzyme.

[0162] In some embodiments, a CRISPR enzyme bound to a guide sequence (and optionally complexed with a guide sequence) is delivered to a cell. Typically, CRISPR / Cas9 technology can be used to knock down genetic expression of Suv39h1 in engineered cells. For example, a Cas9 nuclease and a guide RNA specific for the Suv39h1 gene can be introduced into a cell, for example, using a lentiviral delivery vector or any of a variety of known delivery methods or media for transfer to a cell, such as any of a variety of known methods or media for delivering Cas9 molecules and guide RNAs (see also below).

[0163] Delivery of nucleic acids encoding gene disrupting molecules and complexes

[0164] 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 transfer methods can be used to introduce nucleic acids encoding components of a CRISPR, ZFP, ZFN, TALE, and / or TALEN system into cultured cells.

[0165] In some embodiments, since the polynucleotide encoding the polypeptide is introduced into the cell, the polypeptide is synthesized in situ in the cell. In some aspects, the polypeptide can be produced extracellularly and then introduced into the cell.

[0166] 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.

[0167] In some embodiments, the polynucleotide can be introduced into the cell by, for example, a recombinant viral vector (e.g., retrovirus, adenovirus), liposomes, etc. Transient transformation methods include microinjection, electroporation, or particle bombardment. The nucleic acid is administered in the form of an expression vector. Preferably, the expression vector is a retroviral expression vector, an adenoviral expression vector, a DNA plasmid expression vector, or an AAV expression vector.

[0168] Non-viral delivery methods for nucleic acids include lipofection, nucleofection, microinjection, bio-munitions, virosomes, liposomes, immunoliposomes, polycations or lipids, nucleic acid conjugates, naked DNA, artificial virosomes, and agent-enhanced DNA uptake. Lipofection is described in, for example, U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355, and commercially available lipofection reagents (e.g., Transfectam TM Lipofectin TM ). Cationic lipids and neutral lipids suitable for efficient receptor recognition lipid transfection of polynucleotides include those of Feigner WO 91 / 17424; WO 91 / 16024. Delivery to cells (eg, in vitro or ex vivo administration) or target tissues (eg, in vivo administration).

[0169] RNA or DNA viral-based systems include retrovirus, lentivirus, adenovirus, adeno-associated virus, and herpes simplex virus vectors for gene transfer.

[0170] For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, 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., in Current Topics in Microbiology and Immunology Doerfler and Bohm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0171] Reporter genes including but not limited to glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and autofluorescent proteins including blue fluorescent protein (BFP) can be introduced into cells to encode gene products as markers for measuring changes or modifications in the expression of gene products.

[0172] Cell preparation

[0173] Cell separation includes one or more preparation-based and / or non-affinity cell separation steps according to techniques well known in the art. For example, in some embodiments, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to a particular reagent. In some embodiments, cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity, and / or resistance to a particular component.

[0174] In some embodiments, cell preparation includes a step of freezing (e.g., cryopreserving) cells before or after separation, incubation, and / or engineering. In some aspects, any of a variety of known freezing solutions and parameters can be used.

[0175] Typically, cells are incubated under conditions prior to or associated with genetic engineering and / or Suv39h1 inhibition.

[0176] The incubating step may include culturing, incubating, stimulating, activating, amplifying and / or proliferating.

[0177] Inhibition of Suv39h1 according to the present invention may also be achieved in vivo following injection of cells into a target patient. Typically, inhibition of suv39h1 is performed using a pharmacological inhibitor as described above.

[0178] Suv39h1 inhibition can also be performed according to the aforementioned methods during stimulation, activation and / or amplification steps. For example, before adoptive transfer to the patient, PBMC 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. In some embodiments, the composition or cells are incubated in the presence of stimulating conditions or stimulants. Such conditions include those designed to induce proliferation, amplification, activation and / or survival of cells in the population, simulate antigen exposure and / or induce cells to be genetically engineered (such as for introducing genetically engineered antigen receptors).

