IL-2-expressing myeloid cells and their use for rapid anti-cancer therapy

Abstract

The present invention relates to modified cells containing a transgene encoding interleukin-2 (IL-2). The modified cells contain a first vector containing a sequence encoding IL-2 under the control of an inducible or constitutive promoter, which allows, inter alia, localized expression of IL-2. The present invention also relates to therapeutic uses thereof.

Description

[Technical Field]

[0001] The present invention relates to modified cells containing a transgene encoding interleukin-2 (IL-2). The modified cells, preferably modified myeloid cells, contain a first vector containing a sequence encoding IL-2 under the control of an inducible or constitutive promoter, which allows, inter alia, locally restricted expression of IL-2. The present invention also relates to therapeutic uses thereof. [Background technology]

[0002] Solid tumors and their metastases are the most common and therapeutically challenging types of cancer today. The tumor microenvironment (TME) is a complex and heterogeneous mixture of cell populations that interact with each other and with tumor cells. The TME is immunosuppressive, both in terms of evading the immune system and preventing therapeutic intervention from effectively eliminating malignant cells. Myeloid cells within the TME play an important role in contributing to immune escape by exhibiting potent immunosuppressive as well as pro-tumorigenic properties.

[0003] TAMs (tumor-associated macrophages) are key cellular components of the TME in various cancers. The general consensus is that tumor-derived cytokines direct myeloid cell recruitment at the monocyte stage, after which the TME influences their progression into polarized macrophages. TAMs can represent a significant portion of the tumor mass, up to 50% in some breast tumors. They develop into immunosuppressive macrophages, which express antitumor CD8 +They prevent T cells from infiltrating tumors and attract or induce regulatory T cells (Tregs). TAMs secrete growth factors such as VEGF or TGFβ, which promote tumor growth and invasive behavior. Although recent studies have shown that their prognostic impact may vary depending on their localization and polarization (Ramos et al., 2022, Cell 185, pp. 1-19; Tissue-resident FOLR2+ macrophages associate with tumor-infiltrating CD8+ T cells and with increased survival of breast cancer patients), they are generally associated with a poor prognosis.

[0004] Autologous immune cell-based therapies have recently achieved impressive success in treating certain malignancies. The most cutting-edge approach relies on T lymphocytes that have been genetically modified to express chimeric receptors known as CAR (chimeric antigen receptor) T cells, which combine antigen-binding and T cell-activating activities in a single receptor. Adoptively transferred CAR-T cells have shown considerable promise in combating hematological malignancies.

[0005] However, CAR-T cells have so far failed to treat solid tumors. These failures likely result from a combination of factors. First, identifying antigens as strictly tumor-specific remains challenging, raising concerns about potential off-target effects. Second, before reaching cancer cells within tumor tissue, CAR-T cells may encounter physical barriers in the form of tumor-associated membrane membranes (TAMs) and cancer-associated fibroblasts, which produce vast amounts of extracellular matrix. Third, CAR-T cells fail to successfully invade the TME due to a lack of metabolic resources or signals provided by TME cellular components. Finally, due to the persistent antigenic stimulation they receive via tumor cells, CAR-T cells become "exhausted" or dysfunctional, losing their effector function and failing to evolve into effector memory T cells.

[0006] Macrophages are antigen-presenting cells that can stimulate T cells locally and thus promote adaptive anti-tumor responses. Macrophages produce proteases that can dramatically alter the extracellular matrix within tumor masses and, therefore, the structure of tumor tissue. Macrophages also have anti-tumor capabilities, such as the ability to phagocytose whole tumor cells or perform antibody-dependent cellular phagocytosis. These intrinsic characteristics of macrophages make them ideal candidates for overcoming the limitations of CAR-T cells.

[0007] However, macrophages also tend to have difficulty reaching tumor tissue, and therefore there is a need to improve access of immune cells to tumor tissue. In this regard, monocytes are considered better candidates for cell modification due to their greater ability to invade tumor tissue before differentiating into macrophages.

[0008] Systemic administration of IL-2 to boost T cell responses promotes antitumor immunity in mouse tumor models. The cytokine IL-2 produces pleiotropic effects that activate T cells and NK cells. However, systemic administration of IL-2 requires repeated injections because IL-2 has a short half-life in vivo. These repeated injections have been shown to produce adverse effects. Therefore, IL-2 has been neglected in therapeutic use, but local expression of IL-2 may be of great interest.

[0009] Therefore, there is a need for specific expression of IL-2 in tumors (in situ), which may help to recruit local immune cells.

[0010] The present invention meets these needs. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] WO2004004771 [Patent Document 2] WO2004056875 [Patent Document 3] WO2006121168 [Patent Document 4] WO2008156712 [Patent Document 5] WO2009014708 [Patent Document 6] WO2009114335 [Patent Document 7] WO2013043569 [Patent Document 8] WO2014047350 [Patent Document 9] US5,773,578 [Patent Document 10] US6,984,720 [Patent Document 11] US8,017,114 [Patent Document 12] US7,109,003 [Patent Document 13] US8,143,379 [Patent Document 14] WO1997020574 [Patent Document 15] WO2007123737 [Patent Document 16] US8,491,895 [Patent Document 17] US20130177557

Non-licensed literature

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[0013] Accordingly, the present invention relates to modified cells, wherein the cells are selected from myeloid cells, induced pluripotent stem cells (iPS), and hematopoietic stem cells (HSC), and the cells are (i) a first vector comprising a sequence encoding at least one cytokine, preferably at least one interleukin, under the control of an inducible or constitutive promoter, and a second vector comprising a sequence encoding a chimeric antigen receptor (CAR); or (ii) comprises only a first vector encoding both at least one cytokine, preferably at least one interleukin, and a CAR.

[0014] Thus, the present invention relates to modified cells that express interleukin-2 (IL-2), wherein the cells are selected from myeloid cells, induced pluripotent stem cells (iPS), and hematopoietic stem cells (HSC), and the cells comprise a first vector comprising a sequence encoding IL-2 under the control of an inducible or constitutive promoter, said cells being referred to according to the present invention as modified myeloid cells, modified iPS, or modified HSC.

[0015] Preferably, the modified cells are modified myeloid cells that contain a first vector that includes a sequence encoding IL-2 under the control of an inducible or constitutive promoter.

[0016] Preferably, the modified cell further comprises a second vector comprising a sequence encoding a chimeric antigen receptor (CAR).

