IMMUNE CELLS DEFECTIVE FOR ZNF217 AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates to the field of oncology and in particular cellular adoptive therapy. The present invention provides engineered immune cells defective for ZNF217 with enhanced proliferation, persistence and polyfunctionality in vivo. These defective cells, expressing no or less ZNF217 protein than the wild-type cells, also exhibit a conserved central memory phenotype, resistance to tumor-induced exhaustion, stem-like features and pool reconstitution potential after adoptive transfer overtime. Furthermore, at least partially inhibiting ZNF217 in several tumor cell types reduces or abolishes their invasiveness and proliferation. Thus, the present invention relates to products involving ZNF217 defective immune cells as well as to uses thereof. These cells may be combined with a ZNF217 inhibitor, in particular in a pharmaceutical composition.
BACKGROUND OF THE INVENTION
Adoptive T-Cell Therapy (TCT) using T cells armed with recombinant T Cell Receptor (TCR), as well as Chimeric Antigen Receptor (CAR) technologies and Tumor infiltrating T cells (TILs) are emerging as powerful curative treatments against malignancies (Lim W. A & June C. H., 2017; June, C. H. & Sadelain, M., 2018; Melenhorst, J. J. et al., 2022). However, cell-intrinsic barriers restrict adoptive T-cell fitness, persistence and efficacy, which prevent their widespread use in patients, particularly for solid tumors (Rafiq, S., et al., 2020). During in vitro expansion and upon repeated antigen exposure in vivo, T cells rapidly undergo epigenetic remodeling leading to terminal differentiation and exhaustion (dysfunctional T cell properties) (Gattinoni, L. et al., 2005; Sen, D. R. et al., 2016), which hinders durable antitumor response (Pauken, K. E. et al., 2016). Genetic engineering of epigenetic regulators [EZH2 (Weber, E. W. et al., 2021), DNMT3A (Prinzing, B. et al., 2021), TET2 (Fraietta, J. A. et al., 2018)] have shown substantial improvement of CAR-T cells antitumor activity and resistance to exhaustion (Akbari, B. et al., 2021). Using genome-wide (GW) CRISPR screen (Laprie-Sentenac,et al., 2022), inventors previously identified SOCS1 as major checkpoint inhibitor of CD4+ T cells, whose targeting restores human CAR-T cells composition and efficacy (Sutra Del Galy, A. et al. , 2021). However, inventors’ transcriptomic analysis of sgSOCSl human CAR-T cells demonstrates the significant upregulation of other SOCS family members overtime, especially SOCS3 and CISH. Inventors discovered that other SOCS family members can compensate for the loss of individual SOCS protein and that further targeting these genes could enhance the fitness and antitumor activity of TCT.
N6-methyladenosine (m6A) is the most abundant mRNA modification and is catalyzed by the methyltransferase complex, in which methyltransferase-like 3 (METTL3) is the sole catalytic subunit (Delaunay, S. & Frye, M., 2019). The deposition of chemical modifications into RNA is a crucial regulator of temporal and spatial gene expression programs, leading to mRNA degradation or stabilization (Wang, X. et al., 2017). In naive CD4+ T cells, it has been shown to promote the decay of several SOCS family members transcripts (SOCS1, SOCS3 and CISH). Interestingly, METTL3 can regulate IL7/STAT5 pathway (Li, H.-B. et al. , 2017), which is involved in T cells sternness and polyfunctionality (expression of IFNy, IL-2, IL-21, TNFa and granzyme B). More recently, using METTL3 conditional deletion in naive CD4+ T cells, Yao et al. demonstrated that METTL3 expression is essential for T follicular helper (Tfh) differentiation and functionality in lymphocytic choriomeningitis virus model (Yao, Y. et al., 2021). However, the impact of METTL3 overexpression on TCT against cancer has never been explored before.
Interestingly, ZNF217 (human protein, zfp217 in mouse), a multi-zinc finger protein was reported to sequester METTL3, which modulate m6A RNA modification of key pluripotency factors (Liu, Q. et al. , 2019; Aguilo, F. et al., 2015). ZNF217 is also a transcriptional repressor, as a core component of protein complexes including histone deacetylase (HD AC), CoREST and CTBPs (Banck, M. S. et al., 2009). A recent CRISPR screen identified ZNF217 as a regulator of CD 127 (IL7R) expression in human CD8+ T cells (McCutcheon et al., 2023). The IL7R is a surface marker associated with T cell survival, long-term persistence, and positive clinical response to ACT (Haradhvala et al., 2022). However, at the junction between epigenetic and epitranscriptomic networks, the role played by ZNF217 in immune cells fate and functionality, and particularly in controlling T cells has never been explored in vivo before. This is of important interest as RNA modification modulates many aspects of cells behavior including survival, differentiation and migration adding another regulatory layer of complexity which need to be unraveled.
In several cancer cells, including glioblastoma, breast cancers, prostate cancers, ovarian cancers, ZNF217 overexpression promotes proliferation, invasiveness and differentiation toward a less differentiated phenotype (Kuo et al., 2010; Mao et al., 2011; Littlepage et al., 2012; Thollet et al., 2010; Fahme et al., 2022; Szczyrba et al., 2013). In breast cancer patients, ZNF217 amplification and expression correlate with shorter overall, disease-specific, and relapse-free survival (Littlepage, L. E. et al., 2012). ZNF217 overexpression has been shown to increase cellular immortalization, antiapoptosis, telomerase repression and metastatic potential (None et al., 2001; Huang et al., 2005).
Inventors’ data demonstrate that at least partially deleting ZNF217 in T-cells surprisingly promotes their proliferation (KI67, cell cycle progression) in vivo, in the context of both CD4 TCR-transgenic therapeutic transfer (MB49 tumors) and in human CAR19bbz. Inventors in particular observed that ZNF217 targeting induces a T-cell stem-like phenotype associated with improved antitumor response. Among significant observations, they also report upregulation and maintenance overtime of markers associated with memory precursors or T progenitor exhausted (Tpex) cells phenotype including CD62L, CD38, CD127 (IL7Ra), CD27, CCR7, TCF1, Ki67 and METTL3, suggesting a de-differentiation (i.e., rejuvenation) of exhaustion-imprinted T cells. Of note, they observed a strong and reproductible downregulation of CD4 in human CAR-T cells after re-stimulation (PMA-ionomycin-Golgiplug). These CD3+, CD4-, CD8- double negative (DN) T cells exhibit a potent functionality as shown by the expression of both IFNy and TNFa cytokines. Interestingly, a recent study has shown that late-persisting CAR-T cells from children in remission from acute lymphoblastic leukemia comprised a population that did not express CD8 or CD4 co-receptors (Anderson, N. D. et al., 2023).
Altogether, inventors’ experiments demonstrate that (METTL3+) ZNF217 defective immune cells strongly impacts the differentiation program, phenotype, functionality and persistence of TCT effect in vivo, with potent therapeutic applications, which have never been described, considered or imagined before.
SUMMARY OF THE INVENTION
Inventors identified and herein describe a new relevant immunotherapeutic target allowing adoptive T-cell therapy (TCT) to resist to the immunosuppressive tumor microenvironment (TME) and enhance its persistence and metabolic fitness in vivo.
A first object herein described is an immune cell (in particular a T cell, a NK cell, a macrophage or a B cell, preferably a T cell), in particular an engineered immune cell, which is defective for ZNF217. Also herein described is a population of cells comprising such an engineered immune cell. Inventors discovered, and herein reveal for the first time, that the inhibition of ZNF217 leverages antitumor immune responses and decreases tumor cells proliferation and viability, probably through nonoverlapping mechanisms.
In a particular aspect, the engineered immune cell is Ki67+, CD62L+, CD 127+ (IL7Ra+), METTL3+, CD44, CD38+, CD27+, CCR7+, TCF1+, Nanog+, KLF4+, SOX2+, JAK2+, phospho- STAT3+ and/or phospho-STAT5+.
In another particular aspect, the engineered immune cell is Ki67+, CD62L+, CD 127+ (IL7Ra+), METTL3+, CD44, CD38+, CD27+, CCR7+ and/or TCF1+.
In a preferred aspect, the engineered ZNF217 defective immune cell, preferably ZNF217 defective immune T cell, is METTL3+ or is METTL3+ and CD62L+, and also possibly in addition TCF-1+, CD 127+ (IL7Ra+), CD27+ and/or CCR7+, in particular TCF-1+.
In a particular aspect, the engineered immune T cell is a ZNF217 biallelic knock-out (KO) T cell, and the population of cells of the invention comprises such an engineered immune T cell.
In another particular aspect, the engineered immune T cell i) is a ZNF217 biallelic knock-out (KO) T cell and ii) expresses METTL3 and CD62L, and also possibly in addition TCF-1+, CD127+ (IL7Ra+), CD27+ and/or CCR7+, in particular TCF-1+, and the population of cells of the invention comprises such an engineered immune T cell. In related aspects, the population of cells is enriched for cells, e.g., engineered immune T cells, NK cells, macrophages or B cells, co-expressing METTL3 and CD62L. In some aspects, the population of cells is enriched for cells, e.g., engineered immune T cells, NK cells, macrophages or B cells, that express, preferably in addition to METTL3, CD62L and CD27 on the surface, or CD62L and CD27 and CD127 on the surface, or CD62L and CCR7 on the surface, or CD62L and TCF-1 on the surface. The population may comprise, e.g., at least or more than 60%, 70%, 80%, or 90% of cells, e.g., T cells, NK cells, macrophages or B cells, that express CD62L on the surface, or CD62L and CD27 on the surface, or CD62L and CD27 and CD127 on the surface, or CD62L and CCR7 on the surface, or CD62L and TCF-1 on the surface.
Also herein described for the first time are a combination of i) a ZNF217 defective immune cell (typically an engineered immune cell) according to the invention as herein described such as METTL3+ and CD62L+, and possibly TCF-1+, CD127+ (IL7Ra+), CD27+ and/or CCR7+, in particular TCF-1+, ZNF217 defective immune T cell, or of a population of cells comprising such a ZNF217 defective immune cell, and ii) a ZNF217 inhibitor, as well as a composition, typically a pharmaceutical composition, comprising i) a ZNF217 defective immune cell (typically an engineered immune cell) according to the invention as herein described such as METTL3+ and CD62L+, and possibly TCF-1+, CD127+ (IL7Ra+), CD27+ and/or CCR7+, in particular TCF-1+, ZNF217 defective immune T cell, or a population of cells comprising such a ZNF217 defective immune cell, ii) a ZNF217 inhibitor and iii) a pharmaceutically acceptable support.
Also herein described is a kit comprising) a ZNF217 defective immune cell (typically an engineered immune cell) according to the invention as herein described such as a METTL3+ and CD62L+, and possibly TCF-1+, CD127+ (IL7Ra+), CD27+ and/or CCR7+, in particular TCF-1+, ZNF217 defective immune T cell, or a tool for genetically modifying an immune cell in order for said cell to become defective for ZNF217, and ii) a ZNF217 inhibitor.
In a particular aspect, the engineered ZNF217 defective immune cell according to the invention (for example the METTL3+ and CD62L+, and possibly TCF-1+, CD127+ (IL7Ra+), CD27+ and/or CCR7+, in particular TCF-1+, engineered ZNF217 defective immune T cell), or a population of cells comprising said engineered ZNF217 defective immune cell, is for use as a medicament, in particular for use for preventing or treating a cancer. In a particular aspect, the cancer is a cancer in which a lack of infiltration of the tumor by (tumor-infiltrating) immune cells (TILs), including immune T cells, is observed. Preferably, the cancer is a pediatric cancer, in particular a neuroblastoma, Diffuse Intrinsic Pontine Glioma (DIPG, also identified as Diffuse midline glioma), high-grade glioma, or Acute lymphoblastic Leukemia (ALL). In some aspects, the cancer is a solid cancer.
In another particular aspect, the combination of, or composition comprising, i) a ZNF217 defective immune cell according to the invention as herein described such as METTL3+, CD62L+, and possibly TCF-1 +, CD127+ (IL7Ra+), CD27+ and/or CCR7+, in particular TCF-1+, ZNF217 defective immune T cell, or a population of cells comprising such a ZNF217 defective immune cell, and ii) a ZNF217 inhibitor, of the invention, is for use as a medicament, in particular for preventing or treating a cancer.
In another particular aspect, the ZNF217 defective immune cell (for example the METTL3+ and CD62L+, and possibly TCF-1+, CD127+ (IL7Ra+), CD27+ and/or CCR7+, in particular TCF-1+, engineered ZNF217 defective immune T cell), combination or composition of the invention is for use in adoptive cellular therapy of cancer or in allogeneic cellular therapy of cancer.
Also herein described is an ex vivo method of preparing an engineered immune cell defective for ZNF217 comprising introducing a mutation, insertion or deletion in the ZNF217 gene that reduces the activity of ZFN217, optionally followed by use of the engineered immune cell prepared by the method in cancer therapy.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, terms have the meanings ascribed to them unless specified otherwise.
“Treatment”, or “treating” as used herein, is defined as the application or administration of cells as per the invention, or of a combination or a composition comprising said cells, to a patient in need thereof with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease such as cancer, or any symptom of the disease (e.g., cancer). In particular, the terms “treat or treatment” refers to reducing or alleviating at least one adverse clinical symptom associated with the disease such as the cancer, e.g., pain, swelling, low blood count, etc.
With reference to cancer treatment, the term “treat or treatment” also refers to slowing or reversing the progression neoplastic uncontrolled cell multiplication, i.e. shrinking existing tumors and/or halting tumor growth. The term “treat or treatment” also refers to inducing apoptosis in cancer or tumor cells in the subject.
The terms “subject” or “patient” are used interchangeably and refer to mammals, such as humans and nonhuman primates, as well as experimental animals such as a rabbit or a rodent, for example a rat or a mouse. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.
In some aspects, the primate is a monkey or an ape. In some aspects, the subject is a non-primate mammal, such as a rodent. In some examples, the patient or subject is a validated animal model for disease, adoptive cell therapy, and/or for assessing toxic outcomes such as cytokine release syndrome (CRS). In some aspects of the invention, the subject has a cancer, is at risk of having a cancer, or is in remission of a cancer. Typically, the subject may be a subject who needs a cell therapy (preferably an adoptive cell therapy) and/or who will receive a cell therapy.
In some aspects, the subject is suffering from or is at risk of 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 aspects, the disease or condition is an autoimmune or inflammatory disease or condition, such as arthritis, e.g., rheumatoid arthritis (RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease multiple sclerosis, asthma, and/or a disease or condition associated with transplant.
The “cancer” may be a solid cancer or a “liquid tumor” such as cancers affecting the blood, bone marrow and lymphoid system, also known as tumors of the hematopoietic and lymphoid tissues, which notably include leukemia and lymphoma. Liquid tumors include for example acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), in particular pediatric ALL, and chronic lymphocytic leukemia (CLL), (including various lymphomas such as mantle cell lymphoma and non-Hodgkins lymphoma (NHL). Typically, the cancer is, or is associated, with a leukemia, in particular the B Cell Acute Lymphoblastic Leukemia (B-ALL).
Solid cancers notably include cancers affecting one of the organs selected from the group consisting of colon, rectum, skin, endometrium, lung (including non-small cell lung carcinoma, “NSCLC”), uterus, bones (such as Osteosarcoma, Chondrosarcomas, Ewing's sarcoma, Eibrosarcomas, Giant cell tumors, Adamantinomas, and Chordomas), liver, kidney, esophagus, stomach, bladder, pancreas, cervix, brain (such as Meningiomas, Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, Pituitary Tumors, Schwannomas, and Metastatic brain cancers), ovary, breast, head and neck region, testis, prostate and the thyroid gland.
In a particular aspect, the solid cancer according to the invention is selected from lung cancer (in particular NSCLC), breast cancer, ovary cancer, prostate cancer, bladder cancer and brain cancer (in particular glioblastoma). Preferably, a solid cancer according to the invention is selected from lung cancer (in particular NSCLC), breast cancer and bladder cancer.
In another preferred aspect, the cancer is a cancer in which a lack of infiltration of the tumor by immune cell, in particular by immune T cell, is observed, for example a pediatric cancer.
In another preferred aspect, the cancer is a pediatric cancer, in particular a neuroblastoma, Diffuse Intrinsic Pontine Glioma (DIPG, also identified as Diffuse midline glioma), high-grade glioma, or Acute lymphoblastic Leukemia (ALL). “Cell therapy” (also called cellular therapy, cell transplantation, or cytotherapy) is a therapy in which viable cells are injected, grafted or implanted into a patient in order to effectuate a medicinal effect, for example by transplanting immune cells capable of fighting cancer cells via cell-mediated immunity in the course of immunotherapy.
“T-cell therapy” (or “TCT”, also called “T-cell transfer therapy”, “adoptive cell therapy”, “adoptive immunotherapy”, Adoptive T-Cell Therapy or “ATCT”, and “immune cell therapy”) is a type of immunotherapy that makes the immune T cells of a patient better able to attack (recognize and eliminate) cells that have become infected or damaged as well as those that have become cancerous.
In the case of cancer, immune cells known as “killer” T cells are particularly powerful against cancer, due to their ability to recognize (bind) to markers known as antigens on the surface of cancer cells and eliminate said cells in a very precise way. The existence of these T cells alone, however, isn’t always enough to guarantee that they will be able to carry out their mission to eliminate tumors. One potential roadblock is that these T cells must first become activated before they can effectively kill cancer cells, and then they must be able to maintain that activity for a sufficiently long time to sustain an effective anti-tumor response. Another is that these T cells might not exist in sufficient numbers.
Cellular immunotherapies take advantage of this natural ability of T cells and can be deployed in different ways: tumor-infiltrating lymphocytes (or TILs) therapy, engineered T Cell Receptor (TCR) therapy, Chimeric Antigen Receptor (CAR) T cell (CAR-T) therapy.
Some of these approaches involve collecting (isolating) patient’s own immune cells and simply expanding their numbers in the lab before being given back to the patient through a needle in their vein, whereas others involve genetically engineering our immune cells (via gene therapy) to enhance their cancer-fighting capabilities.
In the context of TIL therapy, naturally occurring T cells that have already infiltrated the patient’ s tumor are harvested and then activated and expanded. Then, large numbers of these activated T cells are reinfused into the patient where they can seek out and destroy tumors.
Unfortunately, not all patients have T cells that have already recognized their tumors. Others patients might, but for a number of reasons, these T cells may not be capable of being activated and expanded to sufficient numbers to enable rejection of their tumors. For these patients, doctors may employ an approach known as TCR therapy. This approach also involves taking T cells from patients, but instead of just activating and expanding the available anti-tumor T cells, the T cells can also be equipped with a new T cell receptor that enables them to target specific cancer antigens. By allowing doctors to choose an optimal target for each patient’s tumor and distinct types of T cell to engineer, the treatment can be further personalized to individuals and, ideally, provide patients with greater hope for relief. The previously mentioned TIL and TCR therapies can only target and eliminate cancer cells that present their antigens in a certain context (when the antigens are bound by the major histocompatibility complex, or MHC). Advances in cell-based immunotherapy have enabled doctors to overcome this limitation with CAR T Cell therapy where scientists equip a patient’s T cells with a synthetic receptor (chimeric antigen receptor) known as a CAR. A key advantage of CARs is their ability to bind to cancer cells even if their antigens aren’t presented on the surface via MHC, which can render more cancer cells vulnerable to their attacks. However, CAR T cells can only recognize antigens that are themselves naturally expressed on the cancer cell surface, so the range of potential antigen targets is smaller than with TCRs.
