RU2784531C2 - IMMUNE CELLS DEFECTIVE BY Suv39h1 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to a constructed immune cell, where the specified immune cell is selected from T cells, NK cells, and their precursors, which is defective by Suv39h1, and to its use. In addition, a method for the production of a genetically constructed immune cell defective by Suv39h1 is presented, where the method includes a stage of inhibition of expression and/or activity of Suv39h1 in T cell, NK cell, or their precursor, so that the resulting constructed T cell, NK cell, or their precursor have increased survival rate. A pharmaceutical composition for adoptive cell therapy is also disclosed, containing at least one constructed cell defective by Suv39h1.

EFFECT: invention allows for its use in adoptive therapy, first of all for the effective treatment of cancer.

25 cl, 4 dwg

Description

Technical field

The present invention relates to the field of adoptive therapy. The present invention provides Suv39h1-deficient immune cells with increased survival, recovery potential, and central memory phenotype after adoptive transfer.

Introduction

Adoptive T cell therapy (ATCT) using T cells equipped with T cell receptors (TCRs) and chimeric antigen receptors (CARs) shows very promising activity against several cancers in the early stages of clinical trials conducted at a number of institutions ( Kershaw MH, Westwood JA, Darcy PK Nat Rev Cancer 2013; 13:525–541).

The perspective of the topic is related to the fact that effective engraftment and long-term persistence of therapeutic T-cells correlates with positive therapeutic effects. Some preclinical studies have shown that naïve and first-line differentiated T cells have an increased capacity for 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 elicit strong antitumor responses (Gattinoni L et al., J Clin Investig. 2005 ; 115:1616-1626; Lugli E et al. J Clin Invest. 2013; 123:594-599). In addition, increased persistence of adoptively transferred cells appears to depend on the generation of central memory T cell (TCM) populations (Powell DJ et al., Blood. 2005; 105(1):241–50; Huang J, Khong HT et al J Immunother 2005:28:258–267).

Typically, sustained gene transfer in the clinical setting is accomplished by using gamma retroviral vectors to transduce polyclonal CAR T cells (see, for example, Guest RD et al., Cancer Immunol Immunother. 2014;63:133–145) and TCR ( see in particular Johnson LA et al., Blood 2009;114(3):535-46) and patients with engrafted CAR T cells did not experience adverse symptoms regarding the safety of these engineered cells for more than 10 years (Scholler J et al., Sci Transl Med. 2012;4:132ra153).

For efficient transduction using retroviral or lentiviral vectors, primary T cells must be actively proliferating (Stacchini A et al., Leuk Res. 1999; 23:127–136), which in most cases is achieved by mitogenic stimulation of resting primary T cells. cells.

However, upon activation, T cells develop in an irreversible linear pathway into an effector (TE) phenotype (Mahnke YD et al., Eur J Immunol. 2013; 43:2797–2809; Farber DL. Semin Immunol. 2009; 21:84-91) . Mitogenic activation for retroviral or lentiviral transduction therefore triggers T cell differentiation from naïve towards the TE phenotype. When combined with ex-vivo culture protocols to increase the number of transduced T cells to the number required for clinical use (about 10 ⁹ -10 ¹¹ ), T cells are "guided" towards a more differentiated phenotype that is suboptimal for persistence. generally.

Thus, while adoptive T-cell therapy, including CAR-based T-cell therapy, has shown remarkable therapeutic success over the past few years, in particular in the treatment of certain hematological cancers, only a small number of blood cancer types have been shown to be effective. and some types of solid tumors. It is hypothesized that the low efficacy of the treatment may be the result of limited survival of T cells after adoptive transfer.

Therefore, there remains a need in the art for modified or engineered T cells that exhibit an enhanced central memory phenotype and increased survival after adoptive transfer. In particular, there remains a need to provide immune cells, primarily T-cells, suitable for use in adoptive therapy, which greatly contributes to effective and large-scale antitumor therapy.

Disclosure of invention

In the course of this work, the inventors surprisingly found that Suv39h1 defective T cells carry an improved central memory phenotype and have increased survival after adoptive transfer. In particular, the inventors have shown that Suv39h1 defective T cells accumulate and are more efficiently reprogrammed into long-lived central memory T cells expressing both CD44 and CD62L. In this regard, the present invention relates to modified or engineered immune cells, primarily modified T cells, in which Suv39h1 is inactivated.

These modified or engineered immune cells are of great interest for their use in adoptive therapy. Thus, the present invention particularly relates to an engineered or modified Suv39h1-deficient immune cell, preferably the immune cell further comprising a genetically engineered antigen receptor that specifically binds to a target antigen.

Typically, the engineered immune cell of the invention is a T cell or NK cell, namely a CD4+ or CD8+ T cell. Preferred cells may be selected from T _N cells, TSC _M , TC _M or TE _M cells, or combinations thereof.

Also, in most cases, the engineered immune cell is isolated from the subject. Preferably, said subject is suffering from or at risk of developing cancer.

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.

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

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

The present invention also relates to a method for producing a genetically engineered immune cell, comprising the step of inhibiting the expression and/or activity of Suv39h1 in the immune cell; and optionally the step of introducing into the immune cell a genetically engineered antigen receptor that specifically binds to the target antigen.

Preferably, inhibition of Suv39h1 activity and/or expression comprises at least contacting or contacting the cell with a substance that inhibits Suv39h1 expression and/or activity and/or disrupting the Suv39h1 gene. Said substance or agent may be selected from small molecular weight inhibitors; antibody derivatives, aptamers, nucleic acid molecules that block transcription or translation, or genome editing agents.

The present invention also relates to an engineered immune cell as described herein, or a composition comprising said engineered immune cell, for use in adoptive cell therapy, especially adoptive cancer therapy.

Detailed description

Definitions

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

"Antibody fragment" refers to a molecule other than an intact antibody, containing a portion of an intact antibody that binds to the same antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabody; linear antibodies; heavy chain variable regions (VH), single chain antibody molecules such as scFvs and single VH single domain antibodies; and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibodies are single chain antibody fragments containing a heavy chain variable region and/or a light chain variable region, such as scFvs.

“Single domain antibodies” are antibody fragments containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In some embodiments, the single domain antibody is a human single domain antibody.

As used herein, "repression" of gene expression refers to the suppression or reduction in the expression of one or more gene products encoded by a given gene in a cell, compared to the level of expression of the gene product in the absence of repression. The characteristic products of a gene include the mRNA and protein products encoded by the gene. Repression is in some cases temporary or reversible, while in other cases it is permanent. The repression in some cases is the repression of a functional or full-length protein or mRNA, despite the fact that a truncated or non-functional product may be produced. In some embodiments herein, the activity or function of a gene, as opposed to expression, is repressed. Repression of a gene is usually caused by artificial means, ie, by adding or introducing a compound, molecule, complex or composition, and/or by destroying the gene or nucleic acid associated with this gene, for example, at the DNA level. Typical gene repression techniques include silencing, knockdown, knockout, and/or gene disruption techniques such as genome editing. Examples include antisense technologies such as those based on RNAi, siRNA, shRNA and/or ribozymes, which in most cases result in a temporary decrease in expression, as well as genetic engineering methods that result in inactivation or destruction of the target gene, such as by introducing breaks. and/or homologous recombination.

When used herein, "disruption" of a gene refers to a change in the sequence of a gene at the DNA level. Examples include insertions, mutations and deletions. Destruction in most cases results in repression and/or complete lack of expression of the normal or wild-type product encoded by the gene. Typical examples of such disruption of genes are insertions, frameshifts or missense mutations, deletions, knockins, and knockouts of a gene or part of a gene, including deletions of an entire gene. Such disruptions can occur in a coding region, eg, in one or more exons, resulting in an inability to produce a full-length product, a functional product, or any product, eg, by stop codon insertion. Such disruption can also be accomplished by disruption at a promoter or enhancer or other site affecting transcriptional activation to prevent transcription of the gene. Destruction of a gene includes targeting a gene, including inactivation of a target gene by homologous recombination.

Invention Cells

The cells according to the invention are in most cases eukaryotic cells such as mammalian cells (also referred to in the present invention as animal cells), for example human cells.

More specifically, the cells of the 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 lymphoid cells. including lymphocytes, usually T cells and/or NK cells.

In particular, the cells of the invention are preferably lymphocytes, including T cells, B cells and NK cells.

The cells of the invention may also be immune progenitors such as lymphoid progenitors and more preferably T cell progenitors.

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

Cells in most cases are primary cells, such as cells isolated directly from the subject and/or isolated from the subject and frozen.

In relation to the subject to be treated, the cells of the invention may be allogeneic and/or autologous.

In some embodiments, the cells include one or more subpopulations of T cells or other cell types, such as complete T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, e.g., populations defined by function, activation state, maturity, potential for differentiation , distribution and recycling, localization and/or persistence potential, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation.

Subtypes and subpopulations of T cells and/or CD4+ and/or CD8+ T cells include naïve T (T _N ) cells, effector T cells (T _EFF ), memory T cells, and their subtypes such as stem T- memory cells (TSC _M ), central memory T cells (TC _M ), effector memory T cells (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 invariant T cells (MAIT), naturally occurring and adaptively regulated T cells (Treg), helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells. Preferably, the cells of the invention are T _EFF cells with stem/memory cell properties and increased reproducing capacity resulting from inhibition of Suv39h1, as well as T _N cells, TSC _M , TC _M , TE _M cells, and combinations thereof.

In some embodiments, one or more T cell populations are enriched or depleted in cells that are positive for or express high levels of one or more certain markers, such as surface markers, or that are negative for or express relatively low levels of one or more markers. In some cases, such markers are markers that are absent or expressed at relatively low levels in some T cell populations (such as non-memory cells), but which are present or expressed at relatively high levels in some other T cell populations ( such as memory cells). In one embodiment, cells (such as CD8 ⁺ cells or T cells, eg CD3 ⁺ cells) are enriched (i.e., positively selected based on) cells that are positive or express high levels of surface CD117, CD135, CD45RO, CCR7, CD28, CD27, CD44, CD127 and/or CD62L, and/or are depleted of (eg, negatively selected based on) cells that are positive or express high levels of superficial CD45RA. In some embodiments, cells are enriched or depleted for cells positive or expressing high levels of surface CD122, CD95, CD25, CD27 and/or IL7-Ra (CD127). In some samples, CD8+ T cells are enriched in cells positive for CD45RO (or negative for CD45RA) and for CD62L.

For example, according to the invention, the cells may include a population of CD4+ T cells and/or a subpopulation of CD8+ T cells, for example, a subpopulation enriched in central memory cells (T _CM ). Alternatively, the cells may be other types of lymphocytes, including natural killer (NK) cells, MAIT cells, natural lymphoid cells (ILCs), and B cells.

Cells and compositions (mixtures) containing cells for engineering according to the invention are isolated from a sample, namely, from a biological sample, for example, obtained from or derived from a subject. In most cases, the subject is in need of cell therapy (adoptive cell therapy) and/or is a subject receiving cell therapy. The subject is preferably a mammal, namely a human. In one embodiment of the invention, the subject has a malignant disease.

Samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps such as isolation, centrifugation, genetic engineering (e.g., transduction using a viral vector), washing, and/or incubation . The biological sample may be a sample obtained directly from a biological source or a sample that is being processed. The biological sample includes, but is 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 the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Representative examples include whole blood, peripheral blood mononuclear cells (PBMC), leukocytes, bone marrow, thymus, tissue biopsy specimens, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosal lymphoid tissue, pancreas, other lymphoid tissues and/or cells derived from them. Samples include, in the context of cell therapy (usually adoptive cell therapy), samples from autologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, such as T cell lines. The cells can also be obtained from a xenogeneic source such as a mouse, rat, non-human primate, or pig. Preferably, the cells are human.

Cells defective in Suv39h1

When used herein, the term "Suv39h1" or "H3K9 histone methyltransferase Suv39h1" has its general meaning in the art and refers to the histone methyltransferase "3-9 (Drosophila) mosaic suppressor homologue 1" that specifically trimethylates the histone Lys-9 residue. H3 using monomethylated H3-Lys-9 as 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)3-9 encode centromere-associated proteins which complex with the heterochromatin component M3 1" EMBO J 1 8 (7): 1923-38.) . This histone methyltransferase is also known as MG44, KMT1A, SUV39H, SUV39H1, histone lysine N-methyltransferase SUV39H1, H3-K9-HMTase 1, OTTHUMP00000024298, Su(var)3-9 homologue 1, lysine N-methyltransferase 1A, histone H3 -K9 methyltransferase 1, homologue 3-9 of the position effect of the mosaic type, histone-lysine N-methyltransferase or H3 lysine-9 specific 1. Human Suv39h1 methyltransferase is designated O43463 in UNIPROT and is encoded by the Suv39h1 gene located on chromosome x (gene ID: 6839 in NCBI). The term Suv39h1 according to the invention also considers all SUV39H1 orthologues such as SU(VAR)3-9.

When used herein, the term “Suv39h1 deficient” according to the present invention refers to inhibition or blocking of Suv39h1 activity (i.e., H3 histone Lys-9 methylation by H3K9 histone methyltransferase) in the cell of the invention.

"Inhibition of Suv39h1 activity" according to the invention refers to a reduction in Suv39h1 activity of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more compared to activity or level of uninhibited Suv39h1 protein. Advantageously, inhibition of Suv39h1 activity results in the absence of detectable significant Suv39h1 activity in the cell.

Inhibition of Suv39h1 activity can also be achieved by suppressing the expression of the Suv39h1 gene or by disrupting the Suv39h1 gene. According to the invention, said repression reduces the expression of Suv39h1 in a cell, in particular an immune cell of the invention, by at least 50%, 60%, 70%, 80%, 90% or 95% relative to the same cell produced by this method in the absence of repression. Gene disruption can also result in decreased expression of the Suv39h1 protein or in the expression of a non-functional Suv39h1 protein.

By "non-functional" Suv39h1 protein is meant a protein with reduced or no detectable activity as described above.

Thus, inhibitors of Suv39h1 activity in a cell according to the invention can be selected from any compound or natural substance that does not have the ability to inhibit H3 histone H3 Lys-9 methylation by H3K9 histone methyltransferase or that inhibits SUV39H1 H3K9 histone methyltransferase gene expression.

Inhibition of Suv39h1 in an immune cell according to the present invention may be stable and irreversible, or transient or reversible. Preferably, however, the inhibition of Suv39h1 is stable and irreversible. Inhibition of Suv39h1 in a cell can be achieved before or after injection of the cell into the target patient, as described below.

Genetically engineered cells according to the invention

In some embodiments, the cells contain one or more genetically engineered nucleic acids that encode one or more antigen receptors.

As a rule, nucleic acids are heterologous, (ie, for example, those that are not normally found in the cell, when constructed, and/or in the organism from which such a cell is derived). In some embodiments, the nucleic acids are non-naturally occurring, including chimeric combinations of nucleic acids encoding different domains from several different cell types.

Antigen receptors of the invention include genetically engineered T cell receptors (TCRs) and components thereof, as well as functional non-TCR antigen receptors such as chimeric antigen receptors (CARs).

Chimeric antigen receptors (CAR)

In some embodiments, engineered antigen receptors include chimeric antigen receptors (CARs), including activating or stimulating CARs, costimulatory CARs (see WO2014/055668), and/or inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5 (215) (December, 2013)).

Chimeric antigen receptors (CARs) (also known as chimeric immunoreceptors, chimeric T cell receptors, artificial T cell receptors) are engineered receptors that "graft" an arbitrarily chosen specificity onto an immune effector cell (T cell). Typically, these receptors are used to "graft" the specificity of a monoclonal antibody onto a T cell, along with the transfer of their coding sequence using retroviral vectors.

CARs generally comprise an extracellular antigen-binding (or ligand-binding) domain connected to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s). Such molecules typically mimic or approximate a signal using a natural antigen receptor, a signal using such a receptor in combination with a costimulatory receptor, and/or a signal using a costimulatory receptor alone.

In some embodiments, the implementation of the CAR is designed with specificity for a particular antigen (or marker or ligand), such as an antigen expressed on a particular cell type that is targeted by adoptive therapy, for example, a tumor marker. Thus, in most cases, a CAR contains in its extracellular region one or more antigen-binding molecules, such as one or more antigen-binding fragments, domains or regions, or one or more variable domains of an antibody and/or antibody molecules.

Molecules used for antigen binding fall into three main categories, either single chain antibody fragments (scFvs) derived from antibodies, or Fab's selected from libraries, or natural ligands that engage their cognate receptor (for first-generation CARs). ). Good examples in each of these categories are given in Sadelain M, Brentjens R, Riviere I. The basic principles of chimeric antigen receptor (CAR) design. cancer discovery. 2013; 3(4):388-398 (see in particular table 1) and are included in the present application. ScFv's derived from mouse immunoglobulins are commonly used because they can be easily obtained from well-characterized monoclonal antibodies.

Typically, a CAR comprises an antigen-binding region or regions of an antibody molecule, such as a single chain antibody fragment (scFv) derived from the heavy chain variable region (VH) and the light chain variable region (VL) of a monoclonal antibody (mAb).

In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds to an antigen, such as a tumor marker or surface antigen of the cell or disease being treated, such as a tumor cell or cancer cell, such as any of the target antigens described in this document or known in the art.

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

In some embodiments, the CAR contains a TCR-like antibody, such as an antibody or antigen-binding fragment (e.g., scFv), that specifically recognizes an intracellular antigen, such as a tumor-associated antigen, presented on the cell surface as an MHC-peptide complex. In some embodiments, the antibody or antigen-binding site thereof that recognizes the MHC-peptide complex can be expressed on cells as part of a recombinant receptor, such as an antigen receptor. Antigen receptors include functional non-TCR antigen receptors such as chimeric antigen receptors (CARs). In most cases, a CAR containing an antibody or antigen-binding fragment showing TCR-like specificity directed against peptide-MHC complexes may also be referred to as a TCR-like CAR.

In some aspects, the antigen-specific binding or recognition component couples to one or more transmembrane and intracellular signaling domains. In some embodiments, the implementation of the CAR includes a transmembrane domain connected to the extracellular domain of the CAR. In one embodiment, a transmembrane domain is used that is naturally associated with one of the domains in the CAR. In some instances, a transmembrane domain is selected or modified by amino acid substitution to avoid such domains from binding to transmembrane domains of the same or different proteins on membrane surfaces to minimize interactions with other members of the receptor complex.

The transmembrane domain in some embodiments is derived from either a natural or artificial source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions include regions derived from (i.e., contain at least the transmembrane region(s)) of the T cell receptor alpha, beta, or zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154). The transmembrane domain can also be synthetic.

In some embodiments, a short oligo- or polypeptide linker is present, eg, a linker ranging from 2 to 10 amino acids in length, forming a link between the transmembrane domain and the CAR cytoplasmic signaling domain.

In most cases, CAR includes at least one intracellular signaling component or components. The first generation of CARs usually had an intracellular domain from the CD3 ζ chain, which is the primary transmitter of signals from endogenous TCRs. Second generation CARs in most cases additionally contain intracellular signaling domains from various co-stimulatory protein receptors (eg, CD28, 41BB, ICOS) in the cytoplasmic terminal segment of the CAR to provide additional signals to the T cell. Preclinical testing has shown that the second generation improves the antitumor activity of T cells. Recently, third-generation CARs have combined several signaling domains such as CD3z-CD28-41BB or CD3z-CD28-OX40 to increase efficiency.

For example, a CAR may include an intracellular component of the TCR complex, such as the TCR CD3+ chain, which mediates T cell activation and cytotoxicity, such as the CD3 zeta chain. Thus, in some aspects, the antigen-binding molecule binds to one or more signaling modules of the cell. In some embodiments, cell signaling modules include a CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In addition, the CAR may further include a portion of one or more additional molecules such as Fc receptor γ, CD8, CD4, CD25, or CD16.

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

In some embodiments, the intracellular signaling domain(s) includes cytoplasmic T cell receptor (TCR) sequences, and in some aspects also those co-receptors that naturally co-operate with such a receptor to initiate signal transduction upon interaction with an antigen receptor, and/or any derivative or variant of such molecules, and/or any artificial sequence that has a similar functionality.

T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signal sequences: those that initiate antigen-dependent primary activation through the use of TCRs (primary cytoplasmic signal sequences), and those that act in an antigen-independent manner to provide secondary or costimulatory signal (secondary cytoplasmic signal sequences). In some aspects, the CAR includes one or both of these signaling components.

In some aspects, the CAR includes a primary cytoplasmic signal sequence that regulates the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences acting in a stimulatory manner may contain signaling motifs, which are known as immunoreceptor tyrosine activating motifs or ITAMs. Examples of ITAMs containing primary cytoplasmic signal sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, the implementation of the cytoplasmic signaling molecule(s) in the CAR contains(at) a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3 zeta.

CAR may also include the signaling domain and/or transmembrane region of a costimulatory receptor such as CD28, 4-1BB, OX40, DAP10 and ICOS. In some aspects, the same CAR includes both activating and costimulatory components; alternatively, the activating domain is provided by one CAR while the co-stimulatory component is provided by other CARs recognizing a different antigen.

The CAR or other antigen receptor may also be an inhibitory CAR (eg, iCAR) and include intracellular components that attenuate or suppress a response, such as an immune response. 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, the engineered cell contains an inhibitory CAR comprising the signaling domain of such an inhibitory molecule or derived from such an inhibitory molecule such that it serves to attenuate the response. For example, such CARs are used to reduce the chance of off-target effects when an antigen recognized by an activating receptor, such as CAR, is also expressed, or may also be expressed, on the surface of normal cells.

TCR

In some embodiments, the genetically engineered antigen receptors include recombinant T cell receptors (TCRs) and/or TCRs cloned from naturally occurring T cells.

"T cell receptor" or "TCR" refers to a molecule containing variable a and β chains (also known as TCRa and TCRβ, respectively) or variable γ and δ chains (also known as TCRγ and TCRδ, respectively), and which is capable of specific binding to an antigenic peptide associated with the MHC receptor. In some embodiments, the implementation of the TCR is in αβ form. In general, TCRs that exist in αβ and γδ forms are usually structurally similar, but T cells expressing them may have different anatomical locations or functions. TCR can be found on the cell surface or in soluble form. Typically, TCR is found on the surface of T cells (or T lymphocytes), where it is usually responsible for recognizing antigens associated with major histocompatibility complex (MHC) molecules. In some embodiments, the TCR may also contain a constant domain, a transmembrane domain, and/or a short cytoplasmic end segment (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 TCR chain may comprise one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic terminal segment at the C-terminal region. In some embodiments, the TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Unless otherwise indicated, the term "TCR" should be considered to include functional TCR fragments. The term also includes intact or full-length TCRs, including TCRs in αβ form or γδ form.