[0179] Incubation conditions can include one or more specific culture media, temperature, oxygen content, carbon dioxide content, time, reagents (e.g., nutrients, amino acids, antibiotics, ions) and / or stimulatory factors (such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors) and any other reagents designed to activate cells.

[0180] In some embodiments, the stimulating conditions or reagents include one or more reagents, such as ligands, that can activate the intracellular signaling domain of the TCR complex. In some aspects, the reagent opens or starts the TCR / CD3 intracellular signaling cascade in the T cell. Such reagents may include antibodies, such as those specific to TCR components and / or co-stimulatory receptors, such as anti-CD3, anti-CD28, and / or one or more cytokines bound to a solid carrier (such as beads). Optionally, the amplification method may also include a step of adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulating agent includes IL-2 and / or IL-15, for example, IL-2 concentration is at least about 10 units / mL.

[0181] In certain aspects, incubation is performed according to techniques such as those described in US Pat. 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.

[0182] In some embodiments, T cells are expanded by adding feeder cells (such as non-dividing peripheral blood mononuclear cells (PBMC)) to the culture initiation composition (e.g., so that for each T lymphocyte in the initial population to be expanded, the resulting cell population contains at least about 5, 10, 20 or 40 or more PBMC feeder cells); and the culture is incubated (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells can include γ-irradiated PBMC feeder cells. In some embodiments, PBMCs are irradiated with gamma rays of about 3000-3600rads to prevent cell division. In some aspects, feeder cells are added to the culture medium before adding the T cell population.

[0183] In some embodiments, the stimulation conditions include a temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, typically at least about 30 degrees Celsius, and typically at least about 37 degrees Celsius. Optionally, incubation can also include the addition of non-dividing EBV-transformed lymphoblasts (LCL) as feeder cells. The LCL can be irradiated with gamma rays of about 6000-10,000 rads. In some aspects, LCL feeder cells are provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10: 1.

[0184] In embodiments, antigen-specific T cells, such as 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 of cytomegalovirus antigens can be generated by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.

[0185] In some aspects, the method includes assessing the expression of one or more markers on the surface of engineered cells or cells to be engineered. In one embodiment, the method includes assessing the surface expression of one or more target antigens (e.g., antigens recognized by genetically engineered antigen receptors) targeted by adoptive cell therapy, for example, by affinity-based detection methods, such as by flow cytometry.

[0186] Vectors and methods for cell genetic engineering

[0187] In some aspects, genetic engineering involves introducing into a cell a nucleic acid encoding a genetically engineered component or other component for introduction, such as a component encoding a gene disrupting protein or nucleic acid.

[0188] Typically, engineering CAR into immune cells (e.g., T cells) requires culturing cells to allow transduction and expansion. Transduction can utilize a variety of methods, but stable gene transfer is required to achieve sustained CAR expression in cloned expansion and durable engineered cells.

[0189] In some embodiments, gene transfer is achieved by first stimulating cell growth, such as T cell growth, proliferation and / or activation, and then transducing the activated cells and expanding them in culture to numbers sufficient for clinical applications.

[0190] Various methods of introducing genetically engineered components (e.g., antigen receptors, such as CARs) are well known and can be used together with the methods and compositions provided. Exemplary methods include those for transferring nucleic acids encoding receptors, including by viruses (e.g., retroviruses or slow viruses), transduction, transposons, and electroporation.

[0191] In some embodiments, the recombinant nucleic acid is transferred into cells using recombinant infectious viral particles (such as vectors derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV)). In some embodiments, the recombinant nucleic acid is transferred into T cells using recombinant lentiviral vectors or retroviral vectors (such as γ-retroviral vectors) (e.g., see Koste et al. (2014) Gene Therapy 2014Apr 3.; 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. 2011November; 29(11):550-557).