Adoptive cell therapy strategies have begun to incorporate other immune cells, such as Natural Killer (NK) cells (called “Natural Killer (NK) Cell therapy”) or macrophages, including in particular induced pluripotent stem cells (iPSC) -derived macrophages. One application being explored in the clinic involves equipping these cells with cancer-targeting CARs.
In the context of the invention, the term “sample” includes tissues, fluids, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (for example transduction with viral vector), washing, and/or incubation. Therefore, the sample can be a sample obtained directly from a biological source or a sample that is processed (“engineered”), in particular genetically modified. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom. Preferably, the sample from which the cells are derived or isolated is blood or a blood-derived sample or is or, is derived from, an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, and/or cells derived therefrom. Samples include, in the context of cell therapy (typically adoptive cell therapy), samples from autologous source (obtained from or derived from the subject to be treated) and/or allogeneic source. For example, in some aspects, the cells are derived from cell lines, e.g., T cell lines. The cells can also be obtained from a xenogeneic source, such as a mouse, a rat, a non-human primate, or a pig. Preferably, the cells are human cells. As used herein, a “tissue biopsy” refers to an amount of tissue removed from a patient or subject. In a patient with cancer, tissue can be removed from a tumor, allowing the analysis of cells within the tumor. “Tissue biopsy” can refer to any type of biopsy, such as needle biopsy, fine needle biopsy, surgical biopsy, and the like.
The ZNF217 gene product resembles a kruppel-like transcription factor (Collins, C. et al., 1998, Proc Natl Acad Sci U. S. A. 95: 8703-8), localizes predominantly to the nucleus (Collins, C. et al., 2001, Genome Res 11: 1034-42) and coimmunoprecipitates with histone deacetylase 1 (HDAC1) (You, A., et al., 2001, Proc Natl Acad Sci U. S. A. 98: 1454-8), histones demethylases Jaridlb/Plu-1, G9a, ESDI, EZH2, CoREST/CtBPs complex (Banck et al., 2009; Cowger et al., 2007; Quinlan et al., 2006) suggesting it functions as a transcriptional repressor and epigenetic regulator. As used herein the term “ZNF217” refers to the ZNF217 nucleic acid, protein and polypeptide polymorphic variants, alleles, mutants, and inter species homologs that: (1) have an amino acid sequence that has greater than about 60% amino acid sequence identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 50, 100, 200, 500, 1000, or more amino acids, corresponding to the sequence of the naturally occurring ZNF217 gene (also herein identified as “ZNF217”) as, e. g. , provided in Collins, C. et al., 1998, Proc Natl Acad Sci U. S. A. 95: 8703-8; Gray, J. et al., U. S. Patent No. 5,801, 021; Gray, J. et al., U. S. Patent No. 5,892, 010; Gray, J. et al., U. S. Patent No. 6,268, 184 ; Gray, J. et al., W098/02539; and, e. g., in GenBank Accession No.: AF041259, RefSeq Accession ID No. NM-006526 ; (2) bind to antibodies, e. g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence corresponding to the sequence of the naturally occurring ZFN217 gene, and conservatively modified variants thereof as, as, e. g., provided in Collins, C. et al., 1998, Proc Natl Acad Sci U. S. A. 95: 8703-8 ; Gray, J. et al., U. S. Patent No. 5,801,021; Gray, J. et al., U. S. Patent No. 5,892,010; Gray, J. et al., U. S. Patent No. 6,268,184; Gray, J. et al., W098/02539; and, e. g., in GenBank Accession No. AAC39895, Ref.Seq Accession ID No. Nu006517; (3) specifically hybridize under stringent hybridization conditions to the sequence of the naturally occurring ZFN217 gene and conservatively modified variants thereof; (4) have a nucleic acid sequence that has greater than about 80%, preferably about 85% or 90%, preferably greater than about 96%, 97%, 98%, 99%, or higher nucleotide sequence identity, preferably over a region of at least about 50, 100, 200, 500, 1000, or more nucleotides, corresponding to the sequence of the naturally occurring ZFN217 gene as, e. g., provided in Collins, C. et al., 1998, Proc Natl Acad Sci U. S. A. 95: 8703-8; Gray, J. et al., U. S. Patent No. 5,801,021; Gray, J. et al., U. S. Patent No. 5,892,010; Gray, J. et al., U. S. Patent No. 6,268,184 ; Gray, J. et al., W098/02539; and, e.g., in GenBank Accession No. AF041259, RefSeq Accession ID No. NM006526. A ZNF217 polynucleotide or polypeptide sequence is typically from a mammal including, but not limited to, human, rat, mouse, hamster, cow, pig, horse, sheep, or any mammal. A “ZNF217 polynucleotide” and a “ZNF217 polypeptide” are both either naturally occurring or recombinant. A “ZNF217 protein” or “polypeptide” can comprise naturally occurring or synthetic amino acids, e. g., labeled or otherwise modified amino acids or amino acid analogs. A “ZNF217 protein” will typically contain one or more characteristic protein motifs, any of which can be used independently of other elements normally present in a full-length ZNF217 protein, and will have one or more characteristic activities or properties, a “ZNF217 protein” can refer to any naturally occurring or synthetic ZNF217 polypeptide as described above.
The naturally occurring human ZNF217 gene is located at chromosome 20ql3. 2 based on the Human Genome Project draft sequence data, listed at National Center for Biotechnology Information (NCBI) in LOCUSLINK at LOCUSID7764. A cluster of expressed sequence tags (ESTs) for ZNF217 is found at NCBI in UniGene at UniGene ED number Hs. 155040. The ZFN217 gene is annotated to genomic clone rp4-724E16 (GenBank Locus ID No. AL157838). A “full length” ZNF217 protein or nucleic acid refers to a ZNF217 polypeptide or polynucleotide sequence, or a variant thereof, that contains all of the elements normally contained in one or more naturally occurring, wild type ZNF217 polynucleotide or polypeptide sequences.
SEQ ID NO:1 designates the human wild type ZNF217 polypeptide (XP_054179944.1, zinc finger protein 217 isoform XI [Homo sapiens]):
MQSKVTGNMPTQSLLMYMDGPEVIGSSLGSPMEMEDALSMKGTAVVPFRATQEKNVIQIEGYM PLDCMFCSQTFTHSEDLNKHVLMQHRPTLCEPAVLRVEAEYLSPLDKSQVRTEPPKEKNCKENE FSCEVCGQTFRVAFDVEIHMRTHKDSFTYGCNMCGRRFKEPWFLKNHMRTHNGKSGARSKLQQ GLESSPATINEVVQVHAAESISSPYKICMVCGFLFPNKESLIEHRKVHTKKTAFGTSSAQTDSPQG GMPSSREDFLQLFNLRPKSHPETGKKPVRCIPQLDPFTTFQAWQLATKGKVAICQEVKESGQEGS TDNDDSSSEKELGETNKGSCAGLSQEKEKCKHSHGEAPSVDADPKLPSSKEKPTHCSECGKAFRT YHQLVLHSRVHKKDRRAGAESPTMSVDGRQPGTCSPDLAAPLDENGAVDRGEGGSEDGSEDGL PEGIHLDKNDDGGKIKHLTSSRECSYCGKFFRSNYYLNIHLRTHTGEKPYKCEFCEYAAAQKTSL RYHLERHHKEKQTDVAAEVKNDGKNQDTEDALLTADSAQTKNLKRFFDGAKDVTGSPPAKQL KEMPSVFQNVLGSAVLSPAHKDTQDFHKNAADDSADKVNKNPTPAYLDLLKKRSAVETQANN LICRTKADVTPPPDGSTTHNLEVSPKEKQTETAADCRYRPSVDCHEKPLNLSVGALHNCPAISLS KSLIPSITCPFCTFKTFYPEVLMMHQRLEHKYNPDIHKNCRNKSLLRSRRTGCPPALLGKDVPPLS SFCKPKPKSAFPAQSKSLPSAKGKQSPPGPGKAPLTSGIDSSTLAPSNLKSHRPQQNVGVQGAATR QQQSEMFPKTSVSPAPDKTKRPETKLKPLPVAPSQPTLGSSNINGSIDYPAKNGSPWAPPGRDYFC NQSASNTAAEFGEPLPKRLKSSVVALDVDQPGANYRRGYDLPKYHMVRGITSLLPQDCVYPSQA LPPKPRFLSSSEVDSPNVLTVQKPYGGSGPLYTCVPAGSPASSSTLEGLGGCQCLLPMKLNFTSSF EKRMVATEISCDCTVHKTYEESARNTTVV.
The terms “identical” or “percent identity”, in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., the sequence of the naturally occurring ZFN217 gene), when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical”. This definition also refers to the complement of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length. A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, 1991, Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443, by the search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr. , Madison, WI), or by manual alignment and visual inspection (see, e. g., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel et al. , eds. 1995 supplement). Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1977, Nuc. Acids Res. 25: 3389-3402 and Altschul et al. , 1990, J. Mol. Biol. 215: 403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www. ncbi. nlm. nih. gov/).
The terms “recombinant” and “engineered” when used with reference, e. g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or, in the case of cells, to progeny of a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. Lor instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
An “expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent substitutions” or “silent variations”, which are one species of “conservatively modified variations”. Every polynucleotide sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted. Thus, silent substitutions are an implied feature of every nucleic acid sequence which encodes an amino acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. In some aspects, the nucleotide sequences that encode the enzymes are preferably optimized for expression in a particular host cell (e. g. , yeast, mammalian, plant, fungal, and the like) used to produce the enzymes. As to amino acid sequences, one of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
As used herein the term “SOCS” (suppressor of cytokine signaling proteins) refers to a family of genes and corresponding proteins involved in inhibiting the JAK-STAT signaling pathway. The JAK-STAT pathway designates a chain of interactions between proteins in a cell, and is involved in processes such as immunity, cell division, cell death, and tumor formation. The pathway communicates information from chemical signals outside of a cell to the cell nucleus, resulting in the activation of genes through the process of transcription. There are three key parts of JAK-STAT signaling: Janus kinases (JAKs), signal transducer and activator of transcription proteins (STATs), and receptors (which bind the chemical signals).
As used herein the term “SOCS-1” or “Suppressor of cytokine signaling 1” has its general meaning in the art and is part to the SOCS family proteins which form part of a classical negative feedback system that regulates cytokine signal transduction. The SOCS family comprises eight SOCS proteins encoded in the human genome, SOCS- 1-7 and CISH. All eight are defined by the presence of an SH2 domain and a short, C-terminal domain, the SOCS boxl. The SOCS box of all SOCS proteins are found associated with an adapter complex, elongin B,C. This association allows recruitment of an E3 ubiquitin ligase scaffold (Cullin5) to catalyze the ubiquitination of signaling intermediates recruited by their SH2 domains (Kamizono S et al., 2001).
In addition to their ubiquitin ligase activity, SOCS1 and SOCS3 (or “Suppressor of cytokine signaling 3”) are unique in also having the ability to directly inhibit the kinase activity of JAK (Janus Kinases). This activity relies upon a short motif, which is immediately upstream of the SH2 domain, known as the KIR (kinase inhibitory region). The KIR of SOCS1 is a highly evolved inhibitor of JAK and mutation of any residue within this motif, including the histidine residue that mimics the substrate tyrosine, leads to a significant decrease in affinity. SOCS1 is in particular a direct, potent and selective inhibitor of notably JAK1 and JAK2 as well as TYK2 catalytic activity and thus is typically involved in negative regulation of a number of cytokines, including interleukin-4 (IL-4), IL-6, IL-2, interferon (IFN)-alpha, interferon (IFN)- gamma, prolactin, growth hormone, and erythropoietin, that signal through the JAK/STAT3 pathway (see notably for details on SOCS1 activity: Sharma J, Larkin J 3rd. “Therapeutic Implication of SOCS1 Modulation in the Treatment of Autoimmunity and Cancer. Front Pharmacol. 2019; 10:324; Liau NPD, Laktyushin A, Lucet IS, et al. “The molecular basis of JAK/STAT inhibition by SOCST. Nat Commun. 2018;9(1 ): 1558); Sporri B, Kovanen PE, Sasaki A, Yoshimura A, Leonard WJ. “JAB/SOCS1/SSL1 is an interleukin-2-induced inhibitor of IL-2 signaling”. Blood. 2001 ;97(1):221 -226; Alexander WS, Starr R, Fenner JE, et al. “SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine”. Cell. 1999;98(5):597-608 as well as Kamizono S, Hanada T, Yasukawa H, et al. “The SOCS box of SOCS-1 accelerates ubiquitin-dependent proteolysis of TEL- JAK2. ” J Biol Chem. 2001; 276(16): 12530-12538; and Frantsve J, Schwaller J, Sternberg DW, Kutok J, Gilliland DG. “Socs-1 inhibits TEL-JAK2-mediated transformation of hematopoietic cells through inhibition of JAK2 kinase activity and induction of proteasome-mediated degradation.” Mol Cell Biol. 2001 ;21 ( 10) : 3547-3557) . This protein is also known as JAK-binding protein (JAB), STAT-induced STAT inhibitor 1 (SSL1) or Tec-interacting protein 3 (TIP-3).
The human SOCS-1 protein is referenced 015524 in UNIPROT, and is encoded by the gene SOCS-1 located on chromosome 16 (11 ,254,408-11 ,256,204 reverse strand.) and referenced as ENSG000001 85338 in the Ensembl database. The term SOCS-1 also encompasses all SOCS-1 orthologs. In some aspects, the human protein SOCS-1 according to the present invention is of SEQ ID NO: 2 MVAHNQVAADNAVSTAAEPRRRPEPSSSSSSSPAAPARPRPCPAVPAPAPGDTHFRTFRSHADYR RITRASALLDACGFYWGPLSVHGAHERLRAEPVGTFLVRDSRQRNCFFALSVKMASGPTSIRVHF QAGRFHLDGSRESFDCLFELLEHYVAAPRRMLGAPLRQRRVRPLQELCRQRIVATVGRENLARIP LNPVLRDYLSSFPFQI.
The human SOCS-3 protein is referenced 014543 in UNIPROT, and is encoded by the gene SOCS-3 located on chromosome 16 (11 ,254,408-11 ,256,204 reverse strand) and referenced as ENSG00000184557 in the Ensembl database. The term SOCS-3 also encompasses all SOCS-3 orthologs. In some aspects, the human protein SOCS-3 according to the present invention is of SEQ ID NO: 3: MVTHSKFPAAGMSRPLDTSLRLKTFSSKSEYQLVVNAVRKLQESGFYWSAVTGGEANLLLSAEP AGTFLIRDSSDQRHFFTLSVKTQSGTKNLRIQCEGGSFSLQSDPRSTQPVPRFDCVLKLVHHYMPP PGAPSFPSPPTEPSSEVPEQPSAQPLPGSPPRRAYYIYSGGEKIPLVLSRPLSSNVATLQHLCRKTVN GHLDSYEKVTQLPGPIREFLDQYDAPL.
The human CISH protein is referenced Q9NSE2 in UNIPROT, and is encoded by the gene CISH located on chromosome 16 (11 ,254,408-11 ,256,204 reverse strand) and referenced as ENSG00000114737 in the Ensembl database. The term CISH also encompasses all CISH orthologs.
In some aspects, the human protein CISH according to the present invention is of SEQ ID NO: 4: MVLCVQGPRPLLAVERTGQRPLWAPSLELPKPVMQPLPAGAFLEEVAEGTPAQTESEPKVLDPE EDLLCIAKTFSYLRESGWYWGSITASEARQHLQKMPEGTFLVRDSTHPSYLFTLSVKTTRGPTNV RIEYADSSFRLDSNCLSRPRILAFPDVVSLVQHYVASCTADTRSDSPDPAPTPALPMPKEDAPSDP ALPAPPPATAVHLKLVQPFVRRSSARSLQHLCRLVINRLVADVDCLPLPRRMADYLRQYPFQL.
As used herein the term “METTL3” designates the N6-adenosine-methyltransferase 70 kDa subunit. This enzyme is present in the cytosol of cells where it is involved in the post-transcriptional methylation of internal adenosine residues in eukaryotic mRNAs, forming N6-methyladenosine (m6A) (which is an abundant modification in mRNA and DNA).
The human METTL3 protein is referenced Q86U44 in UNIPROT, and is encoded by the gene METTL3 located on chromosome 16 (11 ,254,408-11 ,256,204 reverse strand) and referenced as ENSG00000165819 in the Ensembl database. The term METTL3 also encompasses all METTL3 orthologs.
In some aspects, the human protein METTL3 according to the present invention is of SEQ ID NO: 5: MSDTWSSIQAHKKQLDSLRERLQRRRKQDSGHLDLRNPEAALSPTFRSDSPVPTAPTSGGPKPST ASAVPELATDPELEKKLLHHLSDLALTLPTDAVSICLAISTPDAPATQDGVESLLQKFAAQELIEV KRGLLQDDAHPTLVTYADHSKLSAMMGAVAEKKGPGEVAGTVTGQKRRAEQDSTTVAAFASS LVSGLNSSASEPAKEPAKKSRKHAASDVDLEIESLLNQQSTKEQQSKKVSQEILELLNTTTAKEQS IVEKFRSRGRAQVQEFCDYGTKEECMKASDADRPCRKLHFRRIINKHTDESLGDCSFLNTCFHM DTCKYVHYEIDACMDSEAPGSKDHTPSQELALTQSVGGDSSADRLFPPQWICCDIRYLDVSILGK FAVVMADPPWDIHMELPYGTLTDDEMRRLNIPVLQDDGFLFLWVTGRAMELGRECLNLWGYE RVDEIIWVKTNQLQRIIRTGRTGHWLNHGKEHCLVGVKGNPQGFNQGLDCDVIVAEVRSTSHKP DEIYGMIERLSPGTRKIELFGRPHNVQPNWITLGNQLDGIHLLDPDVVARFKQRYPDGIISKPKNL.
As used herein, the term “FAS” or “Fas Cell Surface Death Receptor” has its general meaning in the art and refers to the receptor for TNFSF6/FASLG. Also known as FAS receptor (FasR), apoptosis antigen 1 (APO-1 or APT), cluster of differentiation 95 (CD95) or tumor necrosis factor receptor superfamily member 6 (TNFRSF6), FAS is a protein that in humans is encoded by the FAS gene. FAS is a death receptor located on the surface of cells that leads to programmed cell death (apoptosis) if it binds its ligand, Fas ligand (FasL), thus forming the death-inducing signaling complex (DISC) and inducing subsequent caspase 8 activation, via the adaptor molecule FADD. It is one of two apoptosis pathways, the other being the mitochondrial pathway. The human Fas is referenced as P25445 (TNR6_FIUMAN) in UNIPROT and is encoded by the gene FAS located on chromosome 10 (88,990,531-89,017,059 forward strand), referenced as ENSG00000026103 in Ensembl database. The term FAS also encompasses all FAS1 orthologs.