Thus, for the purposes of this document, reference to a TCR includes any TCR or functional moiety, such as the antigen-binding site of a TCR, that binds to a specific antigenic peptide bound to an MHC molecule, i.e. MHC-peptide complex. The terms "antigen-binding site" or "antigen-binding fragment" of a TCR, which may be used interchangeably, refer to a molecule that contains a portion of the structural domains of the TCR, but which binds to an antigen (e.g., an MHC-peptide complex) to which the entire TCR binds. in some cases, the antigen binding site contains TCR variable domains, such as the TCR variable chain and the TCR variable β chain, sufficient to form a binding site for binding to a specific MHC-peptide complex, usually such as when each chain contains three complementarity-determining regions .

In some embodiments, the variable domains of the TCR chains are linked to form loops or complementarity determining regions (CDRs), similar to immunoglobulins, which provide antigen recognition and determine peptide specificity by forming a TCR binding site of the molecule and determine the specificity of the peptide. Typically, like immunoglobulins, CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., Pwc. Nat'lAcad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988 ; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the main CDR responsible for recognition of the truncated antigen, although the alpha chain CDR1 is also shown to interact with the N-terminal region of the antigenic peptide, while the beta chain CDR1 interacts with the C-terminal region of the peptide. CDR2 is believed to recognize the MHC molecule. In some embodiments, the β-chain variable region may contain an additional region of hypervariability (HV4).

In some embodiments, the TCR chains contain a constant domain. For example, like immunoglobulins, the extracellular region of TCR chains (eg, a-chains, β-chains) may contain two immunoglobulin domains, a variable domain (eg, Va or Vp; typically amino acids 1-116 according to the Kabat numbering of 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.) at the N-terminus, and one constant domain (e.g., a-chain constant domain or Ca , typically amino acids 117 - 259 according to Kabat, the constant domain of the β-chain or Cp, usually amino acids 117 - 295 according to Kabat) located near the cell membrane.For example, in some cases, the extracellular region of the TCR, formed by two chains, contains two membrane-proximal constant domains and two membrane-distal variable domains containing CDRs.The constant domain of the TCR domain contains short binding sequences in which the cysteine residue forms a disulfide bond. ide, forming a connection between two chains. In some embodiments, the TCR may have an additional cysteine residue in each of the a and β chains such that the TCR contains two disulfide bonds in the constant domains.

In some embodiments, the TCR chains may contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, TCR chains contain a cytoplasmic end segment. In some cases, the structure allows the TCR to bind to other molecules like CD3. For example, TCRs containing constant domains with a transmembrane region can fix a protein in the cell membrane and bind to invariant subunits of the CD3 signaling apparatus or complex.

Typically, CD3 is a multiprotein complex that can have three different chains (γ, δ and ε) in mammals and a ζ chain. For example, in mammals, this complex may contain a CD3y chain, a CD35 chain, two CD3s chains, and a homodimer of CD3ζ chains. The CD3y, CD35 and CD3s chains are closely related cell surface proteins of the immunoglobulin family containing a single immunoglobulin domain. The transmembrane regions of the CD3y, CD35, and CD3s chains are negatively charged, which is the property that allows these chains to bind to positively charged T-cell receptor chains. Each of the intracellular tails of the CD3y, CD35, and CD3s chains contains one conserved motif, known as the immunoreceptor tyrosine activating motif, or ITAM, while each CD3ζ chain has three. Typically, ITAMs are involved in the signaling ability of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in signal propagation from the TCR into the cell. The CD3 and ζ chains, together with the TCR, form what is called the T-cell receptor complex.

In some embodiments, the TCR may be a two-strand heterodimer of a and β (or optionally γ and δ), or it may be a single-stranded TCR construct. In some embodiments, the TCR is a heterodimer containing two different chains (a and β chains or γ and δ chains) that are linked, for example, via a disulfide bond or disulfide bonds.

Representative antigen receptors, including CARs and recombinant TCRs, as well as methods for constructing and introducing these receptors into cells, include those described, for example, in International Patent Application Publication Nos. /071154, WO2013/123061 публикациях патентных заявок США № US2002131960, US2013287748, US20130149337, патентах США №: 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 и 8,479,118, и Европейской патентной application no. EP2537416 and/or those described by Sadelain et al., Cancer Discov. April 2013; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, genetically engineered antigen receptors include the CARs described in US Pat. No. 7,446,190 and those described in International Patent Application Publication No. WO/2014055668 Al.

Antigens

Antigens targeted by genetically engineered antigen receptors include those expressed in association with the disease, condition, or cell type to be targeted by adoptive cell therapy. Such diseases and conditions include proliferative, neoplastic and malignant diseases and disorders, in particular various types of cancer.

The cancer may be solid cancer or "fluid tissue tumor" such as cancers affecting the blood, bone marrow, and lymphoid system, also known as hematopoietic and lymphoid tissue tumors, which include primarily leukemia and lymphoma. Fluid tissue tumors include, for example, acute myelocytic 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's lymphoma (NHL ), adenoma, squamous cell carcinoma, laryngeal carcinoma, gallbladder and bile duct carcinoma, retinal cancers such as retinoblastoma).

In particular, solid cancers include cancers affecting one of the organs selected from the group consisting of colon, rectum, skin, endometrium, lung (including non-small cell lung carcinoma), uterus, bones (such as osteosarcoma, chondrosarcoma, Ewing's sarcoma , fibrosarcoma, giant cell tumor, adamantinoma and chordoma), liver, kidney, esophagus, stomach, bladder, pancreas, cervix, brain (such as meningioma, glioblastoma, poorly differentiated astrocytoma, oligodendrocytoma, pituitary tumors, schwannoma and metastatic brain cancer) , ovary, breast, head and neck, testis, prostate and thyroid.

Preferably, the cancer according to the invention is a cancer affecting the blood, bone marrow and lymphoid system, as described above. In most cases, the cancer is or is associated with multiple myeloma.

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

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 related to the disease or condition, such as tumor cells or pathogenic cells, over normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on engineered cells. In some embodiments, a multi-objective approach and/or a gene disruption strategy is used to improve specificity and/or efficacy, as described herein.

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 various organs. In some embodiments, the universal tumor antigen is expressed in more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or more of human cancers. In some embodiments, a universal tumor antigen is expressed in at least three, at least four, at least five, at least six, at least seven, at least eight, or more different tumor types. . In some cases, a universal tumor antigen may be expressed in non-tumor cells, such as normal cells, but at lower levels than it is expressed in tumor cells. In some cases, the universal tumor antigen is not expressed in all non-tumor cells, for example, it is not expressed in normal cells. Representative universal tumor antigens include, for example, human telomerase reverse transcriptase (hTERT), survivin, mouse double microprotein 2 homologue (MDM2), cytochrome P450 1B1 (CYP1B), HER2/neu, Wilms tumor gene 1 (WT1), livin, alphafetoprotein ( AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, or cyclin (Dl). 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 TCRs or TCR-like CARs (see, for example, PCT Publication Application No. WO2011009173 or WO2012135854 and US Published Application No. US20140065708).

In some aspects, the antigen is expressed in the context of multiple myeloma, such as CD38, CD138, and/or CS-1. other characteristic multiple myeloma antigens include CD56, TIM-3, CD33, CD123 and/or CD44. Antibodies or antigen-binding fragments directed against such antigens are known and include, for example, those described in US Pat. Nos. 8,153,765; 8.603477, 8.008.450; U.S. Published Application No. US20120189622; and PCT International Publication Application Nos. WO2006099875, WO2009080829 or WO2012092612. In some embodiments, such antibodies or antigen-binding fragments thereof (eg, scFv) can be used to generate CARs.

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

In some embodiments provided herein, an immune cell, such as a T cell, can be engineered to suppress or destroy a gene encoding an antigen in the immune cell so that the expressed genetically engineered antigen receptor does not specifically bind to the antigen under conditions its expression on the immune cell itself. Thus, in some aspects, this may help to prevent non-targeted actions, such as engineered immune cells binding to themselves, which can reduce the efficacy of the engineered immune cells, for example, in relation to adoptive cell therapy.

In some embodiments, such as inhibitory CARs, the target is a non-target marker, such as an antigen that is not expressed on an unhealthy or target cell, but that is expressed on a normal or disease-free cell that also expresses a disease-specific a target targeted by an activating or excitatory receptor in the same engineered cell. Examples of such antigens are MHC molecules, such as class I MHC molecules, for example, in connection with the treatment of diseases or conditions in which the number of such molecules is reduced, but they remain expressed in non-target cells.

In some embodiments, engineered immune cells may contain an antigen that targets one or more other antigens. In some embodiments, one or more of the other antigens is a tumor antigen or tumor marker. Another antigen targeted by antigen receptors on exposed immune cells may, in some embodiments, include orphan tyrosine kinase receptor ROR1, tEGFR, Her2, Ll-CAM, CD19, CD20, CD22, mesothelin, CEA, hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3 or 4, FBP, embryonic receptor, GD2, GD3, HMW-MAA, IL-22R-alpha , IL-13R-alpha2, kdr, kappa light chain, Ley antigen, cell adhesion molecule Ll, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligands, NY-ESO-1, MART-1, gplOO, cancer- fetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate-specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrin B2, CD 123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms tumor antigen 1 (WT-1), cyclin such as cyclin Al (CCNA1), and/or biotinylated molecules and/or molecules expressed by HIV, HCV, HBV or other pathogens.

In some embodiments, the CAR binds a pathogen-specific antigen. In some embodiments, the CAR is specific for viral antigens (such as HIV, HCV, HBV, etc.), bacterial antigens, and/or parasitic antigens.

In some embodiments, the cells of the invention are genetically engineered to express two or more genetically engineered receptors on the cell, each of which recognizes a different antigen and typically includes a different intracellular signaling component. Such multi-target strategies are described, for example, in International Patent Application, Publication No: WO 2014055668 Al (describes a combination of activating and costimulatory CARs, e.g. targeting two different antigens, present individually on non-target e.g. normal cells, but present together only on cells related to the disease or condition to be treated) and Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013) (describes cells expressing both an activating and an inhibitory CAR, e.g., those in which the activated CAR binds to a single antigen expressed both on normal or disease-free cells and on cells having related to the disease or condition to be treated and the inhibitory CAR binds to another antigen expressed only on normal or untreated cells).