[0192] In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), such as a retroviral vector derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), spleen focus forming virus (SFFV) or adeno-associated virus (AAV). Most retroviral vectors are derived from mouse retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell source. Typically, retroviruses are amphipathic, which means that they are able to 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. Many exemplary retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, AD (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).

[0193] Methods of lentiviral transduction are also known. For example, 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.

[0194] In some embodiments, the recombinant nucleic acid is transferred into T cells by electroporation (e.g., see Chicayba 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 transferred into T cells by transposition (e.g., see 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 (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. NY); protoplast fusion; cationic liposome-mediated transfection; tungsten particle-promoted microprojectile bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)).

[0195] Other methods and vectors for transferring genetically engineered nucleic acids encoding genetically engineered products are described in, for example, International Patent Application Publication No. WO2014055668 and U.S. Patent No. 7,446,190.

[0196] Compositions of the present invention

[0197] The present invention also includes compositions containing cells described herein and / or produced by the provided methods. Typically, the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy.

[0198] The pharmaceutical composition of the present invention generally comprises at least one engineered immune cell of the present invention and a pharmaceutically acceptable carrier.

[0199] As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc., which are compatible with drug administration. Supplementary active compounds may be further incorporated into the composition. In some aspects, The choice of carrier in the pharmaceutical composition is determined in part by a specific engineered CAR or TCR, a vector or cell expressing the CAR or TCR, and by a specific method for administering the vector or host cell expressing the CAR. Therefore, there are a variety of suitable formulations. For example, The pharmaceutical composition may contain a preservative. For example, a suitable preservative may include methyl paraben, propyl paraben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. Typically, the preservative or a mixture thereof is present in an amount of about 0.0001-about 2% of the total weight of the composition.

[0200] A pharmaceutical composition is formulated to be compatible with its intended route of administration.

[0201] Treatment

[0202] The present invention also relates to the use of the cells defined previously in adoptive therapy, in particular adoptive T-cell therapy, typically in the treatment of cancer in a subject in need thereof.

[0203] As used herein, "treatment" is defined as the application or administration of cells of the present invention or compositions comprising cells to a patient in need thereof, with the intent to cure, heal, alleviate, relieve, alter, remedy, improve, ameliorate or affect a disease (such as cancer) or any symptom of a disease (such as cancer). In particular, the term "treatment" refers to the reduction or alleviation of at least one adverse clinical symptom associated with a disease (such as cancer), e.g., pain, swelling, low blood counts, etc.

[0204] With respect to cancer treatment, the term "treat" also refers to slowing or reversing the progression of neoplastic uncontrolled cell proliferation, ie, shrinking an existing tumor and / or halting tumor growth. The term "treat" also refers to inducing apoptosis in cancer or tumor cells in a subject.

[0205] 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 may be male or female, and may be of any suitable age, including infants, teenagers, adolescents, adults, and elderly subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent. In some embodiments, the patient or subject is a validated animal model for disease, adoptive cell therapy, and / or for evaluating toxicity results (such as cytokine release syndrome (CRS)). In some embodiments of the present invention, the subject suffers from cancer, is at risk of cancer, or is in cancer remission.

[0206] Cancer can be a solid cancer or a "liquid tumor", such as cancers affecting the blood, bone marrow and lymphatic system, also known as tumors of the hematopoietic system and lymphoid tissue, particularly including leukemias and lymphomas. For example, liquid tumors include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic 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 cancer and bile duct cancer, retinal cancer (such as retinoblastoma)).

[0207] Solid cancers particularly include cancers affecting one of the organs selected from the group consisting of colon, rectum, skin, endometrium, lung (including non-small cell lung cancer), uterus, bone (such as osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, giant cell tumor, adamantoma and chordoma), liver, kidney, esophagus, stomach, bladder, pancreas, cervix, brain (such as meningioma, glioblastoma, low grade astrocytoma, oligodendroglioma, pituitary tumor, schwannoma and metastatic brain cancer), ovary, breast, head and neck, testicle, prostate and thyroid.