In some aspects, the human protein FAS as herein intended is of SEQ ID NO: 6:
MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPP GERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCR CKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVKRKE VQKTCRKHRKENQGSHESPTLNPETVAINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEAKI DEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKANLCTLAEKIQTIILKDITSDSEN SNFRNEIQSLV.
The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, variable heavy chain (VFI) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. In a preferred aspect, the antibody fragment is functional, i.e., it has the same activity as the complete antibody from which it comes or is derived. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SFI, F(ab')2; diabodies; linear antibodies; variable heavy chain (VFI) regions, single-chain antibody molecules such as scFvs and single-domain VFI single antibodies; and multispecific antibodies formed from antibody fragments. In particular aspects, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
“Single-domain antibodies” are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain aspects, a singledomain antibody is a human single-domain antibody. As used herein, “repression”, ‘inactivation” or “inhibition” of gene expression refers to the elimination, inhibition or reduction of expression of one or more gene products encoded by the subject gene in a cell, compared to the level of expression of the gene product in the absence of the repression. Exemplary gene products include mRNA and protein products encoded by the gene. Repression in some cases is transient or reversible and in other cases is permanent. Repression in some cases is of a functional or full-length protein or mRNA, despite the fact that a truncated or non-functional product may be produced. In some aspects herein, gene activity or function, as opposed to expression, is repressed. Gene repression is generally induced by artificial methods, i.e., by addition or introduction of a compound, molecule, complex, or composition, and/or by disruption of nucleic acid of or associated with the gene, such as at the DNA level. Exemplary methods for gene repression include gene silencing, knockdown, knockout, and/or gene disruption techniques, such as gene editing.
Examples include antisense technology, such as RNAi, siRNA, shRNA, and/or ribozymes, which generally result in transient reduction of expression, as well as gene editing techniques which result in targeted gene inactivation or disruption, e.g., by induction of breaks and/or homologous recombination. As used herein, a “disruption” of a gene refers to a change in the sequence of the gene, at the DNA level. Examples include insertions, mutations, and deletions. The disruptions typically result in the repression and/or complete absence of expression of a normal or “wild type” product encoded by the gene. Exemplary of such gene disruptions are insertions, frameshift and missense mutations, deletions, knock-in, and knock-out of the gene or part of the gene, including deletions of the entire gene. Such disruptions can occur in the coding region, e.g., in one or more exons, resulting in the inability to produce a full-length product, functional product, or any product, such as by insertion of a stop codon. Such disruptions may also occur by disruptions in the promoter or enhancer or other region affecting activation of transcription, so as to prevent transcription of the gene. Gene disruptions include gene targeting, including targeted gene inactivation by homologous recombination.
The cells according to the invention are typically eukaryotic cells, typically animal cells such as mammalian cells, e.g., human cells.
More particularly, the cells of the invention are derived from the blood, bone marrow, lymph, or lymphoid organs (notably the thymus) and are (white) cells of the immune system (i.e., “immune cells”), as well as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells.
In a particular aspect, the immune cell is selected from a T cell, a NK cell, a B cell and a macrophage. In another particular aspect, the immune cell is selected from a T cell, a NK cell and a macrophage.
Preferably according to the invention, cells are notably lymphocytes including T cells, B cells and/or NK cells, even more preferably lymphocytes are T cells and/or NK cells. Preferably, the immune cell is a T cell.
The T cell may be a cytotoxic T cell such as a “killer” T cell (CD8+ T cell), a helper CD4 T cell, a NKT cell (CD3+ CD8+ T cell) or a tumor infiltrating T cell. More particularly, the T cell is a CD4+ or CD8+ T cell.
In the context of the present invention, the term “immune cell” may designate a single immune cell or a population of immune cells, preferably a population of tumor-infiltrating immune cells, in particular TILs. Tumor-infiltrating lymphocytes (TILs) are white blood cells that have left the bloodstream and migrated towards a tumor. They include T cells and B cells and are part of the larger category of “tumor-infiltrating immune cells” which consist of both mononuclear and polymorphonuclear immune cells [i.e., T cells, B cells, natural killer (NK) cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, basophils, etc.], in variable proportions. Their abundance varies with tumor type and stage.
Preferred cells may be selected from Naive T cells (TN cells), Stem memory T cells (TSCM cells), memory T cells (TCM cells), tumor-infiltrating lymphocytes (TILs), or effector memory T cells (TEM cells) and combination thereof. In a particularly preferred aspect, the immune cell is a tumor-infiltrating immune cell (or tumor-infiltrating lymphocyte (TIL)), for example a tumor-infiltrating T lymphocyte.
Cells according to the invention may also be immune cell progenitors, such as lymphoid progenitors and more preferably T cell progenitors (which typically express a set of consensus markers including CD44, CD117, CD135, and Sca-1 - cf. Petrie HT, Kincade PW, 2005).
The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. With reference to the subject to be treated, the cells of the invention may be autologous, typically when used in the context of ATCT, and/or allogeneic.
In some aspects, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation.
Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells. Preferably, the cells according to the invention are TEFF cells with stem/memory properties and higher reconstitution capacity, as well as TN cells, TSCM, TCM, TEM cells and combinations thereof. In some aspects, one or more of the T cell populations is enriched for, or depleted of, cells that are positive for or express high levels of one or more particular markers, such as surface markers or secreted markers, or that are negative for or express relatively low levels of one or more markers. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (such as memory cells). In one aspect, the cells (such as the CD8+ cells or the T cells, e.g., CD3+ cells) are enriched for (i.e., positively selected for) cells that are positive for, or in other words that express high surface levels of, or that secrete detectable amount of, CD44, CCR7, CD38, CD27, CD62L, IL7-Ra (CD127), KI67, METTL3 and/or TCF1. In a particular aspect, cells may be, possibly in addition, positive for, or expressing high surface levels of, Nanog, KLF4, SOX2, JAK2, phospho-STAT3 and/or phospho-STAT5.
For example, according to the present application, the cells can include a CD4+ T cell population and/or a CD8+ T cell sub-population, e.g., a sub-population enriched for central memory (TCM) cells. Alternatively, the cells can be other types of lymphocytes, including natural killer (NK) cells, MAIT cells, Innate Fymphoid Cells (IFCs) and B cells.
In a particular aspect, the cells and compositions containing the cells for engineering according to the invention are isolated from a sample, e.g., a biological sample obtained from or derived from a subject.
The terms “isolated”, “purified”, or “biologically pure” refer to a biological material (e.g., nucleic acid, protein or cell) that is substantially or essentially free from components which normally accompany it, as found in its native state. Purity and homogeneity are typically determined using antibodies-mediated enrichment from magnetic selection kits or cell sorting using flow cytometry.
Cells defective for ZNF217
The present disclosure encompasses cells, more specifically immune cells defective for ZNF217.
In a particular aspect of the invention, the immune cell is isolated from a subject, preferably from a subject suffering from a cancer or at risk of suffering from a cancer.
The immune cell, present in a subject or isolated from the subject, may be genetically modified (and is herein identified as an “engineered immune cell”).
In a particular aspect of the present invention, the immune cell defective for ZNF217 is an engineered immune cell, in particular an engineered immune cell isolated from a subject, preferably from a subject suffering from a cancer or at risk of suffering from a cancer. In a particular and preferred aspect of the invention, the engineered immune cell has been genetically modified to become defective for ZNF217. In a particular aspect, the ZNF217 defective immune cell of the invention is CD62L+, CD44+, CD38+, CD27+, CD127+ (IL7Ra+), CCR7+, TCF1+, Ki67+, METTL3+, Nanog+, KLF4+, SOX2+, JAK2+, phospho-STAT3+ and/or phospho-STAT5+.
In a particular aspect, the ZNF217 defective immune cell of the invention is a CD62L+, CD38+, CD27+, CD127 (IL7Ra), CCR7+, TCF1+, Ki67+ and/or METTL3+ immune cell.
In another particular aspect, the ZNF217 defective immune cell of the invention is Nanog+, KLF4+, SOX2+, JAK2+, phospho-STAT3+ and/or phospho-STAT5+.
In another particular aspect, the engineered ZNF217 defective immune cell, preferably ZNF217 defective immune T cell, is METTL3+ or is METTL3+ and CD62L+, and also possibly in addition TCF-1+, CD127+ (IL7Ra+), CD27+ and/or CCR7+, in particular TCF-1+.
In another particular aspect, the engineered immune T cell is a Z/VF277 biallelic knock-out (KO) T cell. In such a ZNF217 biallelic knock-out (KO) T cell, the genetic biallelic KO is responsible for as complete a knock-out as technically possible in the cell, or in other words for a total or quasi-total loss-of-function of ZNF217.
In another particular aspect, the engineered immune T cell i) is a ZNF217 biallelic knock-out (KO) T cell and ii) expresses METTL3 and CD62L, and also possibly in addition TCF-1+, CD127+ (IL7Ra+), CD27+ and/or CCR7+, in particular TCF-1+.
In another particular aspect, the ZNF217 defective immune cell of the invention is a polyfunctional immune cell, preferably a polyfunctional T cell expressing or secreting IFNy, IL-2, IL-21, TNFa and/or granzyme B (GZB) molecule(s), and/or a Nanog+, KLF4+, SOX2+, JAK2+, phospho-STAT3+ and/or phospho- STAT5+ polyfunctional T cell is.
A Nanog+, KLF4+, SOX2+, JAK2+, phospho-STAT3+ and/or phosphor-STAT5+ immune cell is a rejuvenated cell which has self-renewal capacities and which is pluripotent.
In another particular aspect, the ZNF217 defective immune cell of the invention is a CD3+, CD4- and CD8- double negative (DN) T cell, in particular a METTL3+ (or METTL3+, CD62L+, and possibly TCF-1+, CD127+ (IL7Ra+), CD27+ and/or CCR7+, in particular TCF-1+) CD3+, CD4-, CD8- double negative (DN) T cell, in particular, a CD3+, CD4- and CD8- DN CAR T cell, and/or a T cell expressing or secreting IFNy, IL-2, IL-21, TNFa and/or GZB.
In some aspects, ZNF217defective cells can be further defective for at least one additional protein, in particular a protein of the SOCS family, preferably several proteins of said family, even more preferably protein(s) selected for example from SOCS1, SOCS3, CISH and any combination thereof. As used herein the expression “defective for ZNF217” according to the present invention refers to the repression, inhibition, or blockade of ZNF217 protein activity (as illustrated in the experimental part of the invention), such as for example the blockage of the binding of ZNF217 to DNA or to histones demethylases Jaridlb/Plu-1, G9a, LSD1, EZH2- or to transcriptional repressor from the CoREST/CtBPs complexes. In some aspects, inhibition of ZNF217 may be obtained by preventing the binding of ZNF217 to METTL3.
As used herein the expression “defective for SOCS1” according to the present invention refers to the repression, inhibition, or blockade of SOCS-1 activity, such as for example the blockage of the binding of SOCS1 on JAK(s) (JAK1/2 and/or TYK2), and/or the blockage of the SH2 binding domain and/or the blockage of the recruitment of an E3 ubiquitin ligase scaffold (Cullin5) through Elongin BC. In some aspects, inhibition of SOCS1 may be obtained by preventing the binding of SOCS1 on the JAKs (including JAK1/2 and/or TYK2), by preventing the SOCS1 Box from binding to Elongin C (an important intermediate of E3 complex recruitment), and/or by preventing interaction of SOCS1 with cavin-1.
As used herein the expression “defective for SOCS3” according to the present invention refers to the repression, inhibition, or blockade of SOCS-3 activity, such as for example the blockage of SOCS3 KIR binding domain to JAK(s) (JAK1/2/3 and/or TYK2), and/or the blockage of the SH2 binding domain and/or the blockage of the recruitment of an E3 ubiquitin ligase scaffold (Cullin5) through Elongin BC. In some aspects, inhibition of SOCS3 may be obtained by preventing the binding of SOCS3 on JAK(s) (including JAK1/2/3 and/or TYK2), and/or by preventing the SOCS3 Box from binding to Elongin C, and/or by preventing interaction of SOCS3 with cavin-1.
As used herein the expression “defective for CISH” according to the present invention refers to the repression, inhibition, or blockade of CISH activity, such as for example the blockage of the SH2 binding domain to PLC-yl and/or the blockage of the recruitment of an E3 ubiquitin ligase scaffold (Cullin5) through Elongin BC. In some aspects, inhibition of CISH may be obtained by preventing the binding of CISH SH2 domain to PLC-yl and/or by preventing the CISH Box from binding to Elongin C.
As used herein the expressions “defective for ZNF217”, “defective for SOCS1”, “defective for SOCS3”, or “defective for CISH” or “defective for any other protein of the SOCS family” according to the present application refers to the repression, inhibition, or blockade of ZNF217 and/or SOCS1 and/or SOCS3 and/or CISH and/or any other protein of the SOCS family activity, as detailed above, in the cell. For example, “inhibition of ZNF217 activity”, “inhibition of SOCS1 activity”, “inhibition of SOCS3 activity”, or “inhibition of CISH activity” as intended in the present application refers to a decrease of ZNF217 activity, of SOCS1 activity, of SOCS3 activity, or of CISH activity of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the activity, or level, of the ZNF217, SOCS1, SOCS3 or CISH protein which is not inhibited in a corresponding wild-type cell. Preferentially, the inhibition of ZNF217, SOCS1, SOCS3 or CISH activity leads to the absence in the cell of substantial detectable activity of ZNF217, SOCS1, SOCS3 or CISH respectively.
It is to be noticed that a cell defective for ZNF217 and/or for example SOCS1 and/or SOCS3 and/or CISH can be obtained by repression or disruption respectively of the ZNF217 and/or SOCS1 and/or SOCS3 and/or CISH gene(s), but also at the post-transcriptional level (ZNF217 mRNA and/or SOCS1 mRNA, and/or SOCS3 mRNA and/or CISH mRNA) as well at the post-translational or protein level of ZNF217 and/or SOCS1 and/or SOCS3 and/or CISH.
Inhibition of ZNF217 and/or for example SOCS1 and/or SOCS3 and/or CISH activity can thus also be achieved through repression of ZNF217 and/or SOCS1 and/or SOCS3 and/or CISH gene expression or though ZNF217 and/or SOCS1 and/or SOCS3 and/or CISH gene disruption. According to the invention, said repression reduces expression of ZNF217 and/or SOCS1 and/or SOCS3 and/or CISH in the cell, notably the immune cell of the invention by at least 50, 60, 70, 80, 90, or 95 % as to the same cell (i.e. corresponding cell) produced by the method in the absence of the repression or in corresponding wild-type cell. Gene disruption may also lead to a reduced expression of the ZNF217 and/or for example SOCS1 and/or SOCS3 and/or CISH protein or to the expression of a non-functional ZNF217 protein, a nonfunctional SOCS1 protein, a non-functional SOCS3 protein and/or a non-functional CISH protein.
By “non-functional protein”, it is herein intended a protein with a reduced activity or a lack of detectable activity as described above.
Inhibitors of the activity of a protein (or polypeptide) as herein described, typically ZNF217 or e.g., SOCS1 and/or SOCS3 and/or CISH, are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of the protein, e. g. , antagonists. These inhibitors also include genetically modified versions of the protein, e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, antagonists, antibodies, anti-sense oligonucleotides, RNA interfering agents such as siRNAs, shRNA, miRNA or ribozymes, gene editing agents, small chemical molecules, and the like.
In a particular aspect, the protein inhibitor is an inhibitor of a polynucleotide encoding the protein, typically a nucleic acid molecule that block transcription or translation such as e.g., an antisense nucleotide sequence, a RNA interfering agent such as a siRNA, a shRNA, a miRNA or a ribozyme, a gene editing agent.
In another particular aspect, the protein inhibitor is an inhibitor of the activity of the protein (or polypeptide), e.g., a peptide, an antibody or any functional fragment or derivative thereof, an aptamer, a small molecule, or a dominant negative protein (or polypeptide), e.g., a form of the protein that itself has no activity and which, when present in the same cell as a functional protein, reduces or eliminates the protein activity of the functional protein. Design of dominant negative forms is well known to those of or ordinary skill in the art and is described, e.g., in Herskowitz, 1987, Nature, 329: 219-22. Also, inactive polypeptide variants (muteins) can be used, e. g., by screening for the ability to inhibit the protein activity. Methods of making muteins are well known to those of skill (see, e.g., U.S. Patent Nos. 5,486,463; 5,422,260; 5,116,943; 4,752,585; 4,518, 504).
In some aspects, inhibitors of ZNF217 activity in a cell according to the invention can be selected from a nucleic acid molecule; a peptide; a small molecule; an antibody, a derivative or any functional fragment thereof; an aptamer; a ribozyme; a gene editing agent; and any combination thereof.
In a preferred aspect, the ZNF217 inhibitor is a miRNA, in particular MIR503 (SEQ ID NO: 7: TGCCCTAGCAGCGGGAACAGTTCTGCAGTGAGCGATCGGTGCTCTGGGGTATTGTTTCCGCT GCCAGGGTA, NCBI Reference Sequence: NC_000023.11).
In some aspects, inhibitors of SOCS1 activity in a cell according to the invention can be selected among any compound or agent natural or not having the ability of preventing binding of SOCS1 to JAK and/or Elongin C, or inhibiting the SOCS1 gene expression. Inhibitors of SOCS1 activity in a cell according to the invention can be selected among any compound or agent natural or not having the ability of inhibiting SOCS1 activity, notably as above mentioned, or inhibiting the SOCS1 gene expression. In some aspects, a peptide mimetic of SOCS1 or the autophosphorylation site pJAK2 (1001-1013) as described in Lilian W Waiboci, Howard M Johnson, James P Martin and Chulbul M Ahmed, J Immunol April 1 , 2007, 178 (1 Supplement) S170; or in Waiboci LW, Ahmed CM, Mujtaba MG, et al. J Immunol. 2007;178(8):5058- 5068, can be used.
Inhibition of ZNF217 and/or for example SOCS1 and/or SOCS3 and/or CISH (at the gene and/or protein level) in the immune cell according to the present invention can be permanent and irreversible or transient or reversible. Preferably however, ZNF217 inhibition and/or SOCS1 inhibition and/or SOCS3 inhibition and/or CISH inhibition is/are permanent and irreversible. Inhibition of ZNF217 and/or for example SOCS1 and/or SOCS3 and/or CISH in the cell may be achieved prior or after injection of the cell in the targeted patient.
In some aspects, the ZNF217 defective immune cell of the invention expresses a ZNF217 nucleic acid encoding a non-functional ZNF217 protein and, optionally further expresses and/or upregulates a functional METTL3. In a particular aspect, the cell has been genetically engineered to express, in particular overexpress (in comparison to the expression observed in a healthy subject) or upregulate the expression of, a functional METTL3. Such a cell is preferably a CD62L+ engineered immune cell, in particular immune T cell. Genetically engineered cells according to the invention
In a particular aspect, the present invention relates to an engineered immune cell of the invention (defective for ZNF217) which further comprises at least one genetically engineered antigen receptor that specifically binds a target antigen, possibly several genetically engineered antigen receptors. As further explained herein below, the target antigen may be an antigen expressed by a cancer cell and/or may be a universal tumor antigen.
Such a cell typically comprises one or more nucleic acids introduced via genetic engineering that encode one or more antigen receptors.