In some cases, overexpression of a stimulatory factor (eg, lymphokine or cytokine) can be toxic to the subject. Thus, in some cases, the engineered cells include gene segments that render the cells susceptible to negative selection in vivo, such as after administration in adoptive immunotherapy. For example, in some aspects, the cells are engineered to be eliminated as a result of an in vivo change in the condition of the patient to whom they are administered. A negatively selectable phenotype may result from the insertion of a gene that confers the property of being sensitive to the introduced agent, eg a compound. Negatively selectable genes include the Herpes simplex type I virus thymidine kinase (HSV-I TK) gene (Wigler et al., Cell II:223, 1977), which confers ganciclovir sensitivity; cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase gene (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

In other embodiments, the cells, e.g., T cells, are not engineered to express recombinant receptors, but instead include naturally occurring antigen receptors specific for the desired antigens, such as tumor infiltrating lymphocytes and/or T cells cultured in vitro or ex vivo, for example, during the stage(s) of incubation, to promote the expansion of cells with a certain specificity for the antigen. For example, in some embodiments, cells for adoptive cell therapy are obtained by isolating tumor-specific T cells, such as autologous tumor infiltrating lymphocytes (TILs). Direct targeting of human tumors using autologous tumor-infiltrating lymphocytes can in some cases mediate tumor regression (see Rosenberg SA, et al. (1988) N Engl J Med. 319: 1676-1680). In some embodiments, lymphocytes are isolated from resected tumors. In some embodiments, such lymphocytes are grown in vitro. In some embodiments, such lymphocytes are cultured with lymphokines (eg, IL-2). In some embodiments, such lymphocytes mediate specific lysis of autologous tumor cells, but not of allogeneic tumor or autologous normal cells.

Additional nucleic acids, such as genes for insertion, include those that improve the effectiveness of therapy, for example, by promoting the viability and/or function of "transferred" cells; genes providing a genetic marker for cell selection and/or evaluation, such as in vivo survival and localization evaluation; genes that improve safety, for example, making the cell susceptible to negative selection in vivo, as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also PCT/US91/08442 and PCT/US94/05601 Lupton et al. describing the use of bifunctional selectable fusion genes resulting from the combination of a dominant positive selectable marker with a negative selectable marker. See, for example, Riddell et al., US Pat. No. 6,040,177 columns 14-17.

Method for obtaining cells according to the invention

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

Preferably, the method for producing cells according to the invention further comprises the step of introducing into said immune cells a genetically engineered antigen receptor that specifically binds to the target antigen.

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

Suv39h1 inhibition

According to the invention, the engineered immune cell can be contacted with at least one agent that inhibits or blocks the expression and/or activity of Suv39h1.

Said agent may be selected from small molecule inhibitors; antibody derivatives, such as intrabodies, nanobodies, or affibodies, which generally block or inhibit the expression or activity of Suv39h1; 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 RNAs (siRNAs), small hairpin RNAs (shRNAs), microRNAs (miRNAs), or piwiRNAs (piRNAs); ribozymes, or combinations thereof.

The at least one agent may also be an exogenous nucleic acid comprising a) an engineered guide RNA from a non-natural CRISPR (regularly spaced short palindromic repeat) system that hybridizes to the Suv39h1 genomic nucleic acid sequence and/or b) a nucleotide sequence encoding a protein CRISPR (typically a type-II Cas9 protein), optionally the cells are transgenic to express the Cas9 protein. The agent may also be a zinc finger (ZFN) or TAL protein.

The term "small organic molecule" refers to a molecule of size comparable to organic molecules commonly used in pharmaceuticals. The term excludes biological macromolecules (eg proteins, nucleic acids, etc.). Preferred small organic molecules range in size from about 5000 Da, more preferably about 2000 Da, and most preferably about 1000 Da.

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

The Suv39h1 inhibitor 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-epidithiopyrrole[1,2-a]pyrazine-7-carbonitrile)om, the racemic analogue of epidithiodiketopiperazine of the alkaloid ketocin A (see WO2014066435 and also see Baumann M, Dieskau AP, Loertscher BM, et al. Tricyclic Analogues of Epidithiodioxopiperazine Alkaloids with Promising In Vitro and In Vivo Antitumor Activity Chemical science (Royal Society of Chemistry : 2010) 2015;6:4451-4457 and Snigdha S, Prieto GA, Petrosyan A, et al H3K9me3 Inhibition Improves Memory, Promotes Spine Formation, and Increases BDNF Levels in the Aged Hippocampus The Journal of Neuroscience 2016;36(12):3611-3622).

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

Inhibition of Suv39h1 in the cell can be achieved before or after injection into the intended patient. In a separate embodiment, the inhibition, as previously defined, is carried out in vivo upon administration of the cell to the subject. Typically, a Suv39h1 inhibitor, as defined herein, may be included in a composition containing the cell. Suv39h1 can also be administered separately before, simultaneously with, or after administration of the cell(s) to the subject.

Typically, inhibition of Suv39h1 according to the invention can be achieved by incubating the cell according to the invention with a composition containing at least one pharmacological inhibitor, as previously described. The inhibitor is turned on during the expansion of antitumor T cells in vitro, thus modifying their reproduction, survival and therapeutic efficacy after adoptive transfer.

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

Intrabodies can be generated by cloning the appropriate cDNA from an existing hybridoma clone, or more conveniently, novel scFvs/Fabs can be selected using in vitro display methods such as phage display, which provides the desired antibody-coding gene from the start and allows more detailed preliminary design of antibody specificity. In addition, a bacterial display, a yeast display, a mammalian cell surface display, and a ribosome display may be used. However, the most commonly used in vitro display system for the selection of specific antibodies is phage display. In a procedure called panning (affinity selection), recombinant phage antibodies are selected by incubating the phage antibody repertoire with an antigen. This process is repeated several times, resulting in a rich repertoire of antibodies containing specific antigen-binding molecules to just about any possible target. To date, in vitro assembled libraries of recombinant human antibodies have already generated thousands of new recombinant antibody fragments. It should be noted that a precondition for knockdown of a specific protein by a cytoplasmic intrabody is that the antigen is neutralized/inactivated by antibody binding. There are five different approaches to obtain suitable antibodies: 1) In vivo selection of functional antibodies in eukaryotes such as yeast and prokaryotes such as E. coli (antigen dependent and independent); 2) obtaining antibody fusion proteins to improve stability in the cytosol; 3) the use of special "scaffolds" to improve stability in the cytosol (for example, by "transplanting" CDRs or introducing synthetic CDRs into stable framework regions of the antibody); 4) use of single domain antibodies for improved stability in the cytosol; and 5) selection of stable intrabodies lacking a disulfide bond. These approaches are, in particular, detailed in Marschall, A. L et al., mAbs 2015, as noted above.

The most commonly used format for intrabodies is scFv, which consists of the H and L chains of an antibody variable domain (VH and VL) held together by a short, flexible linker sequence (often (Gly4Ser)3) to avoid the need to separate expression and assembly of 2 antibody chains to a complete IgG molecule or Fab molecule. Alternatively, a Fab format is used, additionally containing the C1 domain of the heavy chain and the constant region of the light chain. Recently, a new possible intrabody format, scFab, has been described. The scFab format allows for easier subcloning of available Fab genes into an intracellular expression vector, but it remains to be seen if this offers any advantage over the conventional scFv format. In addition to scFv and Fab, bispecific formats are used as intrabodies. The bispecific Tie-2 x VEGFR-2 antibody targeting ER showed an increased half-life compared to monospecific analog antibodies. The bispecific transmembrane intrabody is being developed as a specific format for the simultaneous recognition of intra- and extracellular epitopes of epidermal growth factor, combining different properties of related monospecific antibodies, i.e. inhibition of autophosphorylation and ligand binding.

Another intrabody format particularly suitable for cytoplasmic expression is single domain antibodies (also referred to as nanobodies) derived from camelids or consisting of a single human VH domain or human VL domain. These single domain antibodies often have advantageous characteristics such as high stability; good solubility; convenience of library cloning and selection; high expression yield in E. coli and yeast.

The intrabody gene can be expressed within a target cell after transfection with an expression plasmid or viral transduction with a recombinant virus. Typically, the selection is intended to provide intrabody transfection and production levels. Successful transfection and subsequent intrabody production can be analyzed by detecting the produced antibody by immunoblotting, and co-immunoprecipitation from extracts of HEK 293 cells temporally co-transfected with the appropriate antigen and intrabody expression plasmids can be used to assess the correct intrabody/antigen interaction.

Inhibition of Suv39h1 in a cell according to the invention can also be carried out using aptamers that inhibit or block the expression or activity of Suv39h1. Aptamers are a class of molecules that provide an alternative to antibodies in terms of molecular recognition. Aptamers are oligonucleotide (DNA or RNA) or oligopeptide sequences with the ability to recognize virtually any class of target molecules with high affinity and specificity.

Oligonucleotide aptamers can be isolated by systematic exponential enrichment ligand evolution (SELEX) from a random sequence library as described in Tuerk C. and Gold L., 1990. The random sequence library is generated by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, ultimately chemically modified, of a unique sequence. Possible modifications, applications and advantages of this class of molecules are discussed in Jayasena S.D., 1999.

Peptide aptamers consist of a conformationally fixed antibody variable region presented on a protein platform such as E. coli thioredoxin A, which are selected from combinatorial libraries using two hybrid methods (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).

, Frejd FY. Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett. 2010 Jun 18;584(12):2670-80). Молекулы аффител основаны на сконструированном варианте (Z домен) B-домена в иммуноглобулин-связывающих областях стафилококкового белка A, теоретически со специфичностью связывания с любой имеющейся мишенью. Библиотеки молекул аффител, как правило, создаются путем комбинаторной рандомизации 13 аминокислотных положений в спирали один и два, которые содержат первичную Fc-связывающую поверхность Z-домена. Обычно библиотеки представлены на фагах, с последующим биопэннингом против желательных мишеней. В случае, если аффинность первого увеличивается, созревание аффинности приводит обычно к улучшенному связыванию и может достигаться путем либо перетасовки спирали или выравнивания последовательности в сочетании с направленным комбинаторным мутагенезом. Вновь идентифицированные молекулы с их измененной связывающей поверхностью обычно сохраняют исходную спиральную структуру, а также высокую устойчивость, тем не менее, сообщалось об уникальных исключениях с интересными свойствами. Благодаря их небольшому размеру и свойствам быстрого свертывания, молекулы аффител могут быть получены методами химического пептидного синтеза.Inhibition of Suv39h1 in the cell according to the invention can also be carried out using affitel molecules. Affitels are small proteins designed to bind to a large number of target proteins or peptides with high affinity that mimic monoclonal antibodies and are therefore members of the antibody mimetic family (see review by Löfblom J, Feldwisch J, Tolmachev V, Carlsson J,

, Frejd FY. Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett. 2010 Jun 18;584(12):2670-80). Affitel molecules are based on an engineered variant (Z domain) of the B-domain in the immunoglobulin-binding regions of the staphylococcal A protein, theoretically with binding specificity for any given target. Libraries of affitetel molecules are typically generated by combinatorial randomization of the 13 amino acid positions in helix one and two that contain the primary Fc-binding surface of the Z-domain. Libraries are typically presented on phage, followed by biopanning against desired targets. In case the affinity of the former is increased, affinity maturation generally results in improved binding and can be achieved by either helix shuffling or sequence alignment in combination with combinatorial directed mutagenesis. The newly identified molecules with their modified binding surface usually retain their original helical structure as well as high stability, however, unique exceptions with interesting properties have been reported. Due to their small size and fast folding properties, affitel molecules can be produced by chemical peptide synthesis methods.

In other embodiments, inhibition of Suv39h1 activity can be achieved by gene repression/suppression by gene knockdown using interference RNA (RNAi), such as small interfering RNA (siRNA), short hairpin RNA (shRNA), or ribozymes. The siRNA technology includes RNAi-based technology using double-stranded RNA molecules having a sequence homologous to an mRNA nucleotide sequence that is transcribed from a gene and a sequence complementary to the nucleotide sequence. In most cases, the siRNA is homologous/complementary with one region of the mRNA that is transcribed from the gene, or may be an siRNA comprising multiple RNA molecules that are homologous/complementary with different regions.