[0208] Preferably, the cancer according to the invention is a cancer affecting the blood, bone marrow and lymphatic system as described above. Typically, the cancer is or is associated with multiple myeloma.

[0209] In some embodiments, the subject is suffering from or at risk of an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial and protozoan infections, 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 (e.g., rheumatoid arthritis (RA)), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease, multiple sclerosis, asthma, and / or a disease or condition associated with transplantation.

[0210] The present invention also relates to a method of treatment, in particular adoptive cell therapy, preferably adoptive T cell therapy, which comprises administering the aforementioned composition to a subject in need thereof.

[0211] In some embodiments, the cell or composition is administered to a subject, such as a subject suffering from cancer or any disease as described above or at risk of suffering from cancer or any disease as described above. In some aspects, these methods thus treat (e.g., improve) one or more symptoms of a disease or condition (such as a disease or condition associated with cancer) by alleviating the tumor burden in a cancer expressing an antigen recognized by an engineered cell.

[0212] Methods of administering cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, adoptive T cell therapy is described in, for example, U.S. Patent Application Publication 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). For example, see 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.

[0213] In some embodiments, cell therapy, such as adoptive cell therapy, such as adoptive T cell therapy, is performed by autologous transfer, wherein cells are isolated and / or otherwise prepared from a subject to be treated with the cell therapy or from a sample from such a subject. Thus, in some aspects, the cells are derived from a subject (e.g., a patient) in need of treatment, and after isolation and processing, the cells are administered to the same subject.

[0214] In some embodiments, cell therapy, such as adoptive cell therapy, such as adoptive T cell therapy, is carried out by allogeneic transfer, wherein the subject (e.g., the first subject) is separated and / or otherwise prepared from the subject other than the subject who will receive or eventually receive the cell therapy. In this embodiment, the cells are then administered to different subjects (e.g., the second subject) of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA category or supertype as the first subject.

[0215] At least one cell according to the present invention can be combined with one or more other therapeutic agents or with another therapeutic intervention to a subject in need thereof, to be performed simultaneously or sequentially in any order. In some cases, these cells are co-administered with another therapy that is close enough in time so that the cell population enhances the effect of one or more other therapeutic agents, and vice versa. In some embodiments, the cell population is administered before one or more other therapeutic agents. In some embodiments, the cell population is administered after one or more other therapeutic agents.

[0216] For cancer treatment, the combined cancer treatment may include, but is not limited to, chemotherapeutic agents, hormones, anti-angiogenic agents, radiolabeled compounds, immunotherapy, surgery, cryotherapy, and / or radiation therapy.

[0217] Immunotherapy includes, but is not limited to, immune checkpoint modulators (i.e., inhibitors and / or agonists), monoclonal antibodies, and cancer vaccines.

[0218] Preferably, the administration of cells in the adoptive T cell therapy according to the present invention is combined with the administration of an immune checkpoint regulator. Most preferably, the immune checkpoint regulator comprises an anti-PD-1 and / or anti-PDL-1 inhibitor.

[0219] The present invention also relates to the use of a composition comprising the engineered immune cells described herein in the preparation 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0220] Figure 1 : Suv39h1-deficient mice have increased numbers of central memory CD8+ T cells. A. Representative FACS dot plots of gated splenic CD8+ T cells. B. The percentage of central memory CD8+ T cells was measured in different hematopoietic compartments (black circles, Suv39h1 KO; white circles: WT littermates).

[0221] Figure 2 : Stemness and memory signatures in endogenous antigen-specific Suv39h1 KO CD8+ T cells. Suv39h1-deficient Kb-OVA+CD8+ T cells have stem cell-like and memory gene expression signatures. Gene set enrichment analysis (GSEA) is shown.