[0017] The present invention also relates to modified cells expressing at least one cytokine, preferably an interleukin, wherein the cells are selected from myeloid cells, induced pluripotent stem cells (iPS), and hematopoietic stem cells (HSC), the cells comprising a first vector comprising a sequence encoding said cytokine under the control of an inducible or constitutive promoter, and optionally a second vector comprising a sequence encoding a chimeric antigen receptor (CAR), the CAR comprising: - an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells of the tumor microenvironment (TME); - optionally, a hinge domain; - a transmembrane domain, and - Intracellular signaling domains Includes:

[0018] The present invention also relates to modified cells expressing at least one interleukin selected from IL-10, IL-15, IL-13, IL-7A, IFN alpha, IFN beta, IFN lambda, IFN gamma, IL-1A, IL-1B, IL-12, and IL-21, wherein the cells are selected from myeloid cells, induced pluripotent stem cells (iPS), and hematopoietic stem cells (HSC), and the cells comprise a first vector comprising a sequence encoding the interleukin under the control of an inducible or constitutive promoter, and optionally a second vector comprising a sequence encoding a chimeric antigen receptor (CAR), wherein the CAR is - an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells of the tumor microenvironment (TME); - optionally, a hinge domain; - a transmembrane domain, and - Intracellular signaling domains Includes:

[0019] In some embodiments, at least one cytokine, preferably an interleukin, is linked to another cytokine, preferably another interleukin, antigenic polypeptide, single-chain antibody, or nanobody with a 2A peptide, which allows ribosomal skipping during protein translation. The antigenic polypeptide, single-chain antibody, or nanobody can block or stimulate receptors, allowing targeting of specific cells, preferably tumor cells or cells from the TME, such as cancer-associated fibroblasts (CAFs) or regulatory T cells. Preferably, the antigenic polypeptide has a length of about 8 to about 50 amino acids.

[0020] The present invention also provides a method for producing modified myeloid cells, modified iPS cells, or modified HSCs, comprising the steps of: - providing at least one cell selected from isolated myeloid cells, iPS cells, and HSCs; - transducing said cells with a first vector, preferably a lentiviral vector, comprising a cytokine, preferably an interleukin, under the control of an inducible or constitutive promoter, preferably under the control of the interleukin-6 promoter or the interleukin-8 promoter; and - optionally transducing said cells with a second vector, preferably a lentiviral vector, comprising a nucleic acid sequence encoding a CAR. The present invention relates to a method, comprising:

[0021] The present invention also provides a method for producing modified myeloid cells, modified iPS cells, or modified HSCs, comprising the steps of: - providing at least one cell selected from isolated myeloid cells, iPS cells, and HSCs; - transducing the cells with a first vector, preferably a lentiviral vector, comprising a sequence encoding IL-2 or a sequence encoding at least one interleukin selected from IL-10, IL-15, IL-13, IL-7A, IFN alpha, IFN beta, IFN lambda, IFN gamma, IL-1A, IL-1B, IL-12, and IL-21 under the control of an inducible or constitutive promoter, preferably under the control of an interleukin-6 promoter or an interleukin-8 promoter; and - optionally transducing said cells with a second vector, preferably a lentiviral vector, comprising a nucleic acid sequence encoding a CAR. The present invention relates to a method, comprising:

[0022] The present invention also relates to a pharmaceutical composition comprising the modified myeloid cells, modified iPS cells, or modified HSCs according to the present invention and a pharmaceutically acceptable carrier.

[0023] The present invention also relates to the use of modified myeloid cells, modified iPS cells, or modified HSCs, or pharmaceutical compositions according to the present invention in the treatment of cancer, autoimmune diseases, or inflammatory diseases.

[0024] The present invention further relates to a product containing modified myeloid cells, modified iPS cells, or modified HSCs and CAR-T cells according to the present invention as a combined preparation for simultaneous, separate, or sequential use in the treatment of cancer, autoimmune diseases, or inflammatory diseases.

[0025] The present invention also relates to a product containing modified myeloid cells, modified iPS cells, or modified HSCs according to the present invention and an immune checkpoint inhibitor as a combined preparation for simultaneous, separate, or sequential use in the treatment of cancer, autoimmune diseases, or inflammatory diseases. DETAILED DESCRIPTION OF THE INVENTION

[0026] Surprisingly, as shown in the Examples, the inventors have demonstrated that monocytes transduced with both a specific CAR and a gene encoding IL-2 under the control of a particular promoter can be used as a treatment, especially in cancer therapy.

[0027] Indeed, said monocytes can infiltrate tumors and differentiate into IL-2-expressing macrophages, which bind to predetermined antigens, thereby activating their superior ability to phagocytose tumor cells, and furthermore, upon encountering tumor cells, can also recruit the antigen-presenting and costimulatory capabilities of macrophages, thereby stimulating antitumor immunity.

[0028] The present invention relates to modified cells, wherein the cells are selected from myeloid cells, induced pluripotent stem cells (iPS), and hematopoietic stem cells (HSC), and the cells are (i) a first vector comprising a sequence encoding at least one cytokine, preferably at least one interleukin, under the control of an inducible or constitutive promoter, and a second vector comprising a sequence encoding a chimeric antigen receptor (CAR); or (ii) comprises only a first vector encoding both at least one cytokine, preferably at least one interleukin, and a CAR.

[0029] Modified cells Preferably, the present invention relates to modified myeloid cells that express IL-2, wherein the cells comprise a first vector comprising a sequence encoding IL-2 under the control of an inducible or constitutive promoter.

[0030] Preferably, the present invention also relates to modified induced pluripotent stem cells (iPS) or hematopoietic stem cells (HSC) that express interleukin-2 (IL-2), wherein the cells comprise a first vector comprising a sequence encoding IL-2 under the control of an inducible or constitutive promoter.

[0031] Such cells are also referred to in this application as "modified cells."

[0032] By "myeloid cell" is meant any type of cell derived from bone marrow tissue (bone marrow) or resembling myeloid. Preferably, it is a monocyte, macrophage, or dendritic cell, more preferably a monocyte. The myeloid cell is modified in that it expresses at least IL-2.