Typically, the nucleic acids are heterologous, (i.e., for example they are not ordinarily found in the cell being engineered and/or in the organism from which such cell is derived). In some aspects, the nucleic acids are not naturally occurring, including chimeric combinations of nucleic acids encoding various domains from multiple different cell types. Among the antigen receptors as per the invention are genetically engineered immune cells receptors, in particular T cell receptors (“TCRs”) or NK cell receptors (“NKCRs” or “KIRs” for killer Ig-like receptors), and components thereof, as well as functional non-TCR, or non- NKCR, antigen receptors, such as chimeric antigen receptors (CARs).
In a particular aspect, the genetically engineered antigen receptor is a CAR comprising an extracellular antigen-recognition domain that specifically binds to the target antigen including for example CD19, GD2, CD20, CD22, HER2, or mesothelin, to a T cell receptor (TCR), to a NK cell receptor (NKCR or KIR), or to a tumor-infiltrating lymphocyte (TIL).
Chimeric Antigen Receptors (CARs)
In some aspects, the engineered antigen receptors comprise chimeric antigen receptors (CARs), including activating or stimulatory 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, which graft an arbitrary 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, with transfer of their coding sequence facilitated by retroviral vectors. These receptors may be used also to graft the specificity of a monoclonal antibody onto another type of immune cell such as a NK cell.
CARs generally include an extracellular antigen (or ligand) binding domain linked 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 through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone.
In some aspects, the CAR is constructed with a specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type to be targeted by adoptive therapy, such as a cancer marker. Thus, the CAR typically includes in its extracellular portion one or more antigen binding molecules, such as one or more antigen binding fragment, domain, or portion, or one or more antibody variable domains, and/or antibody molecules.
The moieties used to bind to antigen fall in three general categories, either single-chain antibody fragments (scFvs) derived from antibodies, Fab’s selected from libraries, or natural ligands that engage their cognate receptor (for the first generation of CARs). Successful examples in each of these categories are notably reported in Sadelain M, Brentjens R, Riviere I. The basic principles of chimeric antigen receptor (CAR) design. Cancer discovery. 2013; 3(4):388-398 (see notably table 1) and are included in the present application. scFv’s derived from murine immunoglobulins are commonly used, as they are easily derived from well-characterized monoclonal antibodies.
Typically, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VFI) and variable light (VL) chains of a monoclonal antibody (mAh).
In some aspects, the CAR comprises an antibody heavy chain domain that specifically binds the antigen, such as a cancer marker or cell surface antigen of a cell or disease to be targeted, such as a tumor cell or a cancer cell, such as any of the target antigens described herein or known in the art.
In some aspects, the CAR contains an antibody or an antigen-binding fragment (e.g. scFv) that specifically recognizes an antigen, such as an intact antigen, expressed on the surface of a cell.
In some aspects, the CAR contains a TCR-like antibody, such as an antibody or an antigen-binding fragment (e.g. scFv) that specifically recognizes an intracellular antigen, such as a tumor-associated antigen, presented on the cell surface as a MFIC -peptide complex (HLA-restricted, peptide-specific antigen binding proteins). In some aspects, an antibody or antigen-binding portion thereof that recognizes an MFIC -peptide complex can be expressed on cells as part of a recombinant receptor, such as an antigen receptor. Among the antigen receptors are functional non-TCR or non-NKCR antigen receptors, such as chimeric antigen receptors (CARs). Generally, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity directed against peptide-MFIC complexes also may be referred to as a TCR-like CAR. In some aspects, the antigen-specific binding, or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some aspects, the CAR includes a transmembrane domain fused to the extracellular domain of the CAR. In one aspect, the transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
The transmembrane domain in some aspects is derived either from a natural or from a synthetic source. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154). The transmembrane domain can also be synthetic.
In some aspects, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
The CAR generally includes at least one intracellular signaling component or components. First generation CARs typically had the intracellular domain from the CD3 z-chain, which is the primary transmitter of signals from endogenous TCRs. Second generation CARs typically further comprise intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41 BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. Preclinical studies indicated that the second generation improves the antitumor activity of T cells. More recently, third generation CARs combine multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to augment potency.
For example, the CAR can include an intracellular component of the TCR complex, such as a TCR CD3+ chain that mediates T-cell activation and cytotoxicity, e.g., the CD3 zeta chain. Thus, in some aspects, the antigen binding molecule is linked to one or more cell signaling modules. In some aspects, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. The CAR can also further include a portion of one or more additional molecules such as Fc receptor y, CD8, CD4, CD25, or CD 16.
In some aspects, 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 nonengineered immune cell (typically a T cell or a NK cell). For example, the CAR can induce a function of a T cell such as cytolytic activity or T-helper activity, secretion of cytokines or other factors, or the CAR induce a function of a NK cell such as IFNy, IL15, granzyme B molecules production and lytic activity associated with upregulation of FASL, TRAIL, CD107, NKG7 expression and/or antigen-dependent cellular cytotoxicity (ADCC) mediated by FcR.
In some aspects, the intracellular signaling domain(s) include the cytoplasmic sequences of the T cell receptor (TCR) or of the NK cell receptor (NKCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capability. T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). NK cell activation is in some aspects described as being mediated by IFNy, IL15, granzyme B molecules production and lytic activity associated with upregulation of FASL, TRAIL, CD107, NKG7 expression and/or antigendependent cellular cytotoxicity (ADCC) mediated by FcR.
In some aspects, the CAR includes one or both of such signaling components.
In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the T or NK CR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine -based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling 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 aspects, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
The CAR can also include a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1 BB, 0X40, DAP10, and ICOS. In some aspects, the same CAR includes both the activating and costimulatory components; alternatively, the activating domain is provided by one CAR whereas the costimulatory component is provided by another CAR recognizing another antigen.
In some aspects, the CAR is a CD19 BBz CAR, as typically known in the literature. Typically such CAR comprises the following construct: scFv antiCD19 (FMC63)-CD8 hinge and transmembrane-CD3z intracellular. Optionally the construct comprises a CD8 signal Peptide, as follow: CD8 signal Peptide-scFv anti-CD19 (FMC63)-CD8 hinge and transmembrane -CD3z intracellular.
The CAR or other antigen receptor can also be an inhibitory CAR (e.g. iCAR) and includes intracellular components that dampen 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, 0X2R, TIM-3, TIGIT, LAIR-1, PGE2 receptors, EP2/4 Adenosine receptors including A2AR. In some aspects, the engineered cell includes an inhibitory CAR including a signaling domain of or derived from such an inhibitory molecule, such that it serves to dampen the response of the immune system. Such CARs are used, for example, to reduce the likelihood of off-target effects when the antigen recognized by the activating receptor, e.g., CAR, is also expressed, or may also be expressed, on the surface of normal cells. TCRs
In some aspects, the genetically engineered antigen receptors include recombinant T cell receptors (TCRs) and/or TCRs cloned from naturally occurring T cells.
A “T cell receptor” or “TCR” refers to a molecule that contains a variable a and b chains (also known as TCRa and TCRp, respectively) or a variable g and d chains (also known as TCRy and TCR5, respectively) and that is capable of specifically binding to an antigen peptide bound to a MHC receptor. In some aspects, the TCR is in the ab form. Typically, TCRs that exist in ab and gd forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. A TCR can be found on the surface of a cell or in soluble form. Generally, a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some aspects, a TCR also can contain a constant domain, a transmembrane domain and/or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3 rd Ed., Current Biology Publications, p. 4:33, 1997). For example, in some aspects, each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end. In some aspects, a TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Unless otherwise stated, the term “TCR” should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full-length TCRs, including TCRs in the ab form or gd form.
Thus, for purposes herein, reference to a TCR includes any TCR or functional fragment thereof, such as an antigen-binding portion of a TCR that binds to a specific antigenic peptide bound in an MHC molecule, i.e., MHC -peptide complex. An “antigen-binding portion” or “antigen-binding fragment” of a TCR, which can be used interchangeably, refers to a molecule that contains a portion of the structural domains of a TCR, but that binds the antigen (e.g. MHC -peptide complex) to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable a chain and variable b chain of a TCR, sufficient to form a binding site for binding to a specific MHC -peptide complex, such as generally where each chain contains three complementarity determining regions.
In some aspects, the variable domains of the TCR chains associate to form loops, or complementarity determining regions (CDRs) analogous to immunoglobulins, which confer antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule and determine peptide specificity. Typically, like immunoglobulins, the CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., Pwc. Nat'IAcad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Eefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some aspects, CDR3 is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the beta chain interacts with the C-terminal part of the peptide. CDR2 is thought to recognize the MHC molecule. In some aspects, the variable region of the b-chain can contain a further hypervariability (HV4) region.
In some aspects, the TCR chains contain a constant domain. For example, like immunoglobulins, the extracellular portion of TCR chains (e.g., a-chain, b-chain) can contain two immunoglobulin domains, a variable domain (e.g., Va or Vp; typically amino acids 1 to 116 based on Kabat numbering Kabat et al., “Sequences of Proteins of Immunological Interest”, US Dept. Health and Fluman 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 to 259 based on Kabat, b-chain constant domain or Cp, typically amino acids 117 to 295 based on Kabat) adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the 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 connecting sequences in which a cysteine residue forms a disulfide bond, making a link between the two chains. In some aspects, a TCR may have an additional cysteine residue in each of the a and b chains such that the TCR contains two disulfide bonds in the constant domains.
In some aspects, the TCR chains can contain a transmembrane domain. In some aspects, the transmembrane domain is positively charged. In some cases, the TCR chains contain a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules like CD3. For example, a TCR containing constant domains with a transmembrane region can anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex.
Generally, CD3 is a multi-protein complex that can possess three distinct chains (g, d, and e) in mammals and the z-chain. For example, in mammals, the complex can contain a CD3y chain, a CD35 chain, two CD3s chains, and a homodimer of Ou3z chains. The CD3y, CD35, and CD3s chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3y, CD35, and CD3s chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of the CD3y, CD35, and CD3s chains each contain a single conserved motif known as an immunoreceptor tyrosine -based activation motif or ITAM, whereas each Ou3z chain has three. Generally, ITAMs are involved in the signaling capacity of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in propagating the signal from the TCR into the cell. The CD3- and z-chains, together with the TCR, form what is known as the T cell receptor complex.
In some aspects, the TCR may be a heterodimer of two chains a and b (or optionally Y and d) or it may be a single chain TCR construct. In some aspects, the TCR is a heterodimer containing two separate chains (a and b chains or g and d chains) that are linked, such as by a disulfide bond or disulfide bonds.
Recombinant HLA-independent (or non-HLA restricted) T cell receptors (referred to as “HI-TCRs”) that bind to an antigen of interest in an HLA-independent manner are described in International Application No. WO 2019/157454. Such HI-TCRs comprise an antigen binding chain that comprises: (a) an antigen-binding domain that binds to an antigen in an HLA-independent manner, for example, an antigen-binding fragment of an immunoglobulin variable region; and (b) a constant domain that is capable of associating with (and consequently activating) a CD3z polypeptide. Because typically TCRs bind antigen in a HLA-dependent manner, the antigen-binding domain that binds in an HLA- independent manner must be heterologous. Preferably, the antigen-binding domain or fragment thereof comprises: (i) a heavy chain variable region (VH) of an antibody and/or (ii) a light chain variable region (VL) of an antibody. The constant domain of the TCR is, for example, a native or modified TRAC polypeptide, or a native or modified TRBC polypeptide. The constant domain of the TCR is, for example, a native TCR constant domain (alpha or beta) or a fragment thereof. Unlike chimeric antigen receptors, which typically themselves comprise an intracellular signaling domain, the HI-TCR does not directly produce an activating signal; instead, the antigen-binding chain associates with and consequently activates a Ou3z polypeptide. The immune cells comprising the recombinant TCR provide superior activity when the antigen has a low density on the cell surface of less than about 10,000 molecules per cell.
The Oi)3z polypeptide is, for example, a native Ou3z polypeptide or a modified Ou3z polypeptide. The Ou3z polypeptide is optionally fused to an intracellular domain of a co-stimulatory molecule or a fragment thereof. Alternatively, the antigen binding domain optionally comprises a co-stimulatory region, e.g. intracellular domain, that is capable of stimulating an immunoresponsive cell upon the binding of the antigen binding chain to the antigen. Example co-stimulatory molecules include CD28, 4-1 BB, 0X40, ICOS, DAP- 10, fragments thereof, or a combination thereof. In some aspects, the recombinant HI-TCR is expressed by a transgene that is integrated at an endogenous gene locus of the immunoresponsive cell, for example, a CD3b locus, a CD3s locus, a CD247 locus, a B2M locus, a TRAC locus, a TRBC locus, a TRDC locus and/or a TRGC locus. In most aspects, expression of the recombinant HI-TCR is driven from the endogenous TRAC or TRBC gene locus. In some aspects, the transgene encoding a portion of the recombinant HI-TCR is integrated into the endogenous TRAC and/or TRBC locus in a manner that disrupts or abolishes the endogenous expression of a TCR comprising a native TCR a chain and/or a native TCR b chain. This disruption prevents or eliminates mispairing between the recombinant TCR and a native TCR a chain and/or a native TCR b chain in the immunoresponsive cell. The endogenous gene locus may also comprise a modified transcription terminator region, for example, a TK transcription terminator, a GCSF transcription terminator, a TCRA transcription terminator, an HBB transcription terminator, a bovine growth hormone transcription terminator, an SV40 transcription terminator, and a P2A element.
The recombinant HI-TCR may be further combined with other features in a immune cell of the present invention. For example, the immune cell is a cell wherein the antigen-specific receptor is a modified TCR comprising a heterologous antigen-binding domain and a native TCR constant domain or fragment thereof, and the antigen-specific receptor is capable of activating a CD3 zeta polypeptide. For example, the immune cell may further comprise at least one chimeric costimulatory receptor (CCR) and/or at least one chimeric antigen receptor. Furthermore, in the immune cells, the nucleic acid encoding the antigen-binding domain of the HI-TCR may be inserted into the endogenous TRAC locus and/or TRBC locus of the immune cell. The insertion of the HI-TCR nucleic acid sequence, or another smaller mutation, can disrupt or abolish the endogenous expression of a TCR comprising a native TCR alpha chain and/or a native TCR beta chain. The insertion or mutation may reduce endogenous TCR expression by at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. Because a single gene encodes the alpha chain (TRAC) rather than the two genes encoding the beta chain, the TRAC locus is a typical target for reducing TCRap receptor expression. Thus, the nucleic acid encoding the antigen-specific receptor (e.g., CAR or TCR) may be integrated into the TRAC locus at a location, preferably in the 5’ region of the first exon, that significantly reduces expression of a functional TCR alpha chain. See, e.g., Jantz et al., WO2017/062451; Sadelain et al., WO2017/180989; Torikai et al.,. Blood, 119(2): 5697-705 (2012); Eyquem et al., Nature. 2017 Mar 2;543(7643): 113-117. Expression of the endogenous TCR alpha may be reduced by at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In such aspects, expression of the nucleic acid encoding the antigen-specific receptor is optionally under control of the endogenous TCR-alpha or endogenous TCR-beta promoter.
Optionally, the immune cell may also comprise a modified CD3 with a single active ITAM domain, and optionally the CD3 may further comprise one or more or two or more costimulatory domains. In some aspects, the CD3 comprises two costimulatory domains, optionally CD28 and 4-1 BB. The modified CD3 with a single active ITAM domain can comprise, for example, a modified CD3zeta intracellular signaling domain in which ITAM2 and ITAM3 have been inactivated, or ITAM1 and ITAM2 have been inactivated. In some aspects, a modified CD3 zeta polypeptide retains only IT AMI and the remaining CD3z domain is deleted (residues 90-164). As another example, ITAM1 is substituted with the amino acid sequence of ITAM3, and the remaining Ou3z domain is deleted (residues 90-164).
The modified immune cells of the present invention may thus further comprise combinations of two or more, or three or more, or four or more, of the foregoing aspects.
For example, the modified immune cell is an immune cell wherein (a) the antigen-specific receptor is a modified TCR comprising a heterologous antigen-binding domain and a native TCR constant domain or fragment thereof, and the antigen-specific receptor is capable of activating a CD3 zeta polypeptide, and/or the antigen-specific receptor is a CAR, and optionally (b) the immune cell comprises a modified CD3 with a single active ITAM domain, e.g. in which ITAM2 and ITAM3 have been inactivated, and optionally (c) the TCR is under control of an endogenous TRAC and/or TRBC promoter, and optionally (d) expression of native TCR-alpha chain and/or native TCR-beta chain are disrupted or abolished. In further aspects, the cell may comprise at least one chimeric costimulatory receptor (CCR).
Exemplary antigen receptors, including CARs and recombinant TCRs, as well as methods for engineering and introducing the receptors into cells, include those described, for example, in international patent application publication numbers W02000/14257, WO2013/126726, WO2012/129514, WO201/4031687, WO2013/166321, WO2013/071154, WO2013/123061, U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Patent Nos.: 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2,537,416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the genetically engineered antigen receptors include a CAR as described in U.S. Patent No.: 7,446,190, and those described in International Patent Application Publication No.: WO/2014055668 Al.
Antigens
Among the antigens targeted by the genetically engineered antigen receptors are those expressed in the context of a disease, condition, or cell type to be targeted via the adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, more particularly cancers, thus in some aspects, the one or more antigens are selected from tumor antigen (e.g., expressed by tumor cells, notably specifically expressed by cancer cells).
The “cancer” may be a solid cancer or a “liquid tumor” such as cancers affecting the blood, bone marrow and lymphoid system, also known as tumors of the hematopoietic and lymphoid tissues, which notably include leukemia and lymphoma. Liquid tumors include for example acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), in particular ALL from children and young adults or pediatric ALL, and chronic lymphocytic leukemia (CLL), (including various lymphomas such as mantle cell lymphoma, non-Hodgkins lymphoma (NHL), adenoma, squamous cell carcinoma, laryngeal carcinoma, gallbladder and bile duct cancers, cancers of the retina such as retinoblastoma).
Solid cancers notably include cancers affecting one of the organs selected from the group consisting of colon, rectum, skin, endometrium, lung (including non-small cell lung carcinoma or “NSCLC”), uterus, bones (such as Osteosarcoma, Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors, Adamantinomas, and Chordomas), liver, kidney, esophagus, stomach, bladder, pancreas, cervix, brain (such as Meningiomas, Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, Pituitary Tumors, Schwannomas, and Metastatic brain cancers), ovary, breast, head and neck region, testis, prostate and the thyroid gland.
In a particular aspect, the solid cancer according to the invention is selected from lung cancer (in particular NSCLC), breast cancer, ovary cancer, prostate cancer, bladder cancer and brain cancer (in particular glioblastoma). Preferably, a solid cancer according to the invention is selected from lung cancer (in particular NSCLC), breast cancer and bladder cancer. According to another preferred aspect, the cancer is a cancer in which a lack of infiltration of the tumor by (tumor-infiltrating) immune cells (TILs), including immune T cells, is observed, in particular a pediatric cancer.
The pediatric cancer may be a neuroblastoma, Diffuse Intrinsic Pontine Glioma (DIPG, also identified as Diffuse midline glioma), high-grade glioma, or Acute lymphoblastic Leukemia (ALL).