[0222] Figure 3 : Increased survival and self-renewal of Suv39h1-deficient CD8 T cells. A. Experimental setup showing the transfer of syngeneic CD45.2 Kb-OVA pentamer+CD8+T cells isolated from WT and Suv39h1 KO LM-OVA-infected mice to naive recipient CD45.1 mice (1: classification and transfer of CD8+T subsets (D0); 2: analysis of self-renewal and differentiation (D40-44). 40 days after adoptive transfer, naive mice were challenged with LM-OVA, and 4 days later, Kb-OVA pentamer+CD8+T cells were analyzed by FACS. B. Representative FACS plots of donor and recipient CD8+T cells are shown. C. The total number of recovered CD8+T cells was measured, and the results are the combined data from two independent experiments.

[0223] Figure 4 :Suv39h1-deficient CD8+OT-1 cells control tumor growth. (A) 7 days after inoculation, IL-2 / OVA treated Suv39h1-KO CD45.2 OT-1 and littermate WT cells were intravenously transferred into recipient tumor-bearing mice and anti-PD-1 antibody (Bio X Cell, RMP-14) was injected intraperitoneally. Tumor growth curves of IL-2 / OVA treated littermate WT (B) and Suv39h1-KO OT-1 cells (C) were adoptively transferred into recipient tumor-bearing mice. DETAILED DESCRIPTION

[0224] result

[0225] Materials and methods:

[0226] Littermates and Suv39h1-KO mice were infected intravenously with 5x10 3 CFU, and 40 days later, 2×106 CFU recombinant Listeria monocytogenes challenge. The bacteria were grown in TSB medium (BD Bioscience) until early logarithmic phase and the OD 600 The initial dump was then - CD44 高 K b -OVA + CD8 + T cells and related subpopulations were FACS sorted. For each subpopulation analyzed, we collected 3 or 4 biological replicates. According to the manufacturer's protocol, RNA was extracted using RNeasy Micro Kit (QUIGEN). Column DNA enzyme treatment (QUIGEN) was included. For each case, RNA was used to synthesize cDNA according to the standard Affymetrix protocol. Labeled DNA was hybridized on Affymetrix Mouse Gene 2.1ST and processed on Affymetrix GeneTitan equipment.

[0227] Microarray data analysis

[0228] Microarray data were processed into R (version 3.0.0) using a software package from Bioconductor. Raw data CEL files were used and quality control analysis was performed using the ArrayQualityMetrics software package. Raw data were preprocessed using the RMA method available in the oligo software package. Probes without annotations were removed from the analysis. A moderate t-test was performed using the limma software package, and p values ​​were adjusted using multiple tests using the Benjamini Hochberg method. Finally, if the adjusted p value was less than 5%, we considered it statistically significant.

[0229] Gene set enrichment analysis. Using data from the Molecular Signatures DataBase (MSigDB databasev5.1, http: / / software.broadinstitute.org / gsea / msigdb / index.jsp) Genes from the immunological marker (C7) and C2 (regulatory) gene sets were subjected to gene set enrichment analysis (GSEA). The GMT file with gene symbol information was downloaded. The GCT file consisted of a total of 41,345 probes and was imported into GSEA. GSEA was run with default parameters except for the number of permutations (n ​​= 10,000). BubbleGUM analysis was completed as described previously.

[0230] Analysis of tumor growth in Suv39h1-deficient CD8+ OT-1 cells was performed as follows:

[0231] 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 littermate WT mice were cultured in complete medium (RPMI 1640 supplemented with 10% FBS, penicillin-streptomycin and L-glutamine) for 3 days and then activated with 100 UI / ml IL-2 and 1 ug / ml OVA257-264 peptide for another 3 days.

[0232] For tumor inoculation, 1x10 6 EL4-OVA lymphoma cells were injected subcutaneously into the right flank of CD45.1 / C57BL / 6 male mice, and 7 days later, the mice were injected intravenously with 2x10 6 IL-2 / OVA-treated Suv39h1-KOCD45.2 OT-1 or littermate WT cells.