Antisense oligonucleotides, including antisense RNA molecules and antisense DNA molecules, act to directly block the translation of Suv39h1 H3K9 histone methyltransferase, thereby preventing protein translation or increasing mRNA degradation, thus decreasing the level of H3K9 histone methyltransferase SUV39H1 and thus thus, its activity in the cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA of the transcript encoding the H3K9 histone methyltransferase SUV39H1 can be synthesized, for example, using conventional phosphodiester methods, and administered, for example, by intravenous injection or infusion. . Methods for using antisense technologies to specifically inhibit the expression of genes whose sequence is known are well known in the art (see, for example, US Pat. Nos. 6,566,135; 6,566, 131;

An "RNA interfering 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 interfering agents include, but are not limited to, nucleic acid molecules, including RNA molecules that are homologous to the target gene of the invention (e.g., Suv39h1), or a fragment thereof, small interfering RNAs (siRNAs), and small molecules that interfere with or inhibit the expression of the target nucleic acid by RNA interference (RNAi).

Small interfering RNAs (siRNAs) can also function as expression inhibitors for use in the present invention. Expression of the H3K9-histone methyltransferase SUV39H1 gene can be reduced by contacting the subject or cell with a small double-stranded RNA (dsRNA) or a vector or construct that causes the production of a small double-stranded RNA so that expression of the H3K9-histone methyltransferase SUV39H1 gene is specifically inhibited (i.e., RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequences are known (see, for example, Tuschl, T. et al. (1999); Elbashir, S. M. 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. 01/68836). All or part of the phosphodiester bonds of the siRNA of the invention are preferably protected. This protection is usually carried out chemically using methods known in the art. Phosphodiester bonds may be protected, for example, with a thiol or amine functional group or with a phenyl group. The 5' and/or 3' ends of the siRNAs of the invention are also advantageously protected, for example using the methods described above for protecting phosphodiester bonds. The siRNA sequences preferably contain at least twelve contiguous dinucleotides or derivatives thereof.

When used in the description, the term "siRNA derivatives" in relation to the present nucleic acid sequences refers to a nucleic acid having a percent identity of at least 90% with erythropoietin or a fragment thereof, preferably at least 95%, for example, at least 98%, and more preferably at least 98%.

When used in the description, the expression "percent identity" between two nucleic acid sequences means the percentage of similarity between two compared sequences obtained by better aligning said sequences, this percentage is purely statistical and the differences between the two sequences are randomly distributed over the nucleic acid sequences. When used in the description, "best alignment" or "optimal alignment" means an alignment for which a certain percentage of identity (see below) is the highest. Sequence comparison between two nucleic acid sequences is usually carried out by comparing these sequences that have been previously aligned according to the best alignment; this comparison is carried out by segments of comparison, in order to identify and compare local areas of similarity. Better sequence alignment for comparison can be realized, apart from the manual method, by using the general homology search algorithm developed by SMITH and WATERMAN (Ad. App. Math., vol. 2, p:482, 1981), by using the local homology search algorithm , developed by NEDDLEMAN and WUNSCH (J. Mol. Biol, vol.48, p:443, 1970), by using the similarity method developed by PEARSON and LIPMAN (Proc. Natl. Acd. Sci. USA, vol.85, p:2444 , 1988), by using computer software using similar algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA in the Wisconsin Genetics Software Suite, Genetics Computer Group, 575 Science Dr., Madison, WI USA), by using MUSCLE multiple alignment algorithms (Edgar, Robert C, Nucleic Acids Research, vol. 32, p: 1792, 2004). To obtain the best local alignment, the use of BLAST software is preferred. The percent identity between two nucleic acid sequences is determined by comparing the two optimally aligned sequences. In order to obtain an optimal alignment between these two sequences, it should be taken into account that the nucleic acid sequences may contain insertions or deletions relative to the reference sequence. The percent identity is calculated by determining the number of identical positions between the two sequences, dividing this number by the total number of positions compared, and multiplying the result by 100 to obtain the percent identity between the two sequences.

shRNAs (short hairpin RNAs) can also function as expression inhibitors when used in the present invention. shRNAs in most cases consist of a short (eg, 19-25 nucleotides) antisense strand followed by a loop of 5-9 nucleotides and a similar sense sequence. Alternatively, the sense sequence may precede the loop structure followed by the antisense strand.

Ribozymes can also function as expression inhibitors when used in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of action of ribozymes involves specific hybridization of the ribozyme molecule sequence with a complementary target RNA followed by endonucleolytic cleavage. Thus, engineered hairpin or hammerhead motifs of ribozyme molecules that specifically and efficiently catalyze endonucleotic cleavage of SUV39H1 H3K9 histone methyltransferase mRNA sequences are useful within the scope of the present invention. Cleavage sites for specific ribozymes in any potential target RNA are initially established by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences GUA, GUU and GUC. Once identified, short RNA sequences between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site are evaluated for predicted structural features, such as a secondary structure that would render the oligonucleotide sequence unusable.

And antisense oligonucleotides and ribozymes suitable as expression inhibitors can be obtained by known methods. These methods include chemical synthesis methods such as, for example, solid phase phosphoramidite chemical synthesis. Alternatively, antisense RNA molecules can be produced by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be "introduced" into a wide variety of vectors that include suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. In order to increase intracellular stability and half-life, various modifications can be introduced into the oligonucleotides of the invention. Possible modifications include, but are not limited to, the addition of flanking ribonucleotide or deoxyribonucleotide sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

The antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention can be delivered in vivo alone or together with a vector. Broadly, a "vector" is any vehicle capable of transferring an antisense oligonucleotide, siRNA, shRNA, or ribozyme nucleic acid into cells, and preferably cells expressing the SUV39H1 H3K9 histone methyltransferase. Preferably, the vector transports the nucleic acid into the cell reducing the rate of degradation relative to the rate of degradation that would occur in the absence of the vector. In general, vectors suitable for the invention include, but are not limited to, plasmids, phagemids, viruses, other carriers derived from viral or bacterial sources that are manipulated by insertion or introduction of an antisense oligonucleotide, siRNA, shRNA, or ribozyme nucleic acid sequences. Viral vectors are the preferred type of vector and include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses such as Moloney mouse leukemia virus, Harvey mouse sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; adenovirus; adeno-associated virus; viruses SV40 type; polyomaviruses; Epstein-Barr virus; papillomavirus; herpes virus; vaccinia virus; poliovirus; and R A virus such as a retrovirus. Other vectors not mentioned but known in the art may be used.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes are replaced with desired genes. Non-cytopathic viruses include retroviruses (eg, lentivirus) whose life cycle involves reverse transcription of the viral genomic RNA into DNA followed by integration of the provirus into the host's cellular DNA. Retroviruses have been approved for testing for use in human gene therapy. Most suitable are those retroviruses that are replication defective (ie capable of directing the synthesis of desired proteins but incapable of producing an infectious particle). Such genetically modified retroviral expression vectors are commonly used for highly efficient gene transduction in vivo. Standard protocols for generating replication-deficient retroviruses (including the steps of introducing exogenous genetic material into a plasmid, transfecting packaging cell lines with plasmid, producing recombinant retroviruses by a packaging cell line, harvesting viral particles from tissue culture media, and infecting a target cell with viral particles) are provided. in Kriegler, 1990 and Murry, 1991).

Preferred viruses for certain modes of use are adenoviruses and adeno-associated (AAV) viruses, which are double-stranded DNA viruses that have already been approved for use in human gene therapy. Currently, 12 different AAV serotypes (AAV1 - 12) are known, each with a different tissue tropism (Wu, Z Mol Ther 2006; 14:316-27). Recombinant AAVs are derived from AAV2 dependent parvoviruses (Choi, VW J Virol 2005; 79:6801-07). Adeno-associated virus type 1-12 can be engineered to be non-replicating and capable of infecting a wide variety of cell types and species (Wu, Z Mol Ther 2006; 14:316-27). It furthermore has advantages such as heat resistance and lipid solubility; high frequency of transduction in cells of various lineages, including hematopoietic cells; and the lack of inhibition of superinfection, thereby providing a number of series of transductions. It is reported that adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability in the expression properties of the introduced retroviral infection gene. In addition, adeno-associated virus infections exist in tissue culture for more than 100 passages in the absence of selective pressure, which means that adeno-associated virus-mediated genomic integration is a relatively stable event. Adeno-associated virus can also function in an extrachromosomal mode.

Other vectors include plasmid vectors. Plasmid vectors are described in detail and are well known to those skilled in the art. See, for example, Sambrook et al, 1989. Over the past few years, plasmid vectors have been used as DNA vaccines to deliver antigen-coding genes to cells in vivo. They are exceptionally suited for this because they do not raise the same safety concerns as many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene 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 skilled in the art. In addition, plasmids can be custom designed using restriction enzymes and ligation reactions to remove and add specific DNA fragments. Plasmids can be delivered by a variety of parenteral, mucosal, and topical routes. For example, the plasmid DNA can be administered by intramuscular, intradermal, subcutaneous, or other routes. It can also be given by intranasal spray or drops, rectal suppositories, and orally. It can also be injected into the epidermis or mucosal surface using a gene gun. Plasmids may be provided in aqueous solution, dried on gold particles, or provided in combination with another DNA delivery system including, but not limited to, liposomes, dendrimers, cochlear delivery vehicles, and microencapsulation.

The antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence of the invention is usually under the control of a heterologous regulatory element, eg a heterologous promoter. The promoter may be specific for Mueller glial cells, microglial cells, endothelial cells, pericytes, and astrocytes. For example, specific expression in Mueller glial cells can be obtained by using an appropriate glutamine synthetase gene promoter. The promoter may also be, for example, a viral promoter such as the CMV promoter or any artificial promoters.

Repression or destruction of the Suv39h1 gene

Inhibition of Suv39h1 in a cell according to the invention can also be carried out by repression or disruption of the Suv39h1 gene, for example, a deletion, such as deletion of an entire gene, exon or region and/or replacement with an exogenous sequence, and/or a mutation, for example, a frameshift or a missense mutation. in a gene, usually in the exon of the gene. In some embodiments, after the introduction of a premature stop codon into the gene, disruption occurs, resulting in the Suv39h1 protein not being expressed or being non-functional. Destruction in most cases is carried out at the DNA level. The destruction is usually long-term, irreversible, or not temporary.

In some embodiments, gene disruption or repression is carried out by genome-editing agents such as DNA-targeted molecules, e.g., a DNA-binding protein or a DNA-binding nucleic acid, or a complex, compound, or composition containing the above that specifically binds or hybridizes. with the gene. In some embodiments, the DNA-targeting molecule comprises a DNA-binding domain, e.g., a zinc finger protein (ZFP) DNA-binding domain, a transcription activator-like protein (TAL) or TAL effector-like protein (TALE) DNA-binding domain, a DNA the CRISPR binding domain (short palindromic repeats regularly arranged in groups), or the meganuclease DNA-binding domain.

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

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 assist in the repression or disruption of a gene. For example, in some embodiments, gene disruption is accomplished by fusion proteins that contain DNA binding proteins and a heterologous regulatory domain or functional fragment thereof.