Preferably, a cancer according to the invention is a cancer affecting the blood, bone marrow and lymphoid system as described above. Typically, the cancer is, or is associated, with a leukemia, in particular the B Cell Acute Lymphoblastic Leukemia (B-ALL) from children and young adults.
Diseases according to the invention also encompass 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, e.g., rheumatoid arthritis (RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease multiple sclerosis, asthma, and/or diseases or conditions associated with transplant.
In some aspects, the antigen is a polypeptide. In some aspects, it is a carbohydrate or other molecule. In some aspects, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other aspects, the antigen is expressed on normal cells and/or is expressed on the engineered cells. In some such aspects, a multi-targeting and/or gene disruption approach is used to improve specificity and/or efficacy.
In some aspects, the antigen, is expressed by a cancer cell and/or is a universal tumor antigen.
The term “universal tumor antigen” refers to an immunogenic molecule, such as a protein, that is, generally, expressed at a higher level in tumor cells than in non-tumor cells and also that is expressed in tumors of different origins. In some aspects, 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 aspects, the 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 types of tumors. In some cases, the 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 at all in non-tumor cells, such as not expressed in normal cells. Exemplary universal tumor antigens include, for example, human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1 Bl (CYP1 B), HER2/neu, Wilms' tumor gene 1 (WT 1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53 or cyclin (DI). 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 e.g. published PCT application No. WO2011/009173 or WO2012/135854 and published U.S. application No. US2014/0065708).
In some aspects, the antigen (such as CD19, CD22, CD20) is expressed on B Cell Acute Lymphoblastic Leukemia. Antibodies or antigen-binding fragments directed against such antigens are known by the skilled person. In some aspects, such antibodies or antigen-binding fragments thereof (e.g. scFv) can be used to generate a CAR.
In some aspects, the antigen may be one that is expressed or upregulated on cancer or tumor cells, but that also may be expressed in an immune cell, such as a resting or activated T cell. For example, in some cases, expression of hTERT, survivin and other universal tumor antigens are reported to be present in lymphocytes, including activated T lymphocytes (see e.g., Weng et al. (1996) J Exp. Med., 183:2471-2479; Flathcock 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). Likewise, in some cases, CD38 and other tumor antigens also can be expressed in immune cells, such as T cells, in particular upregulated in activated T cells. For example, in some aspects, CD38 is a known T cell activation marker.
In some aspects as provided herein, an immune cell, such as a T cell, macrophage or NK cell, can be engineered to repress or disrupt the gene encoding the antigen in the immune cell so that the expressed genetically engineered antigen receptor does not specifically bind the antigen in the context of its expression on the immune cell itself. Thus, in some aspects, this may avoid off-target effects, such as binding of the engineered immune cells to themselves, which may reduce the efficacy of the engineered in the immune cells, for example, in connection with adoptive cell therapy.
In some aspects, such as in the case of an inhibitory CAR, the target is an off-target marker, such as an antigen not expressed on the diseased cell or cell to be targeted, but that is expressed on a normal or nondiseased cell which also expresses a disease-specific target being targeted by an activating or stimulatory receptor in the same engineered cell. Examples of such antigens are MHC molecules, such as MHC class I molecules, for example, in connection with treating diseases or conditions in which such molecules become downregulated but remain expressed in non-targeted cells.
In some aspects, the engineered immune cells can contain an antigen that targets one or more other antigens. In some aspects, the one or more other antigens is a tumor antigen or cancer marker. Other antigen targeted by antigen receptors on the provided immune cells can, in some aspects, include orphan tyrosine kinase receptor ROR1, tEGFR, Her2, LLCAM, CD19, CD20, CD22, mesothelin, CEA, hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPFIa2, ErbB2, 3, or 4, FBP, fetal acethycholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R- alpha2, kdr, kappa light chain, Lewis Y, Ll-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY- ESO-1, MART-1, gplOO, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD 123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1 ), a cyclin such as cyclin Al (CCNA1 ), biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens.
For example, the one or more antigens can be selected from tumor antigens from the group comprising pHER95, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD70, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, Erb- B2, Erb-B3, Erb-B4, FBP, Fetal acetylcholine receptor, folate receptor-a, GD2, GD3, FIER-2, hTERT, IL- 13R-a2, k-light chain, KDR, LeY, LI cell adhesion molecule, MAGE-A1, Mesothelin, MAGEA3, p53, MARTI, GP100, Proteinase3 (PR1 ), Tyrosinase, Survivin, hTERT, EphA2, NKG2D ligands, NY-ESO-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1 R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, LILRB4, PRAME, and ERBB. In some aspects, the CAR binds a pathogen-specific antigen. In some aspects, the CAR is specific for viral antigens (such as HIV, FICV, FIBV, etc.), bacterial antigens, and/or parasitic antigens.
In some aspects, the CAR includes encompasses one or more 4-1 BB co stimulatory domain and binds a CD19 antigen (also known as 19BBz CAR in the literature).
In some aspects, the cells of the invention are genetically engineered to express two or more genetically engineered receptors on the cell, each recognizing a different antigen and typically each including a different intracellular signaling component. Such multi-targeting strategies are described, for example, in International Patent Application, Publication No.: WO2014/055668 Al (describing combinations of activating and costimulatory CARs, e.g., targeting two different antigens present individually on off- target, e.g., normal cells, but present together only on cells of the disease or condition to be treated) and Fedorov et al. (Sci. Transl. Medicine, 5(215), December, 2013) describing cells expressing an activating and an inhibitory CAR, such as those in which the activating CAR binds to one antigen expressed on both normal or non-diseased cells and cells of the disease or condition to be treated, and the inhibitory CAR binds to another antigen expressed only on the normal cells or cells which it is not desired to treat.
In some contexts, overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) may be toxic to a subject. Thus, in some contexts, the engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo, such as upon administration in adoptive immunotherapy. For example in some aspects, the cells are engineered so that they can be eliminated as a result of a change in the in vivo condition of the patient to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al. , Cell II :223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and the bacterial cytosine deaminase gene (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).
In other aspects of the invention, the immune cells, e.g., T cells, are not engineered to express recombinant receptors, but rather include naturally occurring antigen receptors specific for desired antigens, such as tumor-infiltrating lymphocytes and/or T cells cultured in vitro or ex vivo, e.g., during the incubation step(s), to promote expansion of cells having particular antigen specificity. For example, in some aspects, the cells are produced for adoptive cell therapy by isolation of tumor-specific T cells, e.g. autologous tumor infiltrating lymphocytes (TILs). The 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 aspects, lymphocytes are extracted from resected tumors. In some aspects, such lymphocytes are expanded in vitro. In some aspects, such lymphocytes are cultured with lymphokines (e.g., IL-2). In some aspects, such lymphocytes mediate specific lysis of autologous tumor cells but not allogeneic tumor or autologous normal cells.
Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by 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 the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., US Patent No. 6,040,177, at columns 14-17.
Method for obtaining cells according to the invention
The present invention also relates to a method of producing a modified or engineered immune cell, comprising a step consisting of repressing, typically inhibiting, the expression and/or activity of ZNL217 and/or for example SOCS1 and/or SOCS3 and/or CISH in the immune cell.
Preferably, the method for obtaining engineered immune cells according to the invention further comprises a step consisting of introducing into said immune cells a genetically engineered antigen receptor that specifically binds to a target antigen, a T cell receptor, a macrophage receptor, or a NK cell receptor.
The repression, typically inhibition, of the expression and/or activity of ZNE217 (and in some embodiments the additional inhibition of the expression and/or activity of for example SOCS1 and/or SOCS3 and/or CISH) and the introduction of a genetically engineered antigen receptor that specifically binds to a target antigen in the immune cell can be carried out simultaneously or sequentially in any order.
Inhibition of ZNF217, and optionally e.g., SOCS1 and/or SOCS3 and/or CISH
The methods as herein described for inhibition of the gene expression or of the activity of the protein apply to the 4 genes/proteins of interest, namely ZNE217 and optionally for example SOCS1, SOCS3 and/or CISH. When the cell is defective for more than ZNL217, the same of different method(s) can be used to render the cell further defective for, for example, SOCS1, SOCS3 and/or CISH. Embodiments as described herein can therefore be combined according to the skilled person knowledge.
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 ZNF217 and optionally in some embodiments with at least one additional agent that inhibits or blocks the expression and/or activity of for example SOCS1, SOCS3 and/or CISH. The present invention also provides aspects wherein another protein such as FAS, is inactivated in the immune cell (notably cells).
Said inhibiting agent can be selected from a nucleic acid molecule (that block transcription or translation); a peptide; a small molecule; an antibody, a derivative (such as intrabodies, nanobodies or affibodies) or any functional fragment thereof; an aptamer; a ribozyme; a gene editing agent, and any combination thereof. The nucleic acid molecule that block transcription or translation may be for example an antisense molecule complementary to ZNF217, SOCS1, SOCS3 or CISH; or a RNA interfering agent such as a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwiRNA (piRNA). The at least one inhibiting agent can also be a gene editing agent in the form of an exogenous nucleic acid (such as a sgZNF217 as described herein in the experimental section - cf. Table 1 in particular) or genetic tool or complex comprising a) one or more engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) guide RNA that hybridize withZNF217, SOCS1, SOCS3 or CISH genomic nucleic acid sequence and/or b) a nucleic acid sequence encoding a CRISPR protein (typically a Type-IIS Cas9 protein), optionally wherein the cells are transgenic for expressing a Cas9 protein.
The agent may also be a Zinc finger protein (ZFN) or a TAE protein.
The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. A small molecule may be, e.g., a peptide, amino acid, nucleotide, lipid, carbohydrate, or any other organic or inorganic molecule.
In some aspects, the small molecule ZNF217 inhibitor is triciribine, triciribine phosphate (TCN-P), triciribine 5'-phosphate (TCN-P), and the DMF adduct of triciribine (TCN-DMF). See, e.g., U.S. Patent No. 6,413,944. TCN may be synthesized as described in Tetrahedron Fetters, vol. 49, pp. 4757-4760 (1971), which is incorporated herein by reference. TCN-P may be prepared as described in U. S. Pat. No. 4,123, 524, which is incorporated herein by reference. TCN-DMF is described in INSERM, vol. 81, pp. 37-82 (1978). In some aspects, the small molecule is selected from the following list or analogs thereof : bis (2- Nitrophenyl) sulfilimine (NSC number 645984); 3-(4-Fluorophenyl)-3- (4-hydroxy-2-methylphenyl) phthalide (NSC number 682335); (5H-Benzocyclohepten-5-one, 4- (acetyloxy)-6, 6-dibro) (NSC number 624771); and N, N-dimethyl-3- ( (4- pyridinylmethyl) imino)-3H-l, 2,4-dithiazol-5-amine hydrobromide (NSC number 661112). These molecules are from the National Cancer Institute chemical depository.
In some aspects, the small molecule ZNF217 inhibitor is a carbonyl cyanide 3-chlorophenyhydrazone, for example the small molecule identified by Zemaitis et al. (2023) as UM171 (CAS ref: 1448724-09-1).
Inhibition of ZNF217 and/or of a protein of the SOCS family, for example SOCS1, SOCS3 and/or CISH in the cell can be achieved before or after injection in the targeted patient. In some aspects, inhibition as previously defined is performed in vivo after administration of the cell to the subject. For example, a ZNF217 inhibitor as herein defined can be included in the composition containing the cell. One or more SOCS1, SOCS3 or CISH inhibitor(s) for example may also be administered separately before, concomitantly of after administration of the cell(s) to the subject.
Typically, inhibition of ZNF217, and/or for example of SOCS1, SOCS3 and/or CISH according to the present description may be achieved with incubation of a cell according to the invention with a composition containing at least one pharmacological inhibitor as previously described. The inhibitor is included during the expansion of the anti-tumor T cells, macrophages or NK cells, in vitro, thus modifying their reconstitution, survival and therapeutic efficacy after adoptive transfer.
Inhibition of ZNF217, and/or for example of SOCS1, SOCS 3 and/or CISH in a cell according to the invention may be achieved with intrabodies. “Intrabodies” are antibodies that bind intracellularly to their antigen after being produced in the same cell (for a review see for example, Marschall AL, DCibel S and Boldicke T “Specific in vivo knockdown of protein function by intrabodies”, MAbs. 2015;7(6): 1010-35. but 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; Flyland S, Beerli RR, Barbas CF, Flynes NE, Weis 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, Tramontane A, Benvenuto E. “Engineering stable cytoplasmic intrabodies with designed specificity”. J Mol Biol. 2003 Jul 4;330(2):323-32.).
Intrabodies can be generated by cloning the respective cDNA from an existing hybridoma clone or more conveniently, new scFvs/Fabs can be selected from in vitro display techniques such as phage display which provide the necessary gene encoding the antibody from the onset and allow a more detailed predesign of antibody fine specificity. In addition, bacterial-, yeast-, mammalian cell surface display and ribosome display can be employed. However, the most commonly used in vitro display system for selection of specific antibodies is phage display. In a procedure called panning (affinity selection), recombinant antibody phages are selected by incubation of the antibody phage repertoire with the antigen. This process is repeated several times leading to enriched antibody repertoires comprising specific antigen binders to almost any possible target. To date, in vitro assembled recombinant human antibody libraries have already yielded thousands of novel recombinant antibody fragments. It is to be noted that the prerequisite for a specific protein knockdown by a cytoplasmic intrabody is that the antigen is neutralized/inactivated through the antibody binding. Five different approaches to generate suitable antibodies have emerged : 1) in vivo selection of functional intrabodies in eukaryotes such as yeast and in prokaryotes such as E.coli (antigendependent and independent); 2) generation of antibody fusion proteins for improving cytosolic stability; 3) use of special frameworks for improving cytosolic stability (e.g., by grafting CDRs or introduction of synthetic CDRs in stable antibody frameworks); 4) use of single domain antibodies for improved cytosolic stability; and 5) selection of disulfide bond free stable intrabodies. Those approaches are notably detailed in Marschall, A. L et al., mAbs 2015 as mentioned above.
The most commonly used format for intrabodies is the scFv, which consists of the H- and L-chain variable antibody domain (VH and VL) held together by a short, flexible linker sequence (frequently (Gly4Ser)3), to avoid the need for separate expression and assembly of the 2 antibody chains of a full IgG or Fab molecule. Alternatively, the Fab format comprising additionally the Cl domain of the heavy chain and the constant region of the light chain has been used. Recently, a new possible format for intrabodies, the scFab, has been described. The scFab format promises easier subcloning of available Fab genes into the intracellular expression vector, but it remains to be seen whether this provides any advantage over the well- established scFv format. In addition to scFv and Fab, bispecific formats have been used as intrabodies. A bispecific Tie-2 x VEGFR-2 antibody targeted to the ER demonstrated an extended half-life compared to the monospecific antibody counterparts. A bispecific transmembrane intrabody has been developed as a special format to simultaneously recognize intra- and extracellular epitopes of the epidermal growth factor, combining the distinct features of the related monospecific antibodies, i.e., inhibition of autophosphorylation and ligand binding. Another intrabody format particularly suitable for cytoplasmic expression are single domain antibodies (also called nanobodies) derived from camels or consisting of one human VH domain or human VL domain. These single domain antibodies often have advantageous properties, e.g., high stability; good solubility; ease of library cloning and selection; high expression yield in E.coli and yeast.
The intrabody gene can be expressed inside the target cell after transfection with an expression plasmid or viral transduction with a recombinant virus. Typically, the choice is aimed at providing optimal intrabody transfection and production levels. Successful transfection and subsequent intrabody production can be analyzed by immunoblot detection of the produced antibody, but, for the evaluation of correct intrabody/antigen-interaction, co-immunoprecipitation from HEK 293 cell extracts transiently cotransfected with the corresponding antigen and intrabody expression plasmids may be used. Inhibition of ZNF217 and/or for example SOCS1 and/or SOCS3 and/or CISH in a cell according to the invention may also be effected with aptamers that repress, i.e., inhibit or block ZNF217, SOCS1, SOCS3 or CISH expression or activity respectively. “Aptamers” are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide (DNA or RNA) or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
Oligonucleotide aptamers may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L, 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999.
Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by 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).
Inhibition of ZNF217, and for example of SOCS1, SOCS3 and/or CISH in a cell according to the invention may also be effected with affibody molecules. “Affibody” are small proteins engineered to bind to a large number of target proteins or peptides with high affinity, imitating monoclonal antibodies, and are therefore a member of the family of antibody mimetics (see for review Lofblom J, Feldwisch J, Tolmachev V, Carlsson J, Stahl S, Frejd FY. Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett. 2010 Jun 18;584(12):2670-80). Affibody molecules are based on an engineered variant (the Z domain) of the B -domain in the immunoglobulin-binding regions of staphylococcal protein A, with specific binding for theoretically any given target. Affibody molecule libraries are generally constructed by combinatorial randomization of 13 amino acid positions in helices one and two that comprise the original Fc-binding surface of the Z-domain. The libraries have typically been displayed on phages, followed by biopanning against desired targets. Should the affinity of the primary be increased, affinity maturation generally results in improved binders and may be achieved by either helix shuffling or sequence alignment combined with directed combinatorial mutagenesis. The newly identified molecules with their altered binding surface generally keep the original helical structure as well as the high stability, although unique exceptions with interesting properties have been reported. Due to their small size and rapid folding properties, affibody molecules can be produced by chemical peptide synthesis.
In other aspects of the invention, inhibition of ZNF217 and/or for example of SOCS1 and/or SOCS3 and/or CISH activity can be achieved by gene repression/suppression via gene knockdown or knock-out using an RNA or DNA, notably a recombinant DNA or RNA, typically using RNA interference (RNAi) such as dsRNA (double-stranded RNA), miRNA (microRNA), short interfering RNA (siRNA) short hairpin RNA (shRNA)n anti-sens RNA or DNA or sequences encoding ribozymes. For the purposes of the invention, the term “recombinant DNA or RNA” refers to a nucleic acid sequence that has been altered, rearranged, or modified by genetic engineering. The term “recombinant” does not refer to alterations of nucleic acid sequences that result from naturally occurring events, such as spontaneous mutations, or from non- spontaneous mutagenesis followed by selective breeding.
As used herein, the term “RNA” refers to a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” is meant a nucleotide with a hydroxyl group at the 2’ position of a beta.-D-ribofuranose moiety. The terms encompass double stranded RNA, single stranded RNA, RNAs with both double stranded and single stranded regions, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA, or analog RNA, that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of an RNA molecule or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the presently disclosed subject matter can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of a naturally occurring RNA. siRNA technology includes that based on RNAi utilizing a double-stranded RNA molecule having a sequence homologous with the nucleotide sequence of mRNA which is transcribed from the gene, and a sequence complementary with the nucleotide sequence. siRNA generally is homologous/complementary with one region of mRNA which is transcribed from the gene, or may be siRNA including a plurality of RNA molecules which are homologous/complementary with different regions.
In certain aspects herein described, the protein activity is downregulated, or entirely inhibited, by the use of an antisense polynucleotide, including anti-sense RNA molecules and anti-sense DNA molecules.