[0033] "Stem cell" refers to a cell that can give rise to specialized cells by successive division. The term "pluripotent stem cell" refers to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm (stomach lining, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), or ectoderm (epidermal tissue and nervous system). Pluripotent stem cells can give rise to any fetal or adult cell type, but they cannot give rise to an entire organism. "Pluripotent stem cells" can be identified by the expression of one or more of the cell markers Klf4, Sox2, Oct4, cMyc, Nanog, and SSEA1. A cell is considered a pluripotent stem cell if it has the ability to generate cells from any of the three germ layers: endoderm, identified by expression of alpha-fetoprotein; mesoderm (identified by expression of desmin and / or alpha-smooth muscle actin); and ectoderm (identified by expression of beta-tubulin III = Tuj1 and / or E-cadherin through N-cadherin). Assays for assessing the pluripotency of cells are known in the art.

[0034] "Induced pluripotent stem cells" or "iPS" refer to pluripotent cells artificially derived from non-pluripotent cells, typically adult somatic cells, by inducing the forced expression of certain genes. "Induced pluripotent stem cells" are defined by the expression of several transcription factors, including one or more of Klf4, Sox2, Oct4, and cMyc. iPS cells are typically derived by transfecting certain stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection is typically achieved through a viral vector, such as a retrovirus, and transfected genes include Oct-3 / 4 (Pou5fl) and Sox2. Additional genes include certain members of the Klf family (Klfl, Klf2, Klf4, and Klf5), the Myc family (c-myc, L-myc, N-myc), and Nanog and LIN28 have been identified to increase induction efficiency. After 3-4 weeks, a small number of transfected cells begin to resemble pluripotent stem cells morphologically and biochemically and are typically isolated through morphological selection, doubling time, or reporter gene and antibiotic selection. Protocols for iPS culture are disclosed in Mochiduki and Okita, 2012. Non-pluripotent cells that can be used to obtain iPS include, but are not limited to, fibroblasts, keratinocytes, and adipocytes. These cells can be obtained from adults using state-of-the-art, well-known methods (Mochiduki and Okita, 2012).

[0035] "Hematopoietic stem cells" (HSCs) have the potential to completely reconstitute the immune system of the lethally irradiated host from which they are derived. Hematopoietic stem cells can give rise to all blood and immune cells.

[0036] Preferably, the cells, particularly myeloid lineage cells of HSC, are obtained from a biological sample from a donor affected by cancer, an autoimmune disease, or an inflammatory disease.

[0037] a first vector encoding at least one cytokine, preferably at least one interleukin, preferably IL-2; Interleukin-2 (IL-2) is an interleukin that induces the proliferation of responding T cells and NK cells. IL-2 enhances activation-induced cell death (AICD). IL-2 also promotes the differentiation of T cells into effector T cells and into memory T cells when primary T cells are also stimulated by antigen, thus helping the body fight off infection. Along with other polarizing cytokines, IL-2 inhibits the proliferation of naive CD4 + While stimulating the differentiation of T cells into Th1 and Th2 lymphocytes, it also blocks the differentiation into Th17 and follicular Th lymphocytes. IL-2 also increases the cell-killing activity of both natural killer cells and cytotoxic T cells.

[0038] Preferably, the IL-2 is human IL-2 (hIL-2).

[0039] Preferably, the IL-2 is encoded by the following nucleic acid sequence:

[0040] [ka]

[0041] Preferably, the IL-2 is encoded by the following amino acid sequence:

[0042] [ka]

[0043] The first vector for the modified myeloid cells, modified iPS cells, or modified HSCs according to the present invention comprises a sequence encoding IL-2 under the control of an inducible or constitutive promoter. Preferably, the sequence encoding IL-2 is inserted into a lentivector, preferably pCDH1, optionally containing a resistance gene, and under the control of an inducible or constitutive promoter. Preferably, the promoter is inducible.

[0044] Constitutive or inducible promoters are usually associated with constitutive or inducible genes, respectively. Constitutive genes are permanently transcribed genes, in contrast to facultative genes. Inducible genes are genes whose expression responds to stimuli such as environmental changes or the position in the cell cycle. Inducible genes are inactive unless an inducer is present, allowing the gene to be expressed. The use of such constitutive or inducible promoters in genetic engineering allows for the control of the transduced gene of interest to be regulated. Inducible promoters are generally preferred over constitutive promoters due to their reversibility and flexibility. In addition, compared to constitutive promoters, inducible promoters are generally more efficient and less likely to cause side effects such as cell death and growth or development retardation.

[0045] Preferably, the constitutive promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked to it. However, other constitutive promoter sequences can also be used, such as the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

[0046] Preferably, the first vector comprises an inducible promoter. Preferably, the inducible promoter is specific, i.e., it activates transcription primarily in response to CAR activation. An inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence to which it is operably linked when such expression is desired, or turning off expression when expression is not desired. Inducible promoters are well known in the art.

[0047] Preferably, the inducible promoter is selected from positive and negative inducible promoters. In the case of a positive inducible promoter, an activator protein binds to the promoter to initiate transcription. In contrast, in the case of a negative inducible promoter, the promoter is inactive because the bound repressor protein actively blocks transcription. Once the inducer binds to the repressor protein, the repressor protein can be removed from the DNA, activating transcription.

[0048] Inducible promoters can be activated in response to a stimulus such as the presence or absence and / or amount of a chemical, or a change in temperature and / or light.

[0049] Chemically controlled promoters are one of the most common inducible promoters. For example, the inducible positive tetracycline-ON (Tet-On) system works by direct activation. In this system, the reverse tetracycline-regulated transactivator (rtTA) is normally inactive and cannot bind to the tetracycline response element (TRE) in the promoter. Tetracycline and its derivatives are used as inducers to enable promoter activation. Inducible promoters can also be chemically inducible promoters, whose administration can be regulated over time or even locally. The administered chemical agent can be selected from a wide range of molecules, such as tetracycline, biotin, or a combination thereof, such as Tet-Off / Tet-On. The possibility of using photoimmunotherapy also allows for localized and time-limited action.

[0050] Cytokine-specific promoters are also known: they are activated once a cytokine binds to the promoter. Preferably, the cytokine-specific promoter is the interleukin-6 promoter or the interleukin-8 promoter.

[0051] Other examples include the negative inducible promoter pLac or the negative inducible promoter pBad.

[0052] Some promoters are temperature-inducible: they show little expression at normal temperatures but can be induced by exposure to heat or cold. For example, the Hsp70 promoter is inducible by heat shock.