[0233] Mice were injected intraperitoneally with anti-PD1 (Bio X Cell, RMP-14) at a dose of 7.5 mg / Kg body weight / dose twice a week for 2 weeks. Tumor growth was measured using a manual caliper.

[0234] result:

[0235] 1) Our results show that early progenitor cells with a "stem cell-like" phenotype accumulate in cell populations obtained from Suv39h1-KO mice and are reprogrammed into long-lived central memory T cells expressing CD44 and CD62L at a higher efficiency than those obtained from wild-type mice. Figure 1 As shown, the proportion and number of central memory T cells (expressing CD44 and CD62L) were increased in Suv39h1-deficient mice.

[0236] 2) Use transcriptomic analysis to understand the mechanism of this special phenotype:

[0237] Wild-type and Suv39h1-deficient mice were immunized with OVA-expressing Listeria monocytogenes, and OVA-specific T cells were isolated using sorting flow cytometry at day 7 after immunization.

[0238] After Affymetrix analysis of the cells, we found that effector T cells from Suv39h1 expressed higher levels of mRNA encoding stem cell-associated proteins (CD8+ T stem cell-like memory markers (86 genes): Abcb1aIrf8 Rest Abcb1b Jarid2 Rif1 Alpl Kat6a Rnf138 Antxr2 Klf4 Rras Arl4c LdhaSall4 Atr Ldhb Satb1 Baalc Ldhc Setbp1 Basp1 Ldhd Setdb1 Bcl6 Lect1 Skil Bub1Lpin1 Smarcad1 Ccr7 Ly6a Sox2 Ccr9 Ly6e Spon1 Cd27 Map3k8 Stat3 Cxcr6 Mapk12Tbx3 Dock9 Mcm3ap Tcf3 Dusp9 Mcoln2 Tcl1 Eomes Myc Tdgf1 Esrrb Nanog Tert EvlNcor2 Tigit Fas Nr0b1 Tnfaip2 Fgf2 Nr1d2 Tnfrsf1b Fut4 P2ry14 Traf1 Gzmk Pax6Traf4 Hand1 Pcgf2 Trib2 Hesx1 Plekha5 Txnip Ier3 Podxl Zfp42 Il2rb Pou5f1 ZfxIl7r Pou6f1 Zic3 Irf4 Prkce).

[0239] We conclude that OVA-specific T cells from Suv39h1-deficient mice stimulated after infection with Listeria monocytogenes express a 'stem cell'-like mRNA signature.

[0240] 3) Suv39h1-deficient CD8+ T cells show increased survival and self-renewal in vivo.

[0241] Syngeneic 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 Kb-OVA pentamer+CD8+T cells were analyzed by FACS 4 days later.

[0242] like Figure 3As shown, the results indicate that Suv39h1-deficient cells express increased levels of "stem cell-like" markers. They can also survive better and re-populate wild-type mice more efficiently after adoptive transfer.

[0243] 4) Suv39h1-deficient CD8+OT-1 cells control tumor growth in vivo

[0244] Figure 4 B and 4C show that mice adoptively transferred with Suv39h1-KO OT-1 cells and treated with anti-PD1 mAb had better control of tumor growth compared with mice that received similar treatment but with WT OT-1 cells. Notably, by day 30 after tumor injection, 4 out of 5 WT OT-1 transferred mice showed growing tumors, while only 1 out of 5 mice injected with Suv39h1-KO OT-1 cells showed growing tumors.

[0245] in conclusion

[0246] In the therapeutic approach based on adoptive T cell transfer, the lack of Suv39h1 activity in CD8+ T cells is associated with better antitumor efficacy.