In a particular aspect, the antisense polynucleotide is a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence or a subsequence thereof. Binding of the antisense polynucleotide to the protein mRNA reduces the translation and/or stability of the protein mRNA. Antisense polynucleotides can comprise naturally-occurring nucleotides, or synthetic species formed from naturally-occurring subunits or their close homologs. Antisense polynucleotides can also have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species which are known for use in the art. All such analogs are comprehended by this invention so long as they function effectively to hybridize with the protein mRNA. Such antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those of skill in the art.
Anti-sense oligonucleotides would act to directly block the translation of ZNF217, and/or for example of SOCS1, SOCS3 and/or CISH and thus prevent protein translation or increase mRNA degradation, thus decreasing the level of ZNF217, and/or for example of SOCS1, SOCS3 or CISH respectively and thus its/their activity in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding ZNF217, SOCS1, SOCS3 or CISH can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (see for example U.S. Pat. Nos. 6,566,135; 6,566, 131 ; 6,365,354; 6,410,323; 6,107,091 ; 6,046,321 ; and 5,981 ,732).
An “RNA interfering agent” as used herein, is defined as any agent, which interferes with or inhibits 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, which are homologous to the target gene of the invention (e.g., ZNF217), or a fragment thereof, short interfering RNA (siRNA), and small molecules which interfere with or inhibit expression of the target nucleic acid by RNA interference (RNAi). “Small inhibitory RNAs” (siRNAs) can also function as inhibitors of expression for use according to the present application. Thus, in certain aspects, the protein activity is downregulated, or entirely inhibited, by the use of siRNA. siRNA refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA expressed in the same cell as the gene or target gene. siRNA thus encompasses the double stranded RNA formed by the complementary strands. The complementary portions of the siRNA that hybridize to form the double stranded molecule typically have substantial or complete identity. In one aspect, a siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA. The sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof. Typically, the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferable about preferably about 20-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. siRNAs can be introduced into animals according to any methods, including those of, e.g., U.S. Applications 2002/0132788 and 2002/0173478.
ZNF217 gene expression, and for example SOCS1 expression, SOCS3 expression, and/or CISH gene expression, can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that ZNF217 gene expression, SOCS1 gene expression, SOCS3 gene expression, or CISH gene expression 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 sequence is known (see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Flannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WOOl/36646, WO99/32619, and WO01/68836). All or part of the phosphodiester bonds of the siRNAs of the invention are advantageously protected. This protection is generally implemented via the chemical route using methods that are known by art. The phosphodiester bonds can be protected, for example, by a thiol or amine functional group or by a phenyl group. The 5'- and/or 3'- ends of the siRNAs of the invention are also advantageously protected, for example, using the technique described above for protecting the phosphodiester bonds. The siRNAs sequences advantageously comprise at least twelve contiguous dinucleotides or their derivatives.
Short hairpin RNAs (“shRNAs”) can also function as inhibitors of expression for use in the present invention. shRNAs are typically composed of a short (e.g., 19-25 nucleotide) antisense strand, followed by a 5-9 nucleotide loop, and the analogous sense strand. Alternatively, the sense strand may precede the nucleotide loop structure and the antisense strand may follow.
As used herein, the term “microRNA” (“miRNA”) refers to a single-stranded RNA molecule of 21 to 23 nucleotides in length, preferably 21 to 22 nucleotides, which is capable of regulating gene expression. The miRNAs are each processed from a longer precursor RNA molecule (“precursor miRNA”). Precursor miRNAs are transcribed from non-protein-encoding genes. The precursor miRNAs have two regions of complementarity that enable them to form a stem-loop- or fold-back-like structure. The processed miRNA (also referred to as “mature miRNA”) becomes part of a large complex to down-regulate a particular target gene.
An example of a particular miRNA usable in the context of the present invention is MIR503.
In some aspects, a recombinant DNA as herein described is a recombinant DNA encoding a ribozyme. Ribozymes can also function as inhibitors of expression for use in the present invention. “Ribozymes” are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of H3K9-histone methyltransferase SUV39H1 mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences: GUA, GUU and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNAse P, and axhead ribozymes (see, e.g., Castanotto et al., 1994, Adv. in Pharmacology 25: 289-317 for a general review of the properties of different ribozymes).
Both antisense oligonucleotides and ribozymes useful as inhibitors of expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5’ and/or 3’ ends of the molecule, or the use of phosphorothioate or 2’-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
Antisense oligonucleotides, siRNAs, shRNAs, miRNA, and ribozymes herein described may be delivered in vivo alone or in association with a vector. In its broadest sense, a “vector” is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells, preferably to the cells expressing ZNF217 or expressing ZNF217, and for example SOCS1, SOCS3 and/or CISH. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and R A virus such as a retrovirus. One can readily employ other vectors not named but known to the art.
Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell line with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990, and in Murry, 1991.
Preferred viruses for certain applications are the adenoviruses and adeno-associated (AAV) viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. Actually 12 different AAV serotypes (AAV1 to 12) are known, each with different tissue tropisms (Wu, Z Mol Ther 2006; 14:316-27). Recombinant AAVs are derived from the dependent parvovirus AAV2 (Choi, VW J Virol 2005; 79:6801-07). The adeno-associated virus type 1 to 12 can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species (Wu, Z Mol Ther 2006; 14:316- 27). It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.
Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al. , 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a genegun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate delivery vehicles and micro encapsulation. The antisense oligonucleotide, siRNA, shRNA or ribozyme or ribozyme encoding nucleic acid sequences according to the invention are generally under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes, for example, a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable. The promoter can also be, as a matter of example, a viral promoter, such as CMV promoter or any synthetic promoters.
Gene repression or disruption of ZNF217 and/or for example SOCS1 and/or SOCS3 and/or CISH
Inhibition of ZNF217, SOCS1, SOCS3 and/or CISH in a cell according to the invention may also be effected via repression or disruption of the ZNF217 gene, SOCS1 gene, SOCS3 gene, or CISH gene respectively, such as by deletion, e.g., deletion of the entire gene, exon, or region, and/or replacement with an exogenous sequence, and/or by mutation, e.g., frameshift or missense mutation, within the gene, typically within an exon of the gene. In some aspects, the disruption results in a premature stop codon being incorporated into the gene, such that the ZNF217, SOCS1, SOCS3 or CISH protein is not expressed or is non-functional. The disruption is generally carried out at the DNA level. The disruption generally is permanent, irreversible, or not transient. In some aspects, inducible and/or reversible gene inactivation of ZNF217 (and/or SOCS1 and/or SOCS3 and/or CISH) can be favored.
Well-suited method to edit immune cells for cancer immunotherapy according to the present application are notably described in Lucibello F, Menegatti S, Menger L. “Methods to edit T cells for cancer immunotherapy”. Methods Enzymol. 2020;631 :107-135.
In some aspects, the gene disruption or repression is achieved using gene editing agents such as a DNA- targeting molecule, such as a DNA-binding protein or DNA-binding nucleic acid, or complex, compound, or composition, containing the same, which specifically binds to or hybridizes to the gene. In some aspects, 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 (TALE) DNA-binding domain, a clustered regularly interspaced short palindromic repeats (CRISPR) DNA- binding domain, or a DNA-binding domain from a meganuclease. Zinc finger, TALE, and CRISPR system binding domains can be “engineered” to bind to a predetermined nucleotide sequence.
In some aspects, the DNA-targeting molecule, complex, or combination contains a DNA-binding molecule and one or more additional domain(s), such as an effector domain to facilitate the repression or disruption of the gene. For example, in some aspects, the gene disruption is carried out by fusion proteins that comprise DNA-binding proteins and a heterologous regulatory domain or functional fragment thereof. Typically, the additional domain is a nuclease domain. Thus, in some aspects, gene disruption is facilitated by gene or genome editing, using engineered proteins, such as nucleases and nuclease-containing complexes or fusion proteins, composed of sequence-specific DNA-binding domains fused to, or complexed with, non-specific DNA-cleavage molecules such as nucleases. These targeted chimeric nucleases or nuclease-containing complexes carry out precise genetic modifications by inducing targeted double-stranded breaks or singlestranded breaks, stimulating the cellular DNA-repair mechanisms, including error-prone nonhomologous end joining (NHEJ) and homology-directed repair (HDR). In some aspects, 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, U.S. Pat. No. 8,697,359; Sander and Joung (2014) Nat. Biotech. 32:347-355; Hale et al. (2009) Cell 139:945-956; Karginov and Hannon (2010) Mol. Cell 37:7; U.S. Pat. Publ. 2014/0087426 and 2012/0178169; Boch et al. (2011 ) Nat. Biotech. 29: 135-136; Boch et al. (2009) Science 326: 1509-1512; Moscou and Bogdanove (2009) Science 326: 1501 ; Weber et al. (2011 ) PLoS One 6:el9722; Li et al. (2011 ) Nucl. Acids Res. 39:6315-6325; Zhang et al. (201 1 ) Nat. Biotech. 29: 149-153; Miller etal. (2011) Nat. Biotech. 29: 143-148; Lin et al. (2014) Nucl. Acids Res. 42:e47). Such genetic strategies can use constitutive expression systems or inducible expression systems according to well-known methods in the art.
ZFPs and ZFNs; TALs, TALEs, and TALENs
In some aspects, the DNA-targeting molecule includes a DNA-binding protein such as one or more zinc finger protein (ZFP) or transcription activator-like protein (TAL), fused to an effector protein such as an endonuclease. Examples include ZFNs, TALEs, and TALENs. See Lloyd et al., Frontiers in Immunology, 4(221), 1-7 (2013).
In some aspects, the DNA-targeting molecule comprises one or more zinc-finger proteins (ZFPs) or domains thereof that bind to DNA in a sequence-specific manner. A ZFP or domain thereof is a protein or domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. Generally, sequence- specificity of a ZFP may be altered by making amino acid substitutions at the four helix positions (1, 2, 3 and 6) on a zinc finger recognition helix. Thus, in some aspects, the ZFP or ZFP-containing molecule is non-naturally occurring, e.g., is engineered to bind to the target site of choice. 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 aspects, the DNA-targeting molecule is or comprises a zinc-finger DNA binding domain fused to a DNA cleavage domain to form a zinc-finger nuclease (ZFN). In some aspects, fusion proteins comprise the cleavage domain (or cleavage half domain) from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. In some aspects, the cleavage domain is from the Type IIS restriction endonuclease Fok I. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as 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, the ZFNs efficiently generate a double strand break (DSB), for example at a predetermined site in the coding region of the targeted gene (i.e., ZNF217). Typical targeted gene regions include exons, regions encoding N-terminal regions, first exon, second exon, and promoter or enhancer regions. In some aspects, transient expression of the ZFNs promotes highly efficient and permanent disruption of the target gene in the engineered cells. In particular, in some aspects, delivery of the ZFNs results in the permanent disruption of the gene with efficiencies surpassing 50%. Many gene-specific engineered zinc fingers are available commercially. For example, Sangamo Biosciences (Richmond, CA, USA) has developed a platform (CompoZr) for zinc-finger construction in partnership with Sigma-Aldrich (St. Louis, MO, USA), allowing investigators to bypass zinc-finger construction and validation altogether, and provides specifically targeted zinc fingers for thousands of proteins. Gaj etal., Trends in Biotechnology, 2013, 31(7), 397-405. In some aspects, commercially available zinc fingers are used or are custom designed (See, for example, Sigma- Aldrich catalog numbers CSTZFND, CSTZFN, CTI1-1 KT, and PZD0020).
In some aspects, the DNA-targeting molecule comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as in a transcription activator-like protein effector (TALE) protein, See, e.g., U.S. Patent Publication No. 20110301073. In some aspects, the molecule is a DNA binding endonuclease, such as a TALE-nuclease (TALEN). In some aspects, the TALEN is a fusion protein comprising a DNA-binding domain derived from a TALE and a nuclease catalytic domain to cleave a nucleic acid target sequence. In some aspects, the TALE DNA-binding domain has been engineered to bind a target sequence within genes that encode the target antigen and/or the immunosuppressive molecule. For example, in some aspects, the TALE DNA-binding domain may target CD38 and/or an adenosine receptor, such as A2AR.
In some aspects, the TALEN recognizes and cleaves the target sequence in the gene. In some aspects, cleavage of the DNA results in double-stranded breaks. In some aspects, the breaks stimulate the rate of homologous recombination or non-homologous end joining (NHEJ). Generally, NHEJ is an imperfect repair process that often results in changes to the DNA sequence at the site of the cleavage. In some aspects, repair mechanisms involve rejoining of what remains of the two DNA ends through direct re ligation (Critchlow and Jackson, Trends Biochem Sci. 1998 Oct;23(10):394-8) or via the so-called microhomology- mediated end joining. In some aspects, repair via NHEJ results in small insertions or deletions and can be used to disrupt and thereby repress the gene. In some aspects, the modification may be a substitution, deletion, or addition of at least one nucleotide. In some aspects, cells in which a cleavage-induced mutagenesis event, i.e., a mutagenesis event consecutive to an NHEJ event, has occurred can be identified and/or selected by well-known methods in the art.
TALE repeats can be assembled to specifically target the Suv39hl gene (Gaj et al., Trends in Biotechnology, 2013, 31 (7), 397-405). A library of TALENs targeting 18,740 human protein-coding genes has been constructed (Kim et al., Nature Biotechnology. 31 , 251-258 (2013)). Custom-designed TALE arrays are commercially available through Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). Specifically, TALENs that target CD38 are commercially available (See Gencopoeia, catalog numbers HTN222870-1 , HTN222870-2, and HTN222870-3, available on the World Wide Web at www. genecopoeia.com/product/search/detail.php ?prt=26&cid=&key=HTN222870).
Exemplary molecules are described, e.g., in U.S. Patent Publication Nos. US 2014/0120622, and 2013/0315884.
In some aspects, the TALENs are introduced as transgenes encoded by one or more plasmid vectors. In some aspects, the plasmid vector can contain a selection marker which provides for identification and/or selection of cells which received said vector.
RNA-guided endonucleases (CRISPR/Cas systems)
The gene repression can be carried out using one or more DNA-binding nucleic acids, such as disruption via an RNA-guided endonuclease (RGEN), or other form of repression by another RNA-guided effector molecule. For example, in some aspects, the gene repression can be carried out using clustered regularly interspaced short palindromic repeats (CRISPR) 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 of, or directing the activity of, CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.
Typically, the CRISPR/Cas nuclease or CRISPR/Cas nuclease system includes a non-coding (guide) RNA molecule, which sequence specifically binds to DNA, and a CRISPR protein, with nuclease functionality (e.g., two nuclease domains). One or more elements of a CRISPR system can derive from a type I, type II, or type III CRISPR system, such as Cas nuclease. Preferably, the CRISPR protein is a cas enzyme such Cas9. Cas enzymes are well-known in the field; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. In some aspects, a Cas nuclease and gRNA are introduced into the cell. In some aspects, the CRISPR system induces DSBs at the target site, followed by disruptions as discussed herein. In other aspects, Cas9 variants, deemed “nickases” can be used to nick a single strand at the target site. Paired nickases can also be used, e.g., to improve specificity, each directed by a pair of different gRNAs targeting sequences. In still other aspects, catalytically inactive Cas9 can be fused to a heterologous effector domain, such as a transcriptional repressor, to affect gene expression.
In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of the target sequence. Typically, in the context of formation of a CRISPR complex, “target sequence” generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. The target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. Generally, a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an “editing template” or “editing polynucleotide” or “editing sequence”. In some aspects, an exogenous template polynucleotide may be referred to as an editing template. In some aspects, the recombination is homologous recombination.
It is to be noted that in some aspects, catalytically dead CAS 9 (dCas9) can be used in conjunction with activator or repressor domains to control gene expression.
In some aspects, one or more vectors driving expression of one or more elements of the CRISPR system are introduced into the cell such that expression of the elements of the CRISPR system direct formation of the CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. In some aspects, CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation. In some aspects, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter. In some aspects, a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding the CRISPR enzyme, such as a Cas protein.
In some aspects, a CRISPR enzyme in combination with (and optionally complexed with) a guide sequence is delivered to the cell. Typically, CRISPR/Cas9 technology may be used to knockdown or knock-out gene expression of ZNF217 in the engineered cells. For example, Cas9 nuclease and a guide RNA specific to the ZNF217 gene can be introduced into cells, for example, using lentiviral delivery vectors or any of a number of known delivery method or vehicle for transfer to cells, such as any of a number of known methods or vehicles for delivering Cas9 molecules and guide RNAs (see also below).
In some aspects, inducible gene repression system, notably inducible CRISPR gene inactivation, may be favored such as described in Chylinski, K., Hubmann, M., Hanna, R.E. et al. CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci. Nat Commun 10, 5454 (2019), or in MacLeod, R.S., Cawley, K.M., Gubrij, I. et al. Effective CRISPR interference of an endogenous gene via a single transgene in mice. Sci Rep 9, 17312 (2019). Delivery of nucleic acids encoding the gene disrupting molecules and complexes
In some aspects, a nucleic acid encoding the DNA-targeting molecule, complex, or combination, is administered or introduced to the cell. Typically, viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding components of a CRISPR, ZFP, ZFN, TALE, and/or TALEN system to cells in culture.
In some aspects, the polypeptides are synthesized in situ in the cell as a result of the introduction of polynucleotides encoding the polypeptides into the cell. In some aspects, the polypeptides could be produced outside the cell and then introduced thereto.
Methods for introducing a polynucleotide construct into animal cells are known and include, as non-limiting examples, stable transformation methods wherein the polynucleotide construct is integrated into the genome of the cell, transient transformation methods wherein the polynucleotide construct is not integrated into the genome of the cell, and virus mediated methods. In some aspects, the polynucleotides may be introduced into the cell by for example, recombinant viral vectors (e.g. retroviruses, adenoviruses), liposome and the like. Transient transformation methods include microinjection, electroporation, or particle bombardment. The nucleic acid is administered in the form of an expression vector. Preferably, the expression vector is a retroviral expression vector, an adenoviral expression vector, a DNA plasmid expression vector, or an AAV expression vector. In mammalian expression vector, it is to be noted that Promoter driving Cas9 expression can be constitutive or inducible. U6 promoter is typically used for gRNA. Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355, and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO91/17424; WO91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). In some aspects, Cas9 RNP (ribonucleoproteins) can be used. Cas9 RNPs consist of purified Cas9 protein in complex with a gRNA. They are assembled in vitro and can be delivered directly to cells using standard electroporation or transfection techniques. Cas9 RNPs are capable of cleaving genomic targets with similar efficiency as compared to plasmid-based expression of Cas9/gRNA. Cas9 RNPs are delivered as intact complexes, are detectable at high levels shortly after transfection, and are quickly cleared from the cell via protein degradation pathways. Cas9 RNP delivery to target cells is typically carried out via lipid-mediated transfection or electroporation (see for details Wang, Ming, et al. “Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles.” Proceedings of the National Academy of Sciences 113.11 (2016): 2868- 2873; Liang, Xiquan, et al. “Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.” Journal of biotechnology 208 (2015): 44-53; Zuris, John A., et al. “Cationic lipid- mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.” Nature biotechnology 33.1 (2015): 73-80 or Kim, Sojung, et al. “Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.” Genome research 24.6 (2014): 1012-1019). RNA or DNA viral-based systems include retroviral, lentivirus, adenoviral, adeno- associated and herpes simplex virus vectors for gene transfer. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, TIBTECH 11 :211-217 (1993); Mitani & Caskey, TIBTECH 11 : 162-166 (1993); Dillon. TIBTECH 11 : 167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51 (1 ):31 -44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bohm (eds) (1995); and Yu et al., Gene Therapy 1 : 13-26 (1994).