In most cases, the additional domain is the nuclease domain. Thus, in some embodiments, gene disruption is accomplished by gene or genome editing using engineered proteins such as nucleases and nuclease-containing complexes or fusion proteins consisting of sequence-specific DNA-binding domains fused to or complexed with non-specific DNA-cleaving molecules such as nucleases.

These targeted chimeric nucleases or nuclease-containing complexes perform precise genetic modifications by inducing targeted double-strand breaks or single-strand breaks that promote cellular DNA repair mechanisms, including error-producing non-homologous end joining (NHEJ) and homology-driven repair (HDR). In some embodiments, the nuclease is an endonuclease, such as a zinc finger nuclease (ZFN), 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 (see, for example, US 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 Patent Publications 2014/0087426 and 2012/0178169, Boch et al. -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 a/ (2011) Nat Biotech 29: 143-148 Lin et al (2014) Nucl Acids Res 42:e47). Such genetic strategies may use constitutive expression systems or inducible expression systems according to methods well known in the art.

ZFP and ZFN; TAL, TALE and TALEN

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

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. ZFP, or a domain thereof, is a protein or domain within a larger protein that binds to DNA in a sequence-specific manner through the use of one or more "zinc finger" amino acid sequence regions within the binding domain, the structure of which is stabilized by coordination with a zinc ion. Typically, ZFP sequence specificity can be altered by making amino acid substitutions at four positions (-1, 2, 3, and 6) on the zinc finger recognition helix. Thus, in some embodiments, the ZFP or ZFP-containing molecule is a molecule of non-natural origin, for example, is designed to bind to a selected target site. See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416.

In some embodiments, the DNA-targeting molecule is or contains a zinc finger DNA binding domain fused to a DNA cleaving domain to form a zinc finger nuclease (ZFN). In some embodiments, the fusion proteins comprise a cleavage domain (or a half-cleavage domain) from at least one type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. In some embodiments, the cleavage domain is from a Fok I type IIS restriction endonuclease. See, for example, US 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.

In some aspects, ZFN effectively causes a double strand break (DSB), for example, at a predetermined location in the coding region of the target gene (ie, Suv39h1). Exemplary target regions of a gene include exons, N-terminal coding regions, first exon, second exon, and promoter or enhancer regions. In some embodiments, transient expression of ZFN promotes highly efficient and long-term disruption of the target gene in the engineered cells. In particular, in some embodiments, ZFN delivery results in long-term gene disruption with an efficiency greater than 50%. Many gene-specific "zinc fingers" are commercially available. For example, Sangamo Biosciences (Richmond, CA, USA) developed a zinc finger construction platform (CompoZr) in partnership with Sigma-Aldrich (St. fingers" and providing specifically targeted "zinc fingers" for thousands of proteins. Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405. In some embodiments, custom made commercially available "zinc fingers" are used. (See, for example, Sigma-Aldrich catalog numbers CSTZFND, CSTZFN, CTI1-1KT and PZD0020).

In some embodiments, the DNA-targeting molecule comprises a naturally occurring or engineered (non-naturally occurring) DNA-binding domain of a transcription activator like protein (TAL), such as in a transcription activator like protein effector (TALE), see e.g. U.S. Patent Publication No. 20110301073. In some embodiments, the implementation of the molecule is a DNA-binding endonuclease, such as TALE-nuclease (TALEN). In some aspects, TALEN is a fusion protein comprising a DNA binding domain derived from TALE and a nuclease catalytic domain for cleaving a target nucleic acid sequence. In some embodiments, a TALE DNA binding domain is designed to bind to a target sequence within a gene encoding a target antigen and/or immunosuppressive molecule. For example, in some aspects of TALE, the DNA binding domain may target CD38 and/or an adenosine receptor such as A2AR.

In some embodiments, TALEN recognizes and cleaves a target sequence in a gene. In some aspects, DNA cleavage results in double-strand breaks. In some aspects, breaks stimulate the rate of homologous recombination or non-homologous end joining (NHEJ). In general, NHEJ is a defective repair process that often results in changes in the DNA sequence at the cleavage site. In some aspects, repair mechanisms involve joining the two remaining DNA ends via direct religation (Critchlow and Jackson, Trends Biochem Sci. 1998 Oct; 23(10):394-8) or via so-called microhomology mediated end joining. In some embodiments, NHEJ repair results in small insertions or deletions and can be used to disrupt the gene and thereby repress the gene. In some embodiments, the modification may be a substitution, deletion, or insertion of at least one nucleotide. In some aspects, cells in which a cleavage-induced mutagenesis event occurs, i. a mutagenesis event following an NHEJ event can be identified and/or selected using methods well known in the art.

TALE repeats can be assembled to specifically target the Suv39h1 gene. (Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405). A library of TALENs targeting 18,740 genes encoding human proteins has been created (Kim et al., Nature Biotechnology. 31, 251-258 (2013)). Custom-designed TALE kits are commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). In particular, CD38-targeting TALENs are commercially available (see Gencopoeia, catalog numbers HTN222870-1, HTN222870-2 and HTN222870-3, available online at www.genecopoeia.com/product/search/detail.php? prt=26&cid=&key=HTN222870). Representative molecules are described, for example, in US Patent Publication Nos. US 2014/0120622 and 2013/0315884.

In some embodiments, TALENs are introduced as transgenes encoded by one or more plasmid vectors. In some aspects, the plasmid vector may contain a selectable marker that allows the identification and/or selection of cells that received the specified vector.

RGEN (CRISPR/Cas systems)

Gene repression can be accomplished by one or more DNA-binding nucleic acids, such as disruption by an RNA-directed endonuclease (RGEN) or other disruption by another RNA-directed effector molecule. For example, in some embodiments, repression of a gene can be carried out using CRISPR (short polyndromic repeats, regularly arranged in groups) and CRISPR-associated proteins. See Sander and Joung, Nature Biotechnology, 32(4): 347-355.

In general, "CRISPR system" refers collectively to transcripts and other elements involved in the expression or control of the activity of CRISPR-associated ("Cas") genes, including sequences encoding the Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. , tracrRNA or active partial tracrRNA), a tracr-mate sequence (comprising a "forward repeat" and a tracrRNA-processed partial direct repeat in the case of an endogenous CRISPR system), a targeting sequence (also called a "spacer" in the case of an endogenous CRISPR system), and/or others sequences and transcripts from the CRISPR locus.

Typically, a CRISPR/Cas nuclease or CRISPR/Cas nuclease system includes a non-coding RNA (guide) RNA molecule that binds sequence-specifically to DNA and a CRISPR protein with nuclease functionality (eg, two nuclease domains). 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 Cas9 protein from S. pyogenes can be found in the SwissProt database under accession number Q99ZW2. In some embodiments, the Cas nuclease and gRNA are introduced into the cell. In some embodiments, the implementation of the CRISPR system induces DSB at the target site, followed by breaks, as discussed in this document. In other embodiments, Cas9 variants considered "nickase" can be used to cut one strand at the target site. Paired nickases can also be used, for example, to improve specificity, with each being driven by a pair of different gRNA targeting sequences. In further embodiments, a catalytically inactive Cas9 may be coupled to a heterologous effector domain, such as a transcriptional repressor, to influence gene expression.

In general, a CRISPR system is characterized by elements that facilitate the formation of a CRISPR complex at the site of a target sequence. Generally, in the context of CRISPR complex formation, "target sequence" generally refers to a sequence to which the generated target sequence is complementary, where hybridization between the target sequence and the target sequence promotes the formation of the CRISPR complex. Complete complementarity is not required as long as there is sufficient complementarity to induce hybridization and promote formation of the CRISPR complex. The target sequence may contain any polynucleotide, such as DNA or RNA polynucleotides. In particular, a sequence or template that can be used for recombination at a target locus containing the target sequences is referred to as an "edit template" or an "edit polynucleotide" or an "edit sequence". In some aspects, the exogenous polynucleotide matrix may be referred to as an editing matrix. In some aspects, the recombination is homologous recombination.

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

In some embodiments, the CRISPR enzyme is delivered to the cell in combination with (and optionally complexed with) a targeting sequence. In most cases, CRISPR/Cas9 technology can be used to knock down Suv39h1 gene expression in engineered cells. For example, Cas9 nuclease and a guide RNA specific for the Suv39h1 gene can be introduced into cells, for example, using lentiviral delivery vectors or using any of a number of known methods of delivery or carriers for transfer to cells, such as any of a number of known methods or carriers for delivery of Cas9 molecules and guide RNAs (also see below).

Delivery of nucleic acids encoding molecules and complexes that destroy a gene.

In some embodiments, a nucleic acid encoding a DNA-targeting molecule, complex, or combination is introduced or introduced into a cell. Typically, viral or non-viral-based gene transfer techniques can be used to introduce nucleic acids encoding components of the CRISPR, ZFP, ZFN, TALE and/or TALEN system into cells in culture.

In some embodiments, the implementation of the polypeptides are synthesized in situ in the cell as a result of the introduction into the cell of polynucleotides encoding polypeptides. In some aspects, the polypeptides can be produced outside the cell and then introduced into it.

Methods for introducing a polynucleotide construct into animal cells are well known and include, but are not limited to, 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.

In some embodiments, the implementation of the polynucleotide can be introduced into the cell, for example, using recombinant viral vectors (eg, retroviruses, adenoviruses), liposomes and the like. Transient transformation methods include microinjection, electroporation, or particle bombardment. The nucleic acid is introduced 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.

Methods for non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolisting, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA capture, and DNA uptake. Lipofection is described, for example, in US Pat. Cationic and neutral lipids suitable for effective receptor-recognizing lipofection of polynucleotides include those mentioned in Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (eg, in vitro or ex vivo administration) or target tissues (eg, in vivo administration).

Gene transfer systems based on RNA or DNA viruses include retroviral, lentiviral, adenovirus, adeno-associated and herpes simplex vectors.

For a review of gene therapy techniques, 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).

Reporter gene including, but not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed , blue fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins, including blue fluorescent protein (BFP), can be "introduced" into a cell to encode a gene product that serves as a marker by which change can be measured or modification of the expression of the gene product.

Getting cells

Isolation of cells includes one or more preparation steps and/or non-affinity based separation steps according to methods well known in the art. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich desired components, lyse, or remove cells susceptible to particular reagents. In some examples, cells are separated based on one or more properties such as density, adhesive property, size, sensitivity, and/or resistance to certain components.

In some embodiments, obtaining cells includes the steps of freezing, eg, cryopreserving, cells, either before or after isolation, incubation, and/or construction. In certain aspects, any of a variety of known freezing solutions and freezing parameters may be used.

In most cases, cells are incubated prior to or in the background of genetic engineering and/or Suv39h1 inhibition.

Incubation steps may include cultivation, incubation, stimulation, activation, expansion and/or reproduction.

Inhibition of Suv39h1 according to the invention can also be achieved in vivo after injection of cells into intended patients. In most cases, inhibition of suv39h1 is carried out using pharmacological inhibitors, as described previously.

Inhibition of Suv39h1 according to the previously described method can also be carried out during the stages of stimulation, activation and/or expansion. For example, PBMCs or purified T cells or purified NK cells or purified lymphoid progenitors are propagated in vitro in the presence of pharmacological inhibitors of Suv39h1 prior to adoptive transfer to patients. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulating agent. Such conditions include conditions designed to cause cells to proliferate, expand, activate, and/or survive in a population, to mimic antigenic challenge, and/or to prepare cells for genetic engineering, such as the introduction of a genetically engineered antigen receptor.