[0053] Light is another means of activating gene expression, and two-component systems used in synthetic biology use light to control transcription. This requires light-sensitive proteins, such as YF1 (known as histidine kinase), that are capable of inducing a transcriptional response, including acting on the synthesis of repressors or inducers.

[0054] Thus, the inducible promoter is preferably selected from chemically regulated promoters, temperature-inducible promoters, and light-inducible promoters. Preferably, the inducible promoter is selected from cytokine-specific promoters (more preferably, interleukin-6 and interleukin-8 promoters), metallothionine promoters, glucocorticoid promoters, progesterone promoters, tetracycline promoters (e.g., Tet-On promoters), pLac, pBad, heat shock protein promoters (more preferably, Hsp70 promoters), and YF1. The inducible promoter can also be selected from artificial promoters containing a response element that is activated via CAR activation. An artificial promoter can be designed from a minimal promoter complemented with multiple binding sites for transcription factors such as NF-KB, AP1, or ISRE (IFN-sensitive response element). Preferably, the artificial promoter contains at least an ISRE. Preferably, the artificial promoter is the mouse INF-beta promoter (including an ISRE), a promoter complemented with an NF-KB response element, or a combination thereof. The promoter can also be promX of SEQ ID NO: 13. A preferred inducible promoter is a cytokine-specific promoter (more preferably, an interleukin-6 promoter, an interleukin-8 promoter, or an interferon beta promoter) that contains a response element (i.e., binding sequence) for NF-KB or an ISRE, or both. Preferably, the inducible promoter is promX of SEQ ID NO: 12, or a cytokine-specific promoter that contains a response element for NF-KB or an ISRE, and preferably, the inducible promoter is promX of SEQ ID NO: 13, or the promoter NF-KB of SEQ ID NO: 14.

[0055] The inducible promoter is preferably activated by the tumor environment surrounding the IL-2-expressing cells. Preferably, the inducible promoter is a cytokine-specific promoter. Preferably, the cytokine-specific promoter is the interleukin-6 promoter or the interleukin-8 promoter, or a synthetic promoter containing various elements capable of binding to transcription factors.

[0056] Inducible promoters are typically activated once the modified cells reach the tumor.

[0057] Preferably, according to the first option, the sequence encoding IL-2 is inserted into a lentivector, preferably pCDH1, under the control of a constitutive promoter. This option does not allow regulation of IL-2 expression.

[0058] Preferably, according to the second option, the sequence encoding IL-2 is inserted into a lentivector, preferably pCDH1, under the control of an inducible promoter.

[0059] The present invention also relates to modified cells expressing at least one cytokine, preferably at least one interleukin selected from IL-10, IL-15, IL-13, IL-7A, IFN alpha, IFN beta, IFN lambda, IFN gamma, IL-1A, IL-1B, IL-12, and IL-21, wherein the cells are selected from myeloid cells, induced pluripotent stem cells (iPS), and hematopoietic stem cells (HSC), and the cells comprise a first vector comprising a sequence encoding said interleukin under the control of an inducible or constitutive promoter, and optionally a second vector comprising a sequence encoding a chimeric antigen receptor (CAR), wherein the CAR is - an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells of the tumor microenvironment (TME); - optionally, a hinge domain; - a transmembrane domain, and - Intracellular signaling domains Includes:

[0060] All of the above embodiments for the first vector are also applicable to the modified cells expressing a cytokine, in which case the sequence of the expressible cytokine is introduced into the first vector in place of the sequence encoding IL-2.

[0061] Preferably, all of the above embodiments for the first vector are also applicable to said modified cells expressing an interleukin (different from IL-2), in which case the sequence of the expressible interleukin is introduced into the first vector instead of the sequence encoding IL-2.

[0062] Preferably, the modified myeloid cells, iPS or HSC according to the present invention comprise a first vector comprising a sequence encoding IL-2 under the control of an inducible or constitutive promoter, and a second vector further comprising a sequence encoding a chimeric antigen receptor (CAR), wherein the CAR is - an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells of the tumor microenvironment (TME); - optionally, a hinge domain; - a transmembrane domain, and - Intracellular signaling domains Includes:

[0063] Such cells are capable of binding to tumor antigens or antigens present on cells of the tumor microenvironment and typically exhibit targeted effector activities such as antigen-dependent phagocytosis of tumor cells.

[0064] A second vector carrying a chimeric antigen receptor (CAR) The modified myeloid cells, modified iPS cells, or modified HSCs of the present invention preferably further comprise a second vector comprising a sequence encoding a chimeric antigen receptor (CAR). The presence of both the first and second vectors is referred to as the "CAR+2 band."

[0065] In another embodiment, the CAR with its promoter may be contained in a first vector (i.e., a vector comprising at least one interleukin sequence). In such cases, the modified myeloid cells, modified iPS, or modified HSC of the present invention comprise only a first vector encoding both an interleukin and a CAR; said vector is referred to as a "CAR2 band." The CAR with its promoter may be upstream or downstream of at least one interleukin sequence. Preferably, the CAR with its promoter is downstream of at least one interleukin sequence. Preferably, the CAR with its promoter is downstream of an IL2 sequence. Preferably, the CAR2 band vector comprises the nucleic acid sequence of SEQ ID NO: 9. Thus, according to this embodiment, preferably, the modified myeloid cells, iPS, or HSC according to the present invention comprise a first vector ("CAR2 band") comprising a sequence encoding IL-2 under the control of an inducible or constitutive promoter and a sequence encoding a chimeric antigen receptor (CAR) under the control of an inducible or constitutive promoter, wherein said CAR is - an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells of the tumor microenvironment (TME); - optionally, a hinge domain; - a transmembrane domain, and - Intracellular signaling domains Includes: Preferably, the CAR2 band comprises a CAR having its promoter downstream of a sequence encoding IL-2, under the control of an inducible or constitutive promoter. In other words, preferably, the CAR2 band comprises a CAR having its promoter 3' to a sequence encoding IL-2, under the control of an inducible or constitutive promoter.

[0066] The CAR of the present invention comprises, from its N-terminus to its C-terminus: - an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells of the TME; - optionally, a hinge domain; - a transmembrane domain; and - Intracellular signaling domains Includes:

[0067] Between each domain, there may be the same or different linkers. Preferably, the CAR does not contain any linkers between different domains. In other words, the CAR is obtained by direct fusion of different domains.