A reporter gene which includes but is not limited to glutathione- 5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and auto-fluorescent proteins including blue fluorescent protein (BFP), may be introduced into the cell to encode a gene product which serves as a marker by which to measure the alteration or modification of expression of the gene product.
Cell preparation
Also herein described is an ex vivo method of preparing an engineered immune cell defective for ZNF217 comprising introducing a mutation, insertion or deletion in the ZNF217 gene that reduces the activity of ZFN217, optionally followed by use of the engineered immune cell prepared by the method in cancer therapy.
Isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps according to well-known techniques in the field. 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 for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
In some aspects, the cell preparation includes steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. Any of a variety of known freezing solutions and parameters in some aspects may be used.
Typically, the cells are incubated prior to or in connection with genetic engineering of ZNF217 (and/or for example of SOCS1 and/or SOCS3 and/or CISH) inhibition. The incubation steps can comprise culture, incubation, stimulation, activation, expansion and/or propagation. In some aspects, inhibition of ZNF217 as per the invention (and/or for example of SOCS1 and/or SOCS3 and/or CISH in some aspects) may also be achieved in vivo after injection the cells to the targeted patients. Typically, inhibition of ZNF217 can be performed using pharmacological inhibitors as previously described.
In other aspects, inhibition of ZNF217 (and/or for example of SOCS1 and/or SOCS3 and/or CISH in some aspects) as per the method as previously described can also be performed during stimulation, activation and/or expansion steps. For example, PBMCs, or purified T cells, or purified NK cells, or purified macrophages, or purified lymphoid progenitors, are expanded in vitro in presence of the pharmacological inhibitor! s) °f ZNF217 and/or for example of SOCS1 and/or SOCS3 and/or CISH before adoptive transfer to patients. In some aspects, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a genetically engineered antigen receptor. The incubation conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
In some aspects, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR or NKCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti- CD28, for example, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some aspects, the stimulating agents include 1 L-2 and/or IL- 15, for example, an IL-2 concentration of at least about 10 units/m L.
In some aspects, incubation is carried out in accordance with techniques such as those described in US Patent No. 6,040,1 77 to Riddell et al., Klebanoff et al., J Immunother. 2012; 35(9): 651-660, Terakura et al., Blood. 2012; 1 :72-82, and/or Wang et al., J Immunother. 2012,35(9):689-701.
In some aspects, the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC) (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some aspects, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.
In some aspects, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25°C, generally at least about 30°C, and generally at or about 37°C. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10: 1.
In some aspects, 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 antigen. Lor example, antigen-specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.
In some aspects, the methods include assessing expression of one or more markers on the surface of the engineered cells or cells being engineered. In one aspect, the methods include assessing surface expression of one or more target antigen (e.g., antigen recognized by the genetically engineered antigen receptor) sought to be targeted by the adoptive cell therapy, for example, by affinity-based detection methods such as by flow cytometry.
Also herein described is a population of engineered immune cells comprising cells defective for ZNL217, e.g., T cells, NK cells, macrophages and/or B cells, wherein said population is enriched for CD62L+ cells or for METTL3+ and CD62L+ cells. Optionally, said population is enriched for cells that express a) CD62L and CD27 on the surface, or (b) CD62L, CD27 and CD127 on the surface, or (c) CD62L and CCR7 on the surface, or (d) CD62L and TCE-1 on the surface.
In a particular aspect, the population of engineered immune cells comprising cells defective for ZNE217 comprises at least, or more than, 60%, 70%, 80%, or 90% of cells, e.g., T cells, NK cells, macrophages and/or B cells, that express (a) CD62L on the surface, or (b) CD62L and CD27 on the surface, or (c) CD62L, CD27 and CD127 on the surface, or (d) CD62L and CCR7 on the surface, or (e) CD62L and TCE-1 on the surface, optionally wherein the immune cell is a T cell, NK cell, macrophage or B cell.
Vectors and methods for cell genetic engineering
In some aspects, the genetic engineering involves introduction of a nucleic acid encoding the genetically engineered component or other component for introduction into the cell, such as a component encoding a gene-disruption protein or nucleic acid.
Generally, the engineering of CARs into immune cells (e.g., T cells or NK cells) requires that the cells be cultured to allow for transduction and expansion. The transduction may utilize a variety of methods, but stable gene transfer is required to enable sustained CAR expression in clonally expanding and persisting engineered cells.
In some aspects, gene transfer is accomplished by first stimulating immune cell growth, proliferation, and/or activation, e.g., T cell’s, macrophage’s or NK cell’s growth, proliferation, and/or activation, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.
Various methods for the introduction of genetically engineered components, e.g., antigen receptors, e.g., CARs, are well known and may be used with the provided methods and compositions. Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.
In some aspects, recombinant nucleic acids are transferred into immune cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some aspects, recombinant nucleic acids are transferred into immune cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3.; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November; 29(11 ): 550-557. In some aspects, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some aspects, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one aspect, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, 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 of lentiviral transduction are also known. Exemplary methods are described in, e.g., 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 aspects, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al., (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some aspects, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21 (4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co- precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).
Other approaches and vectors for transfer of the genetically engineered nucleic acids encoding the genetically engineered products are those described, e.g., in international patent application, Publication No.: WO2014/055668, and U.S. Patent No. 7,446,190.
Combinations, compositions and kits of the invention
The present invention includes the combination of i) a cell of the invention as described herein and/or produced by the methods described herein, in particular an engineered immune cell, or of a population of cells (such as TILs for example) comprising said engineered immune cell, and ii) a ZNF217 inhibitor.
The present invention also includes compositions containing the cells as described herein and/or produced by the described methods. Typically, said compositions are pharmaceutical compositions and formulations for administration, such as for cell therapy, in particular adoptive cell therapy.
A (pharmaceutical) composition of the invention generally comprises at least one engineered immune cell of the invention and a pharmaceutically acceptable carrier or support. A preferred composition further comprises a ZNF217 inhibitor.
As used herein the language “pharmaceutically acceptable carrier (/ support)” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can further be incorporated into the compositions. In some aspects, the choice of carrier in the pharmaceutical composition is determined in part by the particular engineered CAR or TCR, vector, or cells expressing the CAR or TCR, as well as by the particular method used to administer the vector or host cells expressing the CAR. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001 to about 2% by weight of the total composition.
A pharmaceutical composition is formulated to be compatible with its intended route of administration.
A kit is also provided for carrying out the herein disclosed uses and therapeutic methods. This kit comprises at least i) an immune cell defective for ZNF217, a population of cells (or a cell preparation) comprising such a defective immune cell, or a tool for genetically modifying an immune cell in order for said cell to become defective for ZNF217, and ii) a ZNF217 inhibitor, possibly in suitable containers means.
The tool for genetically modifying an immune cell in order for said cell to become defective for ZNF217 may be for example a genetic tool or an expression vector as described herein above.
The kit may also comprise an injection device.
Therapeutic methods
The present invention also relates to the cells as herein above previously described for their use as a medicament, typically in cellular therapy (notably adoptive T cell therapy), in particular in the prevention or treatment of cancer in a subject in need thereof. In some aspects, the cells as herein disclosed can be used in allogenic transfers (allogeneic cellular therapy) notably in the case of cells defective for ZNF217 optionally in combination with inactivation of for example SOCS1, SOCS3, CISH and/or FAS.
In a particular aspect, the cells used in allogenic transfers are cells defective for ZNF217 and FAS, and optionally in addition for SOCS 1 , SOCS3 and/or CISH.
Also herein described is a combination as herein above described of i) a cell of the invention defective for ZNF217, or of a population of cells comprising said defective cell, and ii) a ZNF217 inhibitor for use as a medicament, or for use for preparing a pharmaceutical composition for preventing or treating a disease, typically cancer.
The present invention also relates to a method of treatment and notably a cell therapy, in particular an adoptive cell therapy, preferably an adoptive T cell therapy, comprising the administration to a subject in need thereof of a combination, or of a composition, as previously described.
In some aspects, the cells, combinations, or compositions as herein described are administered to the subject, such as a subject having, or at risk for, a cancer or any one of the diseases as mentioned above. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of, the disease or condition, such as with reference to cancer, by lessening tumor burden in a cancer expressing an antigen recognized by the engineered cell.
Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al. ; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31 (10): 928-933; Tsukahara ef al. (2013) Biochem Biophys Res Commun 438(1 ): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
In some aspects, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject (as such or in the form of a combination or composition of the invention).
In some aspects, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such aspects, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some aspects, the first and second subjects are genetically identical. In some aspects, the first and second subjects are genetically similar. In some aspects, the second subject expresses the same HLA class or supertype as the first subject. In such aspects, the use of cells defective for ZNF217, optionally in combination with SOSC1, SOCS3 and/or CISH inactivation is favored. Administration of at least one cell according to the invention to a subject in need thereof may be combined with one or more additional therapeutic agents, preferably a ZNF217 inhibitor, or in connection with another therapeutic intervention, either simultaneously or sequentially, in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some aspects, the cell populations are administered prior to the one or more additional therapeutic agents. In some aspects, the cell populations are administered after the one or more additional therapeutic agents.
With reference to cancer treatment, a combined cancer treatment can also include but is not limited to chemotherapeutic agents, hormones, anti-angiogens, radiolabelled compounds, immunotherapy, surgery, cryotherapy, and/or radiotherapy.
Immunotherapy includes but is not limited to immune checkpoint modulators (i.e. inhibitors and/or agonists), monoclonal antibodies, cancer vaccines.
Preferably, administration of cell in an adoptive T cell therapy according to the invention is combined with administration of immune checkpoint modulators, notably checkpoint inhibitors. Checkpoint inhibitors include, but are not limited to, PD-1 inhibitors, PD-E1 inhibitors, Eag-3 inhibitors, Tim-3 inhibitors, TIGIT inhibitors, BTEA inhibitors, V-domain Ig suppressor of T-cell activation (VISTA) inhibitors and CTEA-4 inhibitors, IDO inhibitors for example. Co-stimulatory antibodies deliver positive signals through immune- regulatory receptors including but not limited to ICOS, CD137, CD27 OX-40 and GITR. Most preferably, the immune checkpoint modulators comprise a PD-1 inhibitor (such as an anti-PD-1 agent), a PDE1 inhibitor (such as an anti-PDEI agent) and/or a CTEA4 inhibitor. In addition or as an alternative to the combination with checkpoint blockade, the immune cell (notably the immune cell composition) of the present disclosure may also be genetically modified to render them resistant to immune-checkpoints using gene-editing technologies including but not limited to TALEN and Crispr/Cas. Such methods are known in the art, see e.g. US2014/0120622. Gene editing technologies may be used to prevent the expression of immune checkpoints expressed by T cells including but not limited to PD-1, Lag-3, Tim-3, TIGIT, BTLA CTLA-4 and combinations of these. The immune cell as discussed here may be modified by any of these methods.
The immune cell according to the present disclosure may also be genetically modified to express molecules increasing homing into tumours and or to deliver inflammatory mediators into the tumour microenvironment, including but not limited to cytokines, soluble immune -regulatory receptors and/or ligands.
The present description also relates to the use of a combination, or of a composition, comprising the engineered immune cell as herein described for the manufacture of a medicament for treating a cancer, an infectious disease or condition, an autoimmune disease or condition, or an inflammatory disease or condition in a subject.
The present description also encompasses a method for the manufacture of a universal immune cell, in particular universal T cell, usable in allogenic adoptive therapy, for example in the treatment of cancer, comprising a step of repressing of ZNF217 activity (at the gene, mRNA or gene level as previously described), in a T cell optionally in combination with inactivation of for example SOCS1, SOCS3 and/or CISH activity.
The present description also encompasses a method for allogenic adoptive therapy, notably for allogenic cancer adoptive therapy, notably allogenic ATCT, comprising steps of:
- obtaining at least one immune cell, from a subject,
- modifying said at least one immune cell to inhibit or inactivate ZNF217, and,
- administrating said at least one immune cell, typically in the form of a pharmaceutical composition, to another subject in need thereof; optionally wherein said at least one immune cell is further modified to express one or more genetically modified antigen receptor(s) as previously described; optionally wherein said at least one immune cell is further modified to inhibit or inactivate optionally and for example SOCS1, SOCS3 and/or CISH; optionally wherein the at least one cell is a CD4+ T cell, or a mixed population of CD4+/CD8+ T cells as previously described.
This method can also be combined with the aspects previously described. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES
Figure 1: Zfp217 inactivation restores tumor-specific Marilyn CD4 T cells polyfunctionality, sternness and persistence in vivo.
(A) Schematic of Marilyn CD4 T cells (TCT) in C57BL/6 female mice bearing the male DBY- expressing bladder tumor line MB49. s.c., subcutaneous; i.v., intravenous. (B) Tide analysis showing the percentage of NHEJ-mutations in the genomic DNA (gDNA) of primed Marilyn, 4 days after electroporation with sgZfp217. (C) Zfp217 mRNA relative expression to b2m by RT-PCR analysis at day 4 after electroporation in mock (control) and sgZfp217 Marilyn CD4 T cells. (D) Representative flow plots and quantification of control or sgZfp217 Marilyn cells in the tumor draining lymph node (TdLN), tumor, and non-draining LN (Non DLN) at day 12 after transfer. (E) Representative flow plots and percentage of control and sgZfp217 Marilyn cells proliferation (KI67+) in the TdLN at day 6 after transfer. (F) Representative flow plots and percentage of control and sgZfp217 Marilyn cells central memory phenotype (CCR7+, CD62L+) in the TdLN at day 6 and 12 after transfer. (G) Representative flow plot and Mean Fluorescent Intensity (MFI) of TCF1 in control and sgZfp217 Marilyn cells in the TdLN at day 6 and 12 after transfer. (H) Flow plots and percentage of control and sgZfp217 Marilyn cells (TdLN at day 6) capacity to produce cytokines (IFNy, TNFa, IL21) after PMA-Ionomycin- Golgiplug restimulation 4h. (I) Flow plots and quantification of activated B cells (KI67+, IgDlow CD19+), CD8 T cells and NK cells infiltrating the TdLN at day 12 in mock and sgZfp217 TCT groups. (J) MB49 tumor weight at day 12 after mock and sgZfp217 Marilyn cells transfer. Data are shown as means, analyzed by Mann-Whitney U tests (B and D to J); n = 4 to 6 mice per group from two independent experiments.
Figure 2: At least partial inactivation of ZNF217 improves human CAR19 bbz T cells control of B Cell Acute Lymphoblastic Leukemia (B-ALL) disease (NALM6) in NSG mice with increased persistence of CAR4 and upregulation of METTL3 in CAR-T cells. (A) Schema of experimental adoptive transfer of CAR19bbz-T cells in NALM6-bearing NSG mice. (B) Representative flow plots of CD4 and CD8 percentage in CAR19bbz T cells and CAR expression pre-injection. (C) Frequency of NHEJ- mutagenesis 3 days after electroporation with Cas9-sgRNA. (D) Western blot analysis of ZNF217 and Mettl3 expression 4 days after CAR-T cells CRISPR-editing. (E) Spleens weight and size 21 days after CAR-T cells infusion. (F) Whole body tumor radiance and quantification at day 21 after CAR-T cells injection. (G) Representative flow plots and quantification of CAR-T cells infiltrating the bone marrow of NALM6-bearing NSG mice at day 7 and 21 after infusion. (H) Mettl3 expression on CAR4 and CAR8 T cells infiltrating the bone marrow at day 7 post infusion.
Figure 3: At least partial inactivation of ZNF217 induces proliferation and stem-like features in human CAR19 bbz T cells against B-ALL disease.
(A) Representative flow plots and quantification of proliferating (KI67+) CAR4 and CAR8 control and sgZNF217 in the bone marrow (BM) at day 7 post infusion. (B) Representative flow plots and quantification of CD62L and CD27 memory progenitors associated markers on CAR4 and CAR8 control and sgZNF217 in the BM at day 7. (C) Representative flow plots and quantification of CD127 (IL-7R) on CAR4 and CAR8 control and sgZNF217 in the BM at day 7. (D) Percentage of PD1+ and TIM3+ expression on control and sgZNF217 CAR-T cells at day 7 in the spleen and BM. (E) Absolute number of control and sgZNF217 CAR4 and CAR8 T cells infiltrating the spleen at day 21 after infusion (F) Representative flow plots and quantification of proliferating (KI67+) CAR4 and CAR8 control and sgZNF217 in the BM 21 days post infusion. (G) Representative flow plots and quantification of TCF1 and CD62L expression on CAR4 and CAR8 control and sgZNF217 in the BM at day 21.
Figure 4: At least partial inactivation of ZNF217 enhances polyfunctionality in human CAR19 bbz T cells against B-ALL disease.
(A) Percentage of CD4 and CD8 expression on CAR-T cells ex vivo after recall (PMA-ionomycin-golgiplug restimulation 4h) and absolute number of CAR4 and DN CAR at day 7 and day 21 in the BM. (B) Representative flow plots and (C) percentage of effector molecules (INFg, TNFa) produced by CAR4, CAR8 and CD3+ CD4- double negative (DN) CAR-T cells and absolute number of INFg and TNFa producing CAR-T cells from BM at day 7. (D) Representative flow plots and (E) percentage of IFNy and IL21 produced by CAR4, CAR8 and DN CAR-T cells from BM at day 7. (F) Representative flow plots and (G) percentage of granzyme B (GZB) produced by CAR4, CAR8 and DN CAR-T cells and absolute number of GZB producing CAR-T cells from BM at day 7. (H) Representative flow plots and (I) percentage of GZB produced by CAR8 and DN CAR-T cells from BM at day 21. n = 4 mice per group.
Figure 5: At least partial inactivation of ZNF217 enhances human CAR19 bbz T cells control of solid tumors (A549-CD19) in NSG mice and CAR8 persistence in vivo.
(A) Schema of experimental adoptive transfer of CAR19bbz-T cells in A549-CD19-bearing NSG mice. (B) Whole body tumor radiance and (C) radiance quantification at day 14 after CAR-T cells injection. (D) Representative flow plots and (E) quantification of CAR-T cells infiltrating the lung of A549-CD19- bearing NSG mice at day 14 after infusion. Figure 6: At least partial inactivation of ZNF217 reduces adenocarcinoma proliferation and invasiveness (A549 non-small cell lung cancer and breast MDA-MB231)
(A) Frequency of NHEJ-induced mutagenesis in A549 lung adenocarcinoma genomic DNA (gDNA) three days after editing. (B) ZNF217 and METTL3 expression in mock (control) and sgZNF217 A549 cells three days after editing. (C) Wound healing assay using mock (control) and sgZNF217 A549 cells for 24h in vitro. (D) Frequency of NHEJ-induced mutagenesis in MDA-MB231 breast adenocarcinoma genomic DNA (gDNA) three days after editing. (E) ZNF217 and METTL3 expression in mock (control) and sgZNF217 MDA-MB231 cells three days after editing. (F) Wound healing assay using mock (control) and sgZNF217 MDA-MB231 cells for 24h in vitro (X10).