Incubation conditions may include one or more of certain media, temperature, oxygen content, carbon dioxide content, time, substances, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, partners. binding, fusion proteins, recombinant soluble receptors, and any other means designed to activate cells.

In some embodiments, the stimulatory conditions or agents include one or more agents, such as a ligand, that is capable of activating an intracellular signaling domain of the TCR complex. In some aspects, the agent "turns on" or initiates the TCR/CD3 intracellular signaling cascade in the T cell. Such agents may include antibodies, such as antibodies specific for the TCR component and/or co-stimulatory receptor, eg anti-CD3, anti-CD28, eg associated with a solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further include the step of adding anti-CD3 and/or anti-CD28 antibodies to the culture medium (eg, at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulatory agent comprises IL-2 and/or IL-15, for example, at an IL-2 concentration of at least about 10 units/mL.

In some aspects, the incubation is carried out in accordance with the methods described in US patent No. 6,040,1 77 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.

In some embodiments, T cells are grown by adding initiating feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to the culture (e.g., such that the resulting cell population contains at least about 5, 10, 20 , or 40 or more PBMC nurse cells for each T-lymphocyte in the initial population to be increased); and by incubating the culture (eg, for a period of time sufficient to increase the number of T cells). In some aspects, non-dividing nurse cells may contain gamma-irradiated PBMC nurse cells. In some embodiments, the implementation of PBMC irradiated with gamma rays in the range of about 3000 to 3600 rad to prevent cell division. In some aspects, the feeder cells are added to the culture medium prior to the addition of the T cell populations.

In some embodiments, the stimulating conditions include a temperature suitable for the growth of human T lymphocytes, such as at least about 25 degrees Celsius, typically at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further include the addition of non-dividing EBV-transformed lymphoblastoid cells (LCL) as nurse cells. LCLs can be exposed to gamma rays ranging from about 6000 to 10,000 rads. LCL feeder cells, in some aspects, are provided in any suitable amount such that the ratio of feeder LCL to original T lymphocytes is at least about 10:1.

In other 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, lines or clones of antigen-specific T cells for cytomegalovirus antigens can be obtained by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.

In some aspects, the methods include assessing the expression of one or more markers on the surface of the engineered cell or engineered cell. In one embodiment, the methods include assessing the surface expression of one or more target antigens (e.g., an antigen recognized by a genetically engineered antigen receptor) to be targeted by adoptive cell therapy, for example, using affinity-based detection methods such as flow cytometry.

Vectors and methods for cell genetic engineering

In some aspects, genetic engineering includes the introduction of a nucleic acid encoding a genetically engineered component or another component for introduction into a cell, such as a component encoding a gene disruptive protein or nucleic acid.

In general, the construction of CARs in immune cells (eg, T cells) requires cell culture to allow transduction and expansion. A variety of methods can be used for transduction, but robust gene transfer is required to ensure long-term expression of CAR in clonal-expanding and persistent engineered cells.

In some embodiments, gene transfer is accomplished first by promoting cell growth, eg, T cell growth, proliferation and/or activation, followed by transduction of the activated cells and expansion in culture to quantities sufficient for clinical use.

Various methods of administering genetically engineered components, eg antigen receptors, eg CARs, are well known and may be used in conjunction with the methods and compositions provided. Typical methods include methods for transferring receptor-encoding nucleic acids, including transfer by viruses, eg, retroviruses or lentiviruses, transduction, transposons, and electroporation.

In some embodiments, recombinant nucleic acids are “transferred” into cells using recombinant pathogenic virus particles, such as, for example, vectors derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are “transferred” into T cells using recombinant lentiviral vectors or retroviral vectors such as retroviral gamma vectors (see e.g. Koste et al. (2014) Gene Therapy 2014 Apr 3.; Carlens et Alonso-Camino et al (2013) Mol Ther Nucl Acids 2, e93 Park et al., Trends Biotechnol 2011 Nov 29(11): 550 -557.

In some embodiments, the retroviral vector has a long terminal repeat (LTR), e.g., a retroviral vector derived from Moloney mouse leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus ( MSCV), spleen necrosis virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any source, avian or mammalian. Retroviruses are in most cases amphotropic, which means that they are able to infect host cells of several species, including humans. In one embodiment, the gene to be expressed is placed in a retroviral gag, pol and/or env sequence. A number of exemplary retroviral systems have been described (e.g., US Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3 : 102-109.

Methods for lentiviral transduction are also well known. Typical methods are described, for example, 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.

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

Other approaches and vectors for transferring genetically engineered nucleic acids encoding genetically engineered products are described, for example, in International Patent Application Publication No. WO2014055668 and US Patent No. 7,446,190.

Composition of the invention

The present invention also relates to compositions containing the cells described in this document and/or obtained using the proposed methods. In most cases, these compositions are pharmaceutical compositions and compositions for administration, for example, in adoptive cell therapy.

The pharmaceutical composition of the invention typically contains at least one engineered immune cell of the invention and a pharmaceutically acceptable carrier.

When used in the description, the phrase "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and agents that slow absorption, and the like, compatible with pharmaceutical use. In addition, additional active compounds can be added to the composition. In some aspects, the choice of carrier in a pharmaceutical composition is determined in part by the particular engineered CAR or TCR, the vector or cells expressing the CAR or TCR, and the particular method used to administer the vector or host cell expressing the CAR. Accordingly, there are a number of suitable formulations. For example, the pharmaceutical composition may contain preservatives. Suitable preservatives may include, for example, methyl paraben, propyl paraben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative, or mixture thereof, is typically present in an amount of from about 0.0001 to 2% by weight of the entire composition.

The pharmaceutical composition is formulated to be compatible with the intended route of administration.

Therapeutic methods

The present invention also relates to cells as previously defined and their use in adoptive therapy (namely, adoptive T-cell therapy), typically in the treatment of cancer in a subject in need thereof.

"Treatment" as used herein is defined as the use or administration of the cells of the invention, or a composition containing the cells, to a patient in need thereof, for the purpose of correcting, curing, alleviating, relieving, transforming, altering, eliminating, ameliorating or affecting a disease such as cancer, or any symptom of a disease (e.g., cancer) In particular, the terms "treat" or "treatment" refers to the reduction or alleviation of at least one adverse clinical symptom associated with a disease, such as cancer, for example, pain, swelling, low content of blood cells, etc.

In relation to the treatment of cancer, the term "treat" or "treatment" also refers to slowing down or eliminating the uncontrolled reproduction of neoplastic cells, i. shrinkage of existing tumors and/or arrest of tumor growth. The term "treat" or "treatment" also refers to the induction of apoptosis in cancer or tumor cells in a subject.

The subject of the invention (ie the patient) is a mammal, in most cases a primate, such as a human. In some embodiments, the primate is a monkey or a great ape. The subject may be male or female, and may be of any suitable age, including children, adolescents, young adults, adults, and the elderly. In some embodiments, the subject is a non-primate mammal, such as a rodent. In some examples, the patient or subject is a validated animal model of the disease to study adoptive cell therapy and/or to evaluate the consequences of toxicity, such as cytokine release syndrome (CRS). In some embodiments, the subject has cancer, the subject is at risk of developing cancer, or the subject is in cancer remission.

The cancer may be solid cancer or "fluid tissue tumor" such as cancers affecting the blood, bone marrow, and lymphoid system, also known as hematopoietic and lymphoid tissue tumors, which include primarily leukemia and lymphoma. Fluid tissue tumors include, for example, acute myelocytic 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's lymphoma (NHL ), adenoma, squamous cell carcinoma, laryngeal carcinoma, gallbladder and bile duct carcinoma, retinal cancers such as retinoblastoma).

In particular, solid cancers include cancers affecting one of the organs selected from the group consisting of colon, rectum, skin, endometrium, lung (including non-small cell lung carcinoma), uterus, bones (such as osteosarcoma, chondrosarcoma, Ewing's sarcoma , fibrosarcoma, giant cell tumor, adamantinoma and chordoma), liver, kidney, esophagus, stomach, bladder, pancreas, cervix, brain (such as meningioma, glioblastoma, poorly differentiated astrocytoma, oligodendrocytoma, pituitary tumors, schwannoma and metastatic brain cancer) , ovary, breast, head and neck, testis, prostate and thyroid.

Preferably, the cancer according to the invention is a cancer affecting the blood, bone marrow and lymphoid system, as described above. In most cases, the cancer is or is associated with multiple myeloma.

In some embodiments, the subject suffers from or is at risk of developing an infectious disease or condition such as, but not limited to, viral, retroviral, bacterial, and protozoal 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 , Graves' disease, Crohn's disease, multiple sclerosis, asthma, and/or a disease or condition associated with a transplant.

The present invention relates to a method of treatment and in particular to adoptive cell therapy, preferably adoptive T cell therapy, comprising administering to a subject in need thereof a previously described composition.

In some embodiments, cells or compositions are administered to a subject, such as a subject having cancer or at risk of developing cancer or any other disease as mentioned above. In some aspects, these methods thereby treat, for example, improve one or more symptoms of a disease or condition, for example in relation to cancer, by reducing the mass of a tumor expressing an antigen recognized by the engineered cell.

Methods for introducing cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. Adoptive T cell therapy methods are described, for example, in US Published Application No. 2003/0170238 Gruenberg et al; U.S. Patent No. 4,690,915 Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, for example, 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.

In some embodiments, cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is performed using autologous transfer, in which cells are isolated and/or otherwise prepared from a subject who is to receive cell therapy, or from a sample. received from such a subject. Thus, in some aspects, cells are obtained from a subject, such as a patient in need of treatment, and the cells, after isolation and processing, are administered to the same subject.

In some embodiments, cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is performed via allogeneic transfer, in which cells are isolated and/or otherwise obtained from a subject who is not the subject to be received or who is in eventually receives cell therapy, for example, the first subject. In such embodiments, the cells are then administered to another subject, such as a 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 class or supertype as the first subject.

Administration of at least one cell of the invention to a subject in need thereof may be combined with one or more additional therapeutic agents, or administered in conjunction with another therapeutic intervention, or simultaneously or sequentially in any order. In some situations, the cells are co-administered with another treatment close enough in time that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, cell populations are administered prior to administration of one or more additional therapeutic agents. In some embodiments, cell populations are administered after one or more additional therapeutic agents.

With regard to cancer treatment, combination cancer treatment may include, but is not limited to, chemotherapeutic agents, hormones, anti-angiogenic agents, radiolabeled compounds, immunotherapy, surgery, cryotherapy, and/or radiation therapy.

Immunotherapy includes, but is not limited to, immune checkpoint modulators (ie, inhibitors and/or agonists), monoclonal antibodies, cancer vaccines.

Preferably, the administration of the cell in the adoptive T cell therapy of the invention is combined with the administration of immune checkpoint modulators. Most preferably, the immune checkpoint modulators contain anti-PD-1 and/or anti-PDL-1 inhibitors.

The present invention also relates to the use of a composition comprising an engineered immune cell as described herein for the manufacture of a medicament for the treatment of cancer, an infectious disease or condition, an autoimmune disease or condition, or an inflammatory disease or condition in a subject.

Figure captions

Figure 1: The number of CD8+ central memory T cells is increased in Suv39h1-deficient mice. A. Representative FACS diagrams of gated spleen CD8+ T cells. B. The percentage of CD8+ central memory T cells was measured in different hematopoietic compartments (black circles, Suv39h1 KO; open circles: wild-type littermates).