[0068] The term "antigen-binding domain" refers to any polypeptide or fragment thereof, such as an antibody fragment variable domain, whether naturally occurring or synthetic, that binds to an antigen. Antigen-binding domains include, inter alia, antibody-derived polypeptides, such as single-chain variable fragments (scFv), Fab, Fab', F(ab')2, Fv fragments, and nanobodies; T-cell receptor (TCR)-derived polypeptides, such as TCR variable domains; and any ligand or receptor fragment that binds to an antigen. The antigen-binding domain has antigen specificity for a tumor antigen or TME antigen. An "antigen-binding domain with antigen specificity for a tumor antigen" is an antigen-binding domain that binds to an antigen on a tumor. An "antigen-binding domain with antigen specificity for a TME antigen" is an antigen-binding domain that binds to an antigen present on cells in the tumor microenvironment (TME). The TME includes tissues and cells surrounding the tumor; it includes, inter alia, surrounding blood vessels, immune cells such as Treg cells or immunosuppressive macrophages, fibroblasts, signaling molecules, and the extracellular matrix.

[0069] Preferably, the tumor antigen is selected from antigens that are expressed on the surface of tumor cells at higher levels than antigens expressed on other cell types. Preferably, the tumor antigen is CD19, MUC16, MUC1, CA1X, carcinoembryonic antigen (CEA), CD8, CD7, CD 10, CD20, CD22, CD30, 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, GD2Ac, GD3, ITER-2, hTERT, IL-13R-a2, K-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, ERBB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, EphA2, NKG2D ligand, NY-ES0-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, integrin B7, ICAM-1, CD70, Tim3, CLEC12A, ER, human telomerase reverse transcriptase (hTERT), mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2 / neu, p95HER2, Wilms tumor gene 1 (WT1), livin, alpha-fetoprotein (AFP), prostate-specific membrane antigen (PSMA), cyclin (DI), mesothelin, B-cell maturation antigen (BCMA), and tumor-associated calcium transducer and activator of plasma calcium (TROP2).

[0070] Preferably, the TME antigen is selected from an antigen expressed by activated CAFs, such as FAP (fibroblast activation protein), an antigen expressed by Tregs, and an antigen expressed by tumor-promoting myeloid cells, such as TREM-2. Preferably, the TME antigen is selected from an antigen expressed by FAP, an antigen expressed by Tregs, and TREM-2.

[0071] Preferably, the tumor antigen or TME antigen is CD 19. More preferably, the extracellular antigen-binding domain that binds to the tumor antigen or TME antigen is an anti-CD19 binding domain, preferably an anti-CD19 scFv.

[0072] Preferably, the extracellular antigen-binding domain comprises the following amino acid sequence:

[0073] [ka]

[0074] By "hinge domain" is meant any hinge domain present in an immunoglobulin or CD molecule.

[0075] Preferably, the hinge domain is that of CD8. CD8 comprises an alpha chain (CD8a) and a beta chain (CD8b). Preferably, the hinge domain is that of the CD8a chain.

[0076] The human version of CD8a can be found in Uniprot under the accession number Q8TAW8. CD8a contains 235 amino acids. The hinge domain is the fragment of amino acids 138 to 182 of said sequence, which corresponds to SEQ ID NO: 4.

[0077] Preferably, the hinge domain is that of CD8a, preferably human CD8a.

[0078] Preferably, the hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 4).

[0079] By "transmembrane domain" is meant a single- or multi-spanning membrane-spanning sequence.

[0080] Single-pass transmembrane domains are found in certain CD molecules, tyrosine kinase receptors, serine / threonine kinase receptors, TGFs, BMPs, activins, and phosphatases. They often contain a signal peptide region and a transmembrane domain of about 20 to about 25 amino acids, many of which are hydrophobic and capable of forming an alpha helix. A short track of positively charged amino acids often follows the transmembrane span, anchoring the protein to the membrane.

[0081] Multispanning transmembrane domains are present in proteins such as ion pumps, ion channels, and transporters and contain two or more helices that span the membrane multiple times.

[0082] Sequences for single-spanning and multi-spanning transmembrane domains are known and can be selected for incorporation into CARs.

[0083] The transmembrane domain can be selected from wild-type transmembrane domains and mutant transmembrane domains. Mutant transmembrane domains can be modified by mutations such as amino acid substitutions (e.g., substitution of typically charged amino acids with hydrophobic residues). Preferably, the transmembrane domain is that of the alpha, beta, or zeta chain of the T cell receptor, CD3-8, CD3 zeta, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD28, CD33, CD38, CD64, CD80, CD86, CD134, CD137, or CD154. Preferably, the transmembrane domain is a CD8 transmembrane domain.

[0084] Transmembrane domains can also be synthesized de novo and contain mostly hydrophobic residues such as leucine and valine.

[0085] According to the present invention, the transmembrane domain is fused at its N-terminus to the extracellular antigen-binding domain of the CAR and at its C-terminus to the intracellular signaling domain.

[0086] In certain embodiments, a short polypeptide linker may form the link between the transmembrane domain and the intracellular signaling domain of the CAR.

[0087] The CAR may further comprise a stalk, i.e., an extracellular region of amino acids between the extracellular antigen-binding domain and the transmembrane domain. For example, the stalk may be the amino acid sequence naturally associated with the selected transmembrane domain.

[0088] Preferably, the CAR comprises a CD8 transmembrane domain. Preferably, the CAR comprises a CD8 transmembrane domain and a CD8 hinge domain. The hinge domain is preferably fused at its C-terminus to the N-terminus of the transmembrane domain (preferably directly).

[0089] Preferably, the transmembrane domain comprises the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 5), which is that of human CD8.

[0090] Preferably, the hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 4).

[0091] "Intracellular domain" refers to the intracellular signaling domain at the C-terminus of the CAR. This intracellular domain can be selected depending on the targeted effector activity directed by the CAR. The intracellular domain can be derived from other receptors and can be designed to induce a predetermined cellular function, such as phagocytosis, inflammatory activation, or TME regulation.

[0092] The intracellular domain can include one or more intracellular signaling domains derived from a phagocytic receptor, a scavenger receptor, or an integrin receptor. For example, the intracellular domain can include one or more intracellular signaling domains that promote phagocytic activity, an inflammatory response, or integrin activation.