Figure 7: At least partial inactivation of ZNF217 abolishes MCF7 breast cancer cells survival
(A) Frequency of NHEJ-induced mutagenesis in MCF7 breast adenocarcinoma genomic DNA (gDNA) three days after editing. (B) Light microscopy images of mock (control) and sgZNF217 MCF7 cells in culture seven days after editing (X10 and X4).
Figure 8: At least partial inactivation of ZNF217 in human and mouse T cells produces a population of stem-like CD62L+, METTL3+ T cells, in which METTL3 expression is essential for the sternness- associated phenotype
(A). Expression of METTL3 protein in CD4 TCR-Tg Marilyn in the tumor-draining lymph node at day 6 by flow analysis. (B). Expression of ZNF217, METTL3 and Actin in human anti-CD19 human CAR-T cells at day 4 after electroporation by Cas9-RNP ZNF217 sgRNA by western blot analysis. (C). Expression of METTL3 in human anti-CD19 human CAR-T cells at day 21, after intravenous injection in NALM6(ALL)-bearing NSG mice. (D, E). Expression of CD62L and other sternness-associated markers in mouse CD4 tumor- specific T cells (control, KO for ZNF217, deleted for ZNF217 and METTL3) at days 6 and 12 after transfer in tumor-bearing C57BL6 mice. (F). Kelly neuroblastoma tumor growth in NSG mice treated with 105 anti-GD2 CART and 105 sgZNF217 anti-GD2 CART.
Other characteristics and advantages of the invention are given in the following experimental section (with reference to figures 1 to 8), which should be regarded as illustrative and not limiting the scope of the present application. EXPERIMENTAL PART
EXAMPLE 1 - Inactivation of epigenetic/epitranscriptomic regulator ZFP217 rejuvenate adoptive T cells therapies
MATERIAL AND METHODS
Cell lines and mice
FFLuc-BFP NALM6 (NALM6) cell line, provided by O. Bernard were maintained in RPML1640 supplemented with 10% FBS. CD45.1Z1 female Marilyn TCR-transgenic Rag2’^“ mice were maintained at Centre d'Exploration et de Recherche Fonctionnelle (CERFE, Evry) France. C57BL/6 and female, male NOD-scid IL Ry7 (NSG) mice were purchased from Charles River Laboratories (L’Arbresle, France). All experiments were conducted with 6-12 weeks old, sex balanced in an accredited animal facility by the French Veterinarian Department following ethical guidelines, approved by the relevant ethical committee (APAFIS #35057-2022013112272631 v4).
Murine T cell culture and adoptive transfer
CD4+ T cells were isolated from Marilyn mice secondary lymphoid organs (spleen, mesenteric, axillary, inguinal, brachial, cervical and lumbar lymph nodes) using Magnisort Mouse CD4 T cell enrichment kit (Invitrogen) according to manufacturer’s instructions. CD4+ T cells were activated using anti-CD3 (l,5pg/mL) coated 6 well plate and anti-CD28 (3pg/mL) (BD Biosciences). IL-2 (20UI/mL), IL-7 (2ng/mL) (Peprotech) were added starting at day 2 and every 2 days in complete RPMI-1640 [RPMI, lOOmM Hepes, 2mM Glutamax, 50pM 2-mercaptoethanol, lOOU/lOOug/ml Penicillin/Streptomycin, IX non-essential amino acids, ImM Sodium Pyruvate (all Gibco, Thermo Fisher), 10% FCS (Eurobio, France)].
Human T cell activation and transduction
Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using Ficoll density gradient centrifugation. T lymphocytes were purified using the Pan T cell enrichment kit (Invitrogen) and activated with Dynabeads human T-Activator CD3/CD28 (1:1 bead:cell) (ThermoFisher) in full RPMI- 1640 (supplemented with 10% FCS (Eurobio, France) and 0.5 mM b-mercaptoethanol) with IL7 (25ng/mL) and IL 15 (25ng/mL) (Peprotech) at density of 106 cells/mL. 48 hours after activation, CD3/CD28 beads were magnetically removed, T cells were electroporated with Cas9-ribonucleoproteins (Cas9-RNP) and then transduced with lentiviral supernatants of an anti-CD19 (FMC63)-CD8tm-4IBB- CD3^ CAR construct (rLV.EF1.19BBz, Flash Therapeutics) at MOI 10 in full RPMI-1640 with 4 pg/mL of Polybrene.
Cas9-RNP electroporation of primary murine or human T cells
For murine T cells, 1 pl Oligos crRNA (lOOnM) and I ,u I tracrRNA (lOOnM) (Table 1) were annealed at 95°C for 5 min and incubated at room temperature 10 min with 10 pg S.p Hifi Cas9 Nuclease V3. For human T cells, 2 pl of single guide RNA (sgRNA) were incubated with 10 pg S.p Hifi Cas9 Nuclease V3 at a 2:1 molecular ratio and incubated for 10 min at room temperature. 2.106 murine T or human T cells were resuspended in 20 pl of nucleofection solution with 3 pl RNP, transferred to Nucleofection cuvette strips (4D-Nucleofector X P3 kit S or L; Lonza) and electroporated using the DN-110 program (for murine T cells), or the EO-115 program (for human T cells) of 4D nucleofector (4D-Nucleofector Core Unit: Lonza, AAF-1002B). Murine CD4+ T cells were incubated two days at 32°C and maintained in complete RPMI with IL-2 (20UI/mL) and IL-7 (2 ng/mL) at 37°C. Human CAR T cells were maintained in full RPM-1640 with IL7 (25ng/mL) and IL 15 (25ng/mL) (Peprotech) at 37 °C. Table 1: Murine and human antibodies + guideRNA sites
Flow cytometry
Single-cell suspensions from lymph nodes, spleens and bone marrows were obtained by mechanical disruption over a 70 pM cell strainer in MACS buffer (PBS IX, EDTA lOrnM, FBS 0.5%). Splenocytes were treated for one minute with red blood lysis buffer. MB49 tumors and A549-CD19 NSCLC -bearing lungs were digested for 30 min at 37°C in RPMI-1640 with 125 pg/mL of liberase TL (Roche) and 125 pg/mL of DNAse-I (Roche). Cells were incubated with the appropriate antibodies or isotype controls. All antibodies are detailed in Table 1. Dead cells were excluded using live/dead cell marker (Fixable Yellow/blue Dead Cell Stain Kit or Fixable Viability Dye Red). All surface antibody stainings were performed in PBS with FBS 0.5% and EDTA 10 mM 4°C for 30 min. CAR expression was assessed using a CD 19 CAR detection reagent (Biotinylated) and cells were incubated for 10 minutes at room temperature. After washing, cells were stained PE-conjugated streptavidin. Cell Sorting V450 Set-up Beads (Life Technologies) were used to quantify and normalize cell numbers between samples and experiments. All samples were acquired on Cytek (Aurora) and analyzed with FlowJo software v.10.6.2 (BD Biosciences). NHEJ analysis
Genomic DNA was isolated from murine T and CAR-T cells 4 days post electroporation using the QIAamp DNA Mini Kit (Qiagen) according to manufacturer’s protocols. 700 bp sequences flanking the guideRNA site were amplified by PCR (Table 1) and amplicons were analyzed by Sanger sequencing (Eurofins).
RESULTS
Inventors have previously developed a syngeneic model to study the intrinsic limitations of CD4 helper T cell therapy (TCT) in vivo (Sutra Del Galy, A. et al., 2021). They challenged female C57BL/6 mice with the immunogenic but aggressive MB49 male bladder carcinoma cells subcutaneously and, 10 days later, transfer Dby (HY)-specific Marilyn CD4 T cells to stimulate a CD8+ T cell- and natural killer (NK) cell-dependent rejection (Fig. 1A). Inventors efficiently inactivated 7 217 in Marilyn CD4 T cells (60% indels) 4 days after electroporation with CRISPR-Cas9 ribonucleoprotein (Fig. IB), as confirmed by the significant decrease of Zfp217 transcriptomic expression in sgZfp217 Marilyn CD4 T cells (Fig. 1C). To determine the impact of Zfp217 inactivation on CD4 T-cells, inventors compared the number, phenotype, and cytokines production of the transferred Marilyn T cells in the TdLNs, in the tumors, and in a distant irrelevant LN (irr-LN) overtime (day 6, 12). They observed that sgZfp217 Marilyn T cells infiltrate tumors and persisted more efficiently in vivo (TdLN, irr-dLN) than mock Marilyn cells (Fig. ID). This is associated with a higher percentage of proliferating sgZfp217 Marilyn cells in TdLN at day 6 (Fig. IE) and a conserved central memory phenotype (CCR7+ CD62L+) overtime (Fig. IF) with increased expression of TCF1 (Fig.lG) as compared to mock Marilyn cells. TCF-1 is essential for the formation and function of a precursor population of T cells with self-renewal capacity, the ability to regenerate themselves and to generate more differentiated cells (stem-cell-like properties) (Zehn et al., 2022). Thus, Zfp217 inactivation is inducing a pool of memory T cells that are able to continuously generate antitumor effector T cells, as evidenced by the increased number of Marilyn T cells in the tumor (Fig. ID).
Analysis of cytokines expression demonstrated that sgZfp217 Marilyn T cells produced THI (IFNy, TNFa) to the same level of control as Marilyn cells but exhibited an enhanced capacity to secrete TFH (IL21) cytokines in the TdLN at day 6 (Fig. 1H). Consistent with sgZfp217 Marilyn T cells polyfunctionality, major differences emerged in the TdLN infiltration by endogenous cells. The group of mice transferred with sgZfp217 Marilyn T cells exhibit a significant increase in activated and proliferating B cells (CD19+, KI67+, IgDl°w, Eas-i- GL7+ (data not shown)), CD8 and NK cells number at day 12 (Fig. II), together with a reduction in tumors weight (Fig. 1 J). Thus, 7 217 deletion enables Marilyn CD4 T cells to undergo rapid expansion and persistence in vivo, infiltrate tumors, and elicit antitumor responses with a polyfunctional molecular signature indicative of THI/TFH progenitors or memory precursors cells (Xia Y. et al. , 2022). The inventors demonstrate that deletion of zfp217 can enhance the helper capacities of CD4 TCT, which then stimulate an endogenous antitumoral response, involving killers cells (CD8 and NK cells) and a B-cell dependent antitumor immune response.
Inventors then assessed the translational relevance of ZNF217 inactivation on human CAR19bbz T cells antitumor activity against NALM6-Luciferase (NALM6-Luc) acute lymphoblastic leukemia (ALL) in NSG mice (Fig. 2A). The CAR19bbz construct has been shown to enhance the expansion and survival of CD8+ CAR-T cells (CAR8) central-memory-like populations (Guedan et al., 2018; Haradhvala et al., 2022) but limit the in vivo life span of CD4+ CAR-T cells (CAR4) (Turtle et al., 2016; Yang et al., 2017). Inventor’s generated CAR19bbz T cells (Fig. 2B) from activated T cells and efficiently deleted (45%) ZNF217 gene using Cas9-RNP electroporation (Fig. 2C), leading to a 40% reduction in ZNF217 protein expression (sgZNF217). Prior to injection, inventors also observed a slight increase in METTL3 expression in sgZNF217 CAR-T cells as compared to control CAR-T cells (Fig. 2D) using western blot analysis. Inventors evaluated the antitumor activity of control and sgZNF217 CAR-T cells against NALM6-Luc using whole body tumor luminescence overtime and spleen weight on day 21. Inventors observed a reduced weight of spleens associated with reduced body tumor luminescence in NSG mice injected with sgZNF217 CAR-T cells as compared to control CAR-T cells (Fig. 2E, F). Infiltration of NSG mice’s bone marrow revealed an increased number of CD4 CAR-T cells (CAR4) at day 7 and day 21 post infusion (Fig. 2G). Interestingly, inventors confirmed the upregulation of METLL3 expression in sgZNF217 CAR-T cells infiltrating the bone marrow at day 7 (Fig. 2H). Altogether, these data demonstrate that the reduction or loss of ZNF217 increase the expression of the m6A methyltransferase METLL3 in CAR4 and CAR8 T cells.
The flow analysis of CAR-T cells infiltrating the BM at day 7 revealed that both CAR4 and CAR8 sgZNF217 exhibit an enhanced expression of KI67, a marker of cell cycle entry as compared to control CAR-T cells (Fig. 3A). Inventors also noted the increased expression of memory markers associated with T cell survival, long-term persistence and positive clinical response to adoptive T cell therapy (Haradhvala et al., 2022), such as CD62L, CD27, CD127 in both CAR4 and CAR8 ZAF2/7-inactivatcd at day 7 (Fig 3B, C). Interestingly, ZNF217 deletion also causes a shift towards a more progenitor exhausted and less terminally exhausted T cell phenotype with lower levels of the exhaustion markers PD- 1 , TIM3 at the tumor site (BM) and in periphery (spleen) at day 7 (Fig. 3D). Inventors also demonstrated that at later time point (day 21), sgZNF217 CAR4 were 10 times more persisting in the BM of NALM6-bearing NSG mice than mock CAR4 (Fig. 3E). Inventors observed that sgZNF217 CAR-T cells conserved proliferative function as compared to control CAR-T cells in the BM (Fig. 3F). Furthermore, associated with an enhanced persistence, inventors observed a maintained expression of T cell factor 1 (TCF1) and CD62L expressions in sgZNF217 CAR4 at day 21 but only enhanced CD62L expression in sgZNF217 CAR8 (Fig. 3G).
Looking at the impact of ZNF217 deletion in CAR-T cells functionality ex vivo, inventors first observed a reproducible and strong reduction in CD4+ CAR-T cells subset, which matched with a progressive increase in CD3+ CD4- double-negative population in sgZNF217 CAR-T cells, after re- stimulation (PMA-Ionomycin, Golgiplug) (Fig. 4A). Inventors evaluated the capacity of these three subpopulations of CAR-T cells to produce cytokines. Inventors showed that ZNF217 deletion induces prominent polyfunctionality in DN sgZNF217 CAR-T cells and CAR8 sgZNF217, highlighted by the production of both IFNy and TNFa as compared to control CAR-T cells (Fig. 4B, C). Furthermore, CAR8 and DN sgZNF217 cells have an increased capacity to produce IFNy and IL21 as compared to control CAR- T cells at the tumor site at day 7 (Fig. 4D, E). CAR8 and DN sgZNF217 cells also exhibit a significantly higher production of cytotoxic granzyme B molecules (Fig. 4F, G). There were also more T cells producing granzyme B in sgZNF217 CAR8 and DN subsets than control CAR-T cells at day 7 (Fig. 4F, G). Inventors finally observed at day 21 that CAR8 and DN sgZNF217 cells presented an enhanced cytotoxic potential as compared to control CAR-T cells overtime (Fig. 4H, I).
Investors also investigated the impact of ZNF217 inactivation on CAR-T cells in a solid tumor model, using xenogeneic A549 lung adenocarcinoma cells transduced with luciferase and CD 19 (A549- CD19). NSG mice were infused with either control or sgZNF217 CAR-T cells (DO) 20 days after i.v. tumor cell injection (Fig. 5A). The whole-body luminescence of A549-CD19 tumors in NSG mice overtime shows that sgZNF217 CAR-T cells better control A549-CD19 growth in vivo (Fig. 5B, C). The flow analysis of lung tumors infiltration at day 14 shows that ZNF217 deletion is enhancing CAR8 subset persistence (Fig. 5D).
With a similar approach, inventors inactivated ZNF217 in several cancer cell lines using Cas9-RNP electroporation (Fig. 6, 7). Inventors demonstrate that ZNF217 deletion (50-60%) in A549 (Fig. 6A), in MDA-MB231 (Fig. 6D) and in MCF7 (Fig. 7A), leading to reduced protein expression (Fig. 6B, E) was reducing the invasiveness (Fig 6C, F) of tumor cells. In the MCF7 cell line, which possesses high endogenous levels of ZNF217 (Thollet et al. , 2010), ZNF217 inactivation even abolished their proliferation and survival in vitro (Fig. 7C).
DISCUSSION
For the first time, inventors showed and herein reveal that ZNF217 deletion unleashed the proliferation and skewed adoptive T cell therapies towards a memory precursor signature, which is maintained overtime in tumor environment. With a significantly higher proportion of persisting central memory T cells with progenitor markers (less-differentiated) (CD62L, CD44, TCF1, CD127 (IL7Ra), CCR7, CD27, CD38, Ki67, and/or METTL3), and polyfunctionality (IL2, IL21, GZB, TNFa, IFNy), inventors herein demonstrate that the at least partial deletion of ZNF217 improves antitumor immune responses of adoptive T cell therapies. Conversely, the at least partial inhibition of ZNF217 is reducing the proliferation and invasiveness of several cancer types, showing the development of a systemic inhibition which further enhances the therapeutic impact of TCT. CONCLUSION
Inventors demonstrate in three different cancer models that ZNF217 deletion leads to T cell reprogramming into ‘Stem-like’ precursors with resistance to tumor-induced exhaustion. Indeed, ZNF217 (zfp217 in mouse) exhibit an enhanced proliferation (Ki67+) in vivo with upregulation and maintenance of markers associated with T cell survival, sternness and persistence [CD62L, CD44, IL7Ra (CD127), CD27, CCR7, TCF1, CD38, Ki67, and/or METTL3] as well as antitumor activity (IFNy, IL2, IL21, TNFa, Granzyme B). ZNF217 inactivation in transferred CD4 T cells therapies is improving endogenous antitumor immune responses, involving NK cells activation, macrophages recruitment and activation at tumor site, memory CD8 T cells formation, and increased B cell-mediated antitumor response.
EXAMPLE 2 - ZNF217 defective, METTL3+ CD62L+ T cells exhibit enhanced anti-tumor activity.
Human and mouse T cells defective for ZNF217 upregulate the expression of METTL3 (cf. Fig. 8 A, B, C). However, inventors observed that the level of expression of METTL3 is not equivalent in each of the detected ZNF217 deficient populations, and that the co-expression of METTL3 and CD62L defines a particularly efficient and persistent ZNF217 defective T cell population advantageously usable as the preferred T cell population in the context of T cell-based therapies. As shown on Figures 8D and 8E, the sternness phenotype associated with ZNF217 deletion is lost when METTL3 is co-inactivated.
As a highly personalized therapeutic option with the benefit of long-term immune protection, cellular immunotherapies represent the most promising method to cure cancer where a lack of immune T cells infiltration is observed, in particular solid tumors or pediatric malignant tumors, that resist conventional care. Indeed, pediatric cancers, including neuroblastoma, Diffuse Intrinsic Pontine Glioma (DIPG, also identified as Diffuse midline glioma), and high-grade glioma, as well as liquid cancers such as Acute lymphoblastic Leukemia (ALL), are associated with immunosuppressive environments and low T-cell infiltration. Inventors have developed an anti-GD2 CAR bbz (41BB-CD3z) construct and studied the impact of ZNE217 deletion on CAR-T antitumor efficacy against pediatric cancers expressing GD2 ganglioside (neuroblastoma and DIPG). While integrating the observed escape, directly linked to the variability inherent in experiments carried out in vivo, these results demonstrate that anti-sgZNE217 GD2 CAR-T cells were able to eradicate the neuroblastoma tumors (Kelly) engrafted in NSG mice (cf. Pig. 8P ). REFERENCES
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