Figure 2: Characterization of stemness and memory in endogenous antigen-specific Suv39h1 KO CD8+ T cells. Suv39h1-deficient Kb-OVA+ CD8+ T cells have a gene expression profile similar to stem cells and memory cells. Gene group enrichment analysis (GSEA) is shown.

Figure 3: Increased survival and self-renewal of Suv39h1 deficient CD8T cells. A. Experimental setup demonstrating the transfer of congenic CD45.2 Kb-OVA pentamers+ CD8+ T cells isolated from WT and Suv39h1 KO LM-OVA infected mice to naïve CD45.1 recipient mice (1: CD8+ T subpopulation sorting and transfer ( D0) 2: Self-renewal and differentiation analysis (D40-44) 40 days after adoptive transfer, recipient naive mice were challenged with LM-OVA, and 4 days after that, Kb-OVA pentamers+ CD8+ T cells were analyzed by FACS. B. Characteristic FACS plots of donor and recipient CD8+ T cells are shown C. The total number of recovered CD8+ T cells was measured The results combine data from two independent experiments.

Fig. 4: Suv39h1 deficient CD8+ OT-1 cells control tumor growth. (A) IL-2/OVA treated Suv39h1-KO CD45.2 OT-1 and wild-type cells from WT littermates carried i.v. a recipient mouse with a tumor 7 days after inoculation and injected intraperitoneally with an anti-PD-1 antibody (Bio X Cell, RMP-14). Tumor growth curves in IL-2/OVA-treated WT littermates (B) and Suv39h1-KO OT-1 cells (C) adoptively "transferred" to a tumor-bearing recipient mouse.

results

Materials and methods:

Single litter mice and Suv39h1 -KO mice were infected iv with 5x10 ³ CFU and 40 days later were challenged with 2x10 ⁶ CFU of recombinant Listeria monocytogenes expressing OVA (LM-OVA derived from wild-type strain 140403s). Bacteria were grown in TSB medium (BD Bioscience) to early logarithmic phase and their growth was assessed with a photometer at OD ₆₀₀ . Naive, dump ^- CD44 ^high K ^b -OVA ⁺ CD8 ⁺ T cells and related subpopulations were sorted by FACS. For the analysis of each subpopulation, we obtained 3 or 4 biological replicates. RNA was extracted using the Rneasy Micro Kit (QUIGEN) according to the manufacturer's protocol. DNase column treatment (QUIGEN) was included. For each mode, RNA was used to synthesize cDNA according to a standard affimetric protocol. Labeled DNA was hybridized on an Affymetrix mouse Gene 2.1 ST and processed on an Affymetrix GeneTitan instrument.

Results of microarray analysis:

Microarray data were processed in R (version 3.0.0) using a package from Bioconductor. CEL raw information files were used and quality control analysis was performed using the ArrayQualityMetrics package. The raw data was pre-processed using the RMA method available in the oligo package. Samples without labeling were removed from the analysis. Moderate t-tests were determined using the limma package, and p-values were adjusted using multiple testing using the Benjamini-Hochberg method. Finally, we considered a value to be statistically significant if the adjusted p-value was less than 5%.

Analysis of the enrichment of gene groups. Gene group enrichment analysis (GSEA) was performed using a gene with immunological scores (C7) and C2 (recommended) from a set of genes from the Molecular Signatures DataBase (MSigDB database v5.1, http://software.broadinstitute.org/gsea /msigdb/index.jsp). The GMT file has been uploaded along with the gene symbol information. The GCT file consisted of only 41345 samples and was imported into the GSEA. GSEA was run using standard parameters except for a few permutations (n = 10000). BubbleGUM analysis was performed as previously described.

Tumor growth analysis with Suv39h1 deficient CD8+ OT-1 cells was performed as follows:

Whole spleen and lymph node from male Suv39h1-KO CD45.2 OT-1 (specific for OVA257-264 peptide SIINFEKL in the context of H2-Kb MHC class I) and mice of the same litter of wild type 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 μg/ml OVA257-264 peptide for another 3 days.

In order to inoculate the tumor, 1x10 ⁶ EL4-OVA lymphoma cells were injected subcutaneously into the right flank of a male CD45.1/C57BL/6 mouse and after 7 days mice were injected iv 2x10 ⁶ IL-2/OVA treated with Suv39h1-KO CD45.2 OT-1 , or wild-type cells from a littermate animal.

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

Results:

1) Our results showed that in a cell population derived from Suv39h1-KO mice, early progenitors with a “stem cell-like” phenotype accumulate and are reprogrammed with increased efficiency into long-lived central memory T cells expressing both CD44 and CD62L, compared with cell population derived from wild-type mice. As illustrated in Figure 1, the proportion and number of central memory T cells (expressing both CD44 and CD62L) are increased in Suv39h1-deficient mice.

2) In order to understand the mechanism of this particular phenotype, a transcriptomic analysis was used:

Wild-type and Suv39h1 deficient mice were immunized with OVA-expressing Listeria m., and OVA-specific T cells were isolated on Day 7 post-immunization using sorting by flow cytometry.

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

We concluded that OVA-specific T cells from Suv39h1-deficient mice stimulated after infection with Listeria m. express a stem cell-like mRNA profile.

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

Congenic CD45.2 Kb-OVA pentamers+ CD8+ T cells isolated from WT mice and Suv39h1 KO LM-OVA infected mice were transferred to naive CD45.1 recipient mice. 40 days after adoptive transfer, naive recipient mice were challenged with LM-OVA and 4 days later, Kb-OVA pentamers+ CD8+ T cells were analyzed by FACS.

The results demonstrated (Figure 3) that the Suv39h1-deficient cell expresses elevated levels of a "stem cell-like" profile. They also survive better and repopulate more efficiently in wild-type mice after adoptive transfer.

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

Figures 4B and C show that mice adoptively transferred with Suv39h1-KO OT-1 cells and treated with anti-PD1 monoclonal antibody had better tumor growth control than similarly treated mice using WT OT-1 cells. Of note, 30 days after tumor injection, 4 out of 5 WT OT-1 transferred mice showed tumor growth compared to only 1 out of 5 mice injected with Suv39h1-KO OT-1 cells.

Conclusion

The absence of Suv39h1 activity in CD8+ T cells is associated with improved antitumor efficacy of the adoptive T cell transfer therapeutic approach.

Claims (25)

1. An engineered immune cell, wherein said immune cell is selected from T cells, NK cells and their progenitors, which is Suv39h1 defective and has increased survival compared to a non-Suv39h1 defective cell. 2. The engineered immune cell of claim 1, which further comprises a genetically engineered antigen receptor that specifically binds to the target antigen. 3. The engineered immune cell according to claim 1 or 2, which is a T cell or its precursor. 4. Constructed immune cell according to any one of paragraphs. 1-3, which is a CD4+ T cell or a CD8+ T cell. 5. Constructed immune cell according to any one of paragraphs. 1-4, which is extracted from the subject. 6. The engineered immune cell of claim 5, wherein the subject is suffering from or at risk of developing cancer. 7. Constructed immune cell according to any one of paragraphs. 1-6, which contains two or more genetically engineered receptors, where each receptor recognizes a different antigen. 8. Constructed immune cell according to any one of paragraphs. 1-7, wherein the activity and/or expression of Suv39h1 in said engineered immune cell is selectively inhibited or blocked. 9. Constructed immune cell according to any one of paragraphs. 1-8, where the Suv39h1 gene is repressed or disrupted by deletion of a gene, exon, or region, replacement with an exogenous sequence, and/or the gene contains a frameshift or missense mutation. 10. Constructed immune cell according to any one of paragraphs. 1-9, wherein said engineered immune cell expresses a Suv39h1 nucleic acid encoding a non-functional Suv39h1 protein. 11. Constructed immune cell according to any one of paragraphs. 2-10, where the target antigen is expressed on cancer cells and/or is a universal tumor antigen. 12. Constructed immune cell according to any one of paragraphs. 2-11, wherein the genetically engineered antigen receptor is a chimeric antigen receptor (CAR), or T cell receptor (TCR). 13. Constructed immune cell according to any one of paragraphs. 2-12, wherein said genetically engineered antigen receptor is a chimeric CAR antigen receptor which contains (a) a primary cytoplasmic signal sequence and/or a secondary cytoplasmic signal sequence containing an intracellular domain from the CD3 zeta chain, FcR gamma, FcR beta, CD3 gamma, CD3 delta , CD3 epsilon, CDS, CD22, CD79a, CD79b and/or CD66d. 14. The engineered immune cell of claim 13, wherein the CAR contains a costimulatory domain from 4-1BB, CD28, ICOS, OX40, or DAP10. 15. The engineered immune cell of claim 13 or 14, wherein the genetically engineered antigen receptor is a CAR that contains (a) an intracellular signaling domain from the CD3 zeta chain, (b) one or more co-stimulatory domains from 4-1BB, CD28, ICOS, OX40 or DAP10, and (c) an antibody heavy chain variable region and/or an antibody light chain variable region. 16. A method of obtaining a genetically engineered immune cell defective in Suv39h1, where the method includes the step of inhibiting the expression and/or activity of Suv39h1 in a T cell, NK cell or their precursor; so that the resulting engineered T cell, NK cell, or precursor thereof has increased survival. 17. A method for producing a genetically engineered Suv39h1-deficient immune cell according to claim 16, which also includes the step of introducing into said genetically engineered Suv39h1-deficient immune cell a genetically engineered antigen receptor specifically binding to a target antigen. 18. A method of obtaining a genetically engineered immune cell defective in Suv39h1 according to any one of paragraphs. 16, 17, according to which the inhibition of the expression and/or activity of Suv39h1 includes 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. 19. A method of obtaining a genetically engineered immune cell defective in Suv39h1 according to claim 18, according to which the agent is selected from small molecular weight inhibitors; antibody derivatives, optionally intrabodies, nanobodies or affitetels, aptamers, nucleic acid molecules that block transcription or translation: small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), or piwiRNAs (piRNAs); antisense nucleotides; ribozymes, or gene-editing agents. 20. Pharmaceutical composition for adoptive cell therapy, containing at least one engineered cell defective in Suv39h1 according to any one of paragraphs. 1-15 or at least one genetically engineered immune cell defective in Suv39h1, obtained by the method according to any one of paragraphs. 16-19, and a pharmaceutically acceptable carrier. 21. The use of an engineered immune cell defective in Suv39h1, according to any one of paragraphs. 1-15 or a genetically engineered immune cell defective in Suv39h1, obtained in the method according to any one of paragraphs. 16-19 or compositions according to claim 20, in adoptive cell therapy. 22. Use according to claim 21, wherein the immune cell is autologous or allogeneic. 23. Use according to claim 21 or 22 where the engineered immune cell defective in Suv39h1, according to any one of paragraphs. 1-15, or obtained in the method according to any one of paragraphs. 16-19 or the compositions of claim 20 are used in the treatment of cancer. 24. Use according to claim 23, where the engineered immune cell defective in Suv39h1, according to any one of paragraphs. 1-15, or obtained in the method according to any one of paragraphs. 16-19 or the compositions of claim 20 are used in combination with a second anti-cancer therapeutic agent. 25. The use of claim 24 wherein the second cancer therapeutic agent is an immune checkpoint inhibitor.

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