[0093] The intracellular signaling domain may be derived from a phagocytic receptor or a tethering receptor, or may comprise a phagocytic activation domain. Preferably, the intracellular signaling domain that promotes phagocytic activity (i.e., also referred to as a phagocytic activation domain) comprises an intracellular signaling domain derived from an FcγR, FcαR, or FcεR. In some embodiments, the intracellular signaling domain is derived from a receptor other than a phagocytic receptor selected from Megf10, MerTk, FcR-alpha, or Bai1. In some embodiments, the intracellular signaling domain is derived from a phagocytic receptor selected from lectin, Dectin 1, CD206, Scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fc-alpha receptor I, CR1, CD35, CR3, CR4, Tim-1, Tim-4, and CD169.

[0094] Preferably, the intracellular signaling domain that promotes an inflammatory response (i.e., also referred to as a pro-inflammatory signaling domain) comprises a PI3 kinase (PI3K) recruitment domain, preferably an intracellular signaling domain of TLR3, TLR4, TLR9, MYD88, TRIF, RIG-1, MDA5, IFN receptor, NLRP-1, NLRP-2, NLRP-3, NLRP-4, NLRP-5, NLRP-6, NLRP-7, NLRP-8, NLRP-9, NLRP-10, NLRP-11, NLRP-12, NLRP-13, NLRP-14, NOD1, NOD2, pyrin, AIM2, NLRC4, and / or CD40.

[0095] Preferably, the intracellular domain comprises at least two intracellular signaling domains comprising any of the following: (i) a first intracellular signaling domain derived from an FcγR or an FcεR, and (ii) a second intracellular signaling domain that includes (A) a PI3K recruitment domain or (B) is derived from CD40; or (i) a first intracellular signaling domain derived from a phagocytic receptor and (ii) a second intracellular signaling domain that includes (A) a PI3K recruitment domain or (B) is derived from CD40.

[0096] Examples of intracellular domains include fragments or domains from one or more molecules or receptors, including, but not limited to, STING, cGAS, TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, CD79a, CD79b, DAP10, DAP 12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80. (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD l id, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAMl, CRTAM, Ly9 (CD229), CD160 (BY55), PSGLl, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9.

[0097] Preferably, the intracellular signaling domain of the present invention comprises, from N- to C-terminus, a first intracellular signaling domain comprising the CD40 cytotail (cytoplasmic tail), which is fused to a second intracellular signaling domain comprising the CD3 zeta intracellular domain.

[0098] "CD40 cytotail" refers to the cytosolic domain of the CD40 molecule. CD40, also called TNFRSF5, is a costimulatory protein found on antigen-presenting cells and is required for their activation. The sequence of human CD40 (hCD40) can be found in Uniprot under accession number P25942. It contains 277 amino acids. The fragment containing amino acids 216 to 277 of said sequence is the cytosolic portion. Said fragment corresponds to SEQ ID NO:6.

[0099] Preferably, the intracellular signaling domain comprises the CD40 cytotail, which is a fragment of human CD40.

[0100] Preferably, the intracellular signaling domain comprises the following amino acid sequence:

[0101] [ka]

[0102] The first intracellular signaling domain is fused at its C-terminus to a second intracellular signaling domain comprising a CD3 zeta intracellular domain. Preferably, the fusion is direct, i.e., without any linker.

[0103] CD3 zeta, also known as OKT3 or CD247, is a member of the T cell receptor (TCR) complex. In humans, in 95% of T cells, the TCR consists of an alpha chain and a beta chain, while in 5% of T cells, the TCR consists of a gamma chain and a delta chain. At the plasma membrane, the alpha and beta chains of the TCR associate with six additional adaptor proteins to form an octameric complex. The complex contains not only both the alpha and beta chains, which form the ligand-binding site, but also one CD3 gamma chain, one CD3 delta chain, two CD3 epsilon chains, and two CD3 zeta chains.

[0104] The sequence of the human CD3 zeta chain (hCD3 zeta) can be found in Uniprot under the accession number P20963. It contains 164 amino acids. The fragment containing amino acids 52 to 164 of said sequence is the cytosolic part. Said fragment corresponds to SEQ ID NO: 7.

[0105] Preferably, the second intracellular signaling domain comprises a human CD3 zeta intracellular domain.

[0106] Preferably, the second intracellular signaling domain comprises the following amino acid sequence:

[0107] [ka]

[0108] Preferably, the intracellular signaling domain is a first intracellular signaling domain comprising the CD40 cytoplasmic tail, preferably of sequence SEQ ID NO: 6, or a first intracellular signaling domain comprising the CD40 cytoplasmic tail, preferably of sequence SEQ ID NO: 6, fused to a second intracellular signaling domain comprising the CD3 zeta intracellular domain, preferably of sequence SEQ ID NO: 7.

[0109] Preferably, the modified cells of the present invention are myeloid cells and comprise a first vector comprising a sequence encoding IL-2 under the control of an inducible or constitutive promoter, and a second vector comprising a sequence encoding a chimeric antigen receptor (CAR), wherein the CAR is - an extracellular antigen-binding domain with antigen specificity for a tumor antigen or a TME antigen; - optionally, a hinge domain; - a transmembrane domain; and - an intracellular signaling domain comprising STING or one of its fragments Includes:

[0110] Such cells are referred to in this application as "modified myeloid cells bearing a CAR comprising STING or one of its fragments."

[0111] The above embodiments and definitions for engineered myeloid cells expressing a CAR, excluding the intracellular signaling domain, also apply to engineered myeloid cells with a CAR comprising STING or one of its fragments.

[0112] Preferably, the modified myeloid cells according to the invention comprise a first vector ("CAR2 band") comprising a sequence encoding IL-2 under the control of an inducible or constitutive promoter, and a sequence encoding a chimeric antigen receptor (CAR) under the control of an inducible or constitutive promoter, said CAR comprising: - an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells of the tumor microenvironment (TME); - optionally, a hinge domain; - a transmembrane domain, and - an intracellular signaling domain comprising STING or one of its fragments Includes:

[0113] Preferably, the CAR2 band comprises a CAR having its promoter downstream of a sequence encoding IL-2, under the control of an inducible or constitutive promoter. In other words, preferably, the CAR2 band comprises a CAR having its promoter 3' to a sequence encoding IL-2, under the control of an inducible or constitutive promoter.

[0114] The intracellular signaling domain of the engineered myeloid cells bearing a CAR comprising STING or one of its fragments comprises STING or one of its fragments.

[0115] The stimulator of interferon genes (STIMULATOR) (STING) protein is an endoplasmic reticulum (ER)-resident protein that plays a central role in innate immunity. Indeed, STING is an adaptor protein that orchestrates the transcriptional activation of type I interferons and proinflammatory cytokines in the presence of pathological nucleic acid species. STING activation depends on the detection of dsDNA, ssDNA, or RNA:DNA hybrids by the cyclic GMP-AMP synthetase (cGAS) pathogen-recognition receptor. Association of cGAS with these nucleic acid species in the cytosol was found to result in the cGAS-dependent synthesis of cyclic GMP-AMP (cGAMP). Interaction of cGAMP with STING activates pathways that ultimately lead to the transcription of proinflammatory cytokines and type I interferons.

[0116] The sequence of human STING can be found in Uniprot under accession number A0A2R3XZB7. It contains 379 amino acids. Preferably, a sequence with several amino acid deletions at the N-terminus is used. STING can be used in its wild-type version or in a mutant form that can attenuate its activity.

[0117] Preferably, a fragment of STING is used, preferably a fragment containing an N-terminal deletion. Preferably, the fragment corresponds to amino acids 137 to 379 of A0A2R3XZB7.

[0118] Preferably, the intracellular signaling domain comprises the following amino acid sequence:

[0119] [ka]

[0120] Preferably, the intracellular signaling domain is encoded by the nucleic acid sequence of SEQ ID NO:10.

[0121] The present invention further provides a modified cell comprising a CAR, wherein said CAR comprises: - an extracellular antigen-binding domain with antigen specificity for a tumor antigen or a TME antigen; - a transmembrane domain; and - Intracellular signaling domain containing a chimeric fragment of STING wherein the modified cell is a myeloid cell.

[0122] The above embodiments and definitions for CAR myeloid cells, excluding the intracellular signaling domain, are also valid for such modified myeloid cells bearing a CAR comprising a chimeric fragment of STING.

[0123] "Chimeric fragment of STING" refers to a modified STING C-terminal tail (CTT). The modified STING CTT contains two sequence motifs known as IRF3 and TBK1, and optionally, an additional fish-specific NF-κB motif. Preferably, the modified STING CTT is a chimeric construct in which the IRF3 motif is derived from human STING, and the TBK1 and NF-κB motifs are derived from human STING or fish STING. Preferably, the fish STING is zebrafish STING. Preferably, the modified STING CTT is a chimeric construct in which the IRF3 and TBK1 motifs are derived from human STING, and the NF-κB motif is derived from zebrafish STING (the CAR is referred to as "CAR-STINGtz"). Preferably, CAR-STINGtz comprises the following amino acid sequence:

[0124] [ka]

[0125] Preferably, CAR-STINGtz is encoded by the following nucleic acid sequence:

[0126] [ka]

[0127] The intracellular signaling domain comprising STING or one of its fragments further comprises a CD40 cytotail, preferably as described above, and / or a CD3 zeta intracellular domain, preferably as described above.

[0128] Preferably, the CAR comprises, from its N-terminus to its C-terminus: - an extracellular antigen-binding domain of sequence SEQ ID NO: 3, - optionally a hinge domain of sequence SEQ ID NO: 4, - a transmembrane domain of sequence SEQ ID NO: 5, - either a first intracellular signalling domain of sequence SEQ ID NO: 6, or a first intracellular signalling domain of sequence SEQ ID NO: 6 fused, preferably directly, with a second intracellular signalling domain of sequence SEQ ID NO: 7; Includes:

[0129] Preferably, the CAR comprises, from its N-terminus to its C-terminus: - an extracellular antigen-binding domain of sequence SEQ ID NO: 3, - optionally a hinge domain of sequence SEQ ID NO: 4, - a transmembrane domain of sequence SEQ ID NO: 5, - a first intracellular signaling domain of sequence SEQ ID NO: 6 fused, preferably directly, with a second intracellular signaling domain of sequence SEQ ID NO: 7; Includes:

[0130] Preferably, the CAR comprises, from its N-terminus to its C-terminus: - an extracellular antigen-binding domain of sequence SEQ ID NO: 3, - optionally a hinge domain of sequence SEQ ID NO: 4, - a transmembrane domain of sequence SEQ ID NO: 5, and - the intracellular signaling domain of sequence SEQ ID NO: 11 Includes:

[0131] The modified myeloid cells, iPS cells, or HSC cells according to the present invention, comprising the first and second vectors, preferably exhibit targeted effector activity. "Targeted effector activity" refers to at least one effector activity selected from phagocytosis, targeted cytotoxic activity, cytokine production, reactive oxygen species (ROS) generation, myeloid activation, antigen processing and presentation to T cells, and the in vivo ability to induce human antigen-dependent tumor regression in NSG mice complemented with human T cells. Preferably, the targeted effector activity is selected from antigen-dependent phagocytosis of tumor cells, antigen-dependent tumor cell cytokine secretion, and the in vivo ability to induce human antigen-dependent tumor regression in NSG mice complemented with human T cells. The human antigen-dependent tumor regression is likely mediated by both macrophage phagocytosis of tumor cells and tumor-specific T cell killing of tumor cells. Antigen-dependent phagocytosis of tumor cells and antigen-dependent tumor cell cytokine secretion can be measured according to methods well known in the art and are illustrated in the Examples. The in vivo ability to induce human antigen-dependent tumor regression in NSG mice complemented with human T cells will be assessed according to the protocol described in the Examples.

[0132] All of the above embodiments for the second vector are also applicable to modified cells that express cytokines.

[0133] All of the above embodiments for the second vector are also applicable to modified cells that express an interleukin (different from IL-2), in which case the sequence of the expressible interleukin is introduced into the first vector in place of the sequence encoding IL-2.

[0134] In another embodiment, the present invention also relates to a modified cell expressing at least one cytokine, preferably an interleukin, wherein the cell is selected from a myeloid cell, an induced pluripotent stem cell (iPS), and a hematopoietic stem cell (HSC), and the cell comprises a first vector comprising a sequence encoding said cytokine under the control of an inducible or constitutive promoter, and optionally a second vector comprising a sequence encoding a CAR, wherein said CAR comprises at least a CD40 cytotail as an intracellular signaling domain. In this embodiment, the modified cell is preferably a macrophage.

[0135] In another embodiment, the present invention also relates to an engineered cell that expresses at least one interleukin selected from IL-2, IL-10, IL-15, IL-13, IL-7A, IFN-alpha, IFN-beta, IFN-lambda, IFN-gamma, IL-1A, IL-1B, IL-12, and IL-21, wherein the cell is selected from a myeloid cell, an induced pluripotent stem cell (iPS), and a hematopoietic stem cell (HSC), and the cell comprises a first vector comprising a sequence encoding the interleukin under the control of an inducible or constitutive promoter, and optionally a second vector comprising a sequence encoding a CAR, wherein the CAR comprises at least a CD40 cytotail as an intracellular signaling domain. In this embodiment, the engineered cell is preferably a macrophage.

[0136] The present invention also relates to a nucleic acid sequence encoding a CAR according to the present invention. The nucleic acid sequence may be a DNA or RNA sequence. The nucleic acid sequence may be used therapeutically, in particular for treating cancer, autoimmune diseases, or inflammatory diseases. Preferably, the nucleic acid sequence is administered to a subject, preferably by injection. Thus, the subject's macrophages receive the nucleic acid sequence and subsequently express a CAR, in particular a CAR comprising STING or one of its fragments, preferably CAR-STINGtz.

[0137] Preferably, the nucleic acid sequence encoding the intracellular domain of CAR-STINGtz is the sequence of SEQ ID NO: 12. Preferably, the nucleic acid sequence comprises the sequence of SEQ ID NO: 12.

[0138] therapeutic use The present invention also relates to the use of the modified myeloid cells, modified iPS cells, or modified HSCs according to the present invention as a pharmaceutical.

[0139] The present invention also relates to a pharmaceutical composition comprising the modified myeloid cells, modified iPS cells, or modified HSCs according to the present invention and a pharmaceutically acceptable carrier.

[0140] The present invention also relates to the use of the modified myeloid cells, modified iPS cells, or modified HSCs according to the present invention, or the pharmaceutical composition described above, in the treatment of cancer, an autoimmune disease, or an inflammatory disease. The inflammatory disease may be an autoimmune disease.

[0141] All of these embodiments also apply to modified cells that express cytokines.

[0142] All of these embodiments also apply to modified cells that express an interleukin (different from IL-2), where the sequence of the interleukin that can be expressed is introduced into the first vector in place of the sequence encoding IL-2.

[0143] Preferably, the myeloid cells are obtained from a blood sample of the patient (donor) to be treated. Preferably, they are modified and reinfused back into the patient (donor), i.e., they are autologous.

[0144] By "autologous," according to the present invention, it is meant that the modified myeloid cells or a therapeutic composition comprising the modified myeloid cells are used as a treatment for the patient who is the source of the myeloid cells.

[0145] The present invention also relates to products containing modified myeloid cells, modified iPS cells, or modified HSC cells and CAR-T cells according to the present invention as a combined preparation for simultaneous, separate, or sequential use in the treatment of cancer, autoimmune diseases, or inflammatory diseases.

[0146] CAR-T cells are well known in the art. Preferably, the CAR-T cells are selected from tisagenlecleucel, axicabtagene ciloreucel, brexcabtagene outrucel, lisocabtagene maraleucel, and idecabtagene bicrueucel.

[0147] The present invention also relates to a product containing modified myeloid cells, modified iPS cells, or modified HSCs according to the present invention and an immune checkpoint inhibitor (ICI) as a combined preparation for simultaneous, separate, or sequential use in the treatment of cancer, autoimmune diseases, or inflammatory diseases.

[0148] An "immune checkpoint inhibitor" refers to any compound that inhibits the function of an immune checkpoint protein. Inhibition includes reduction and complete blocking of function. In particular, the immune checkpoint protein is a human immune checkpoint protein. Thus, the immune checkpoint protein inhibitor is preferably an inhibitor of a human immune checkpoint protein.

[0149] Immune checkpoint proteins that may be cited are CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR (e.g. KIR3DL2, KIR2DL1 / 2 / 3, KIR2L3), TIGIT, VISTA, IDO, CEACAM-1, or A2aR.

[0150] Immune checkpoint inhibitors can be drugs such as small molecules, recombinant forms of ligands or receptors, or preferably antibodies, such as human antibodies. Known inhibitors of immune checkpoint proteins or analogs thereof can be used, particularly chimeric, humanized, or human antibodies.

[0151] Preferably, the ICI is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR (e.g., KIR3DL2, KIR2DL1 / 2 / 3, KIR2L3), TIGIT, VISTA, IDO, CEACAM-1, or A2aR. Preferably, the ICI is an anti-CTLA-4 antibody, more preferably tremelimumab or ipilimumab. In certain embodiments, the ICI is an anti-killer cell immunoglobulin-like receptor (KIR) antibody, more preferably lirilumab and IPH4102. Preferably, the ICI is an anti-PD-1 antibody, more preferably nivolumab (ONO-4538, BMS-936558, MDX1106, GTPL7335, or Opdivo), pembrolizumab (MK-3475, MK03475, lambrolizumab, SCH-900475, or Keytruda), pidilizumab, AMP-514, cemiplimab (REGN2810), CT-011, BMS 936559, MPDL3280A, AMP-224, tislelizumab (BGB-A317), spartalizumab (PDR001 or PDR-001), ABBV-181, JNJ-63723283, BI 754091, MAG012, TSR-042, AGEN2034, and the antibodies described in International Patent Applications WO2004004771, WO2004056875, WO2006121168, WO2008156712, WO2009014708, WO2009114335, WO2013043569, and WO2014047350. Preferably, the PD-L1 inhibitor is durvalumab, atezolizumab, LY3300054, or avelumab. Preferably, the PD-L2 inhibitor is rHIgM12B7. Preferably, the LAG3 inhibitor is IMP321, BMS-986016, or an inhibitor of the LAG3 receptor described in US Patent US 5,773,578. Preferably, the inhibitor of A2aR is PBF-509.Preferably, the CTLA-4 inhibitor is an anti-CTLA-4 antibody, including, but not limited to, ipilimumab (see, e.g., U.S. Patents US6,984,720 and US8,017,114), tremelimumab (see, e.g., U.S. Patents US7,109,003 and US8,143,379), single-chain anti-CTLA4 antibodies (see, e.g., International Patent Applications WO1997020574 and WO2007123737), and antibodies