Claims

1. Use of engineered human immune cells in the preparation of a medicament for adoptive cell therapy, wherein the engineered human immune cells are engineered CD8+T cells or CD8+T cell progenitors, wherein the expression and / or activity of Suv39h1 in the engineered human immune cells is selectively blocked or inhibited, resulting in the absence of substantially detectable H3K9-histone methyltransferase SUV39H1 activity in the immune cells.

2. The use according to claim 1, wherein the engineered human immune cell comprises a genetically engineered antigen receptor that specifically binds to a target antigen.

3. The use according to claim 2, wherein the engineered human immune cell further comprises a second genetically engineered antigen receptor that recognizes a different antigen.

4. The use according to claim 1 or 2, wherein the engineered human immune cells are isolated from a subject.

5. The use according to claim 1 or 2, wherein the inactivation or destruction of the SUV39H1 gene leads to enhanced anti-cancer activity of the immune cells and / or increased cell survival.

6. The use according to claim 1 or 2, wherein the disruption of the SUV39H1 gene is achieved by gene deletion, replacement with an exogenous sequence or mutation.

7. The use according to claim 1 or 2, wherein the inhibition of the SUV39H1 gene is achieved by gene silencing, knockdown or knockout.

8. The use according to claim 2 or 3, wherein the target antigen is expressed in a disease context.

9. Use according to claim 8, wherein the disease is selected from cancer, infectious diseases and autoimmune or inflammatory diseases or disorders.

10. The use according to claim 2 or 3, wherein the antigen is selectively expressed on cells of a disease or disorder.

11. The use according to claim 2 or 3, wherein the antigen is expressed on cancer cells and / or is a universal tumor antigen.

12. The use according to claim 2 or 3, wherein the genetically engineered antigen receptor is a chimeric antigen receptor (CAR) comprising an extracellular antigen recognition domain that specifically binds to a target antigen.

13. The use according to claim 12, wherein the CAR comprises a cytoplasmic (intracellular) signaling sequence.

14. The use according to claim 13, wherein the CAR comprises a primary cytoplasmic signaling sequence and / or a secondary cytoplasmic signaling sequence.

15. The use according to claim 14, wherein the CAR comprises a cytoplasmic signaling sequence that activates or inhibits the TCR complex.

16. The use according to claim 13 or 14, wherein the CAR comprises a cytoplasmic signaling sequence from an immunoreceptor tyrosine-based activation motif (ITAM) derived from TCRζ (CD3z), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b and CD66d.

17. The use according to claim 13 or 14, wherein the CAR comprises a cytoplasmic signaling sequence from TCRζ.

18. The use according to claim 16, wherein the CAR comprises one or more signaling domains from a co-stimulatory receptor.

19. The use according to claim 13 or 14, wherein the CAR comprises one or more signaling domains from a co-stimulatory receptor selected from 4-1BB, CD28, ICOS, OX40 or DAP10.

20. The use according to claim 13 or 14, wherein the CAR comprises (a) an intracellular signaling domain from the CD3ζ chain and (b) one or more co-stimulatory domains of 4-1BB, CD28, ICOS, OX40 or DAP10.

21. The use according to claim 13 or 15, wherein the CAR is an inhibitory CAR.

22. The use according to claim 2 or 3, wherein the genetically engineered antigen receptor is a T cell receptor (TCR).

23. The use according to claim 1, wherein the medicament is for treating a disease selected from cancer, infectious diseases and autoimmune or inflammatory diseases or disorders.

24. The use according to claim 1, wherein the medicament is for treating cancer.

25. The use according to claim 1 or 2, wherein the engineered human immune cells are combined with an immune checkpoint inhibitor.

26. The use according to claim 25, wherein the medicament is for treating cancer.

27. The use according to claim 25, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, CTLA4, LAG3, BTLA, TIM-3, TIGIT, PGE2 receptor or A2AR.

28. The use according to claim 25, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, CTLA4, LAG3, BTLA, TIM-3 or TIGIT.

29. The use according to claim 25, wherein the immune checkpoint inhibitor is anti-PD-1 or anti-PDL-1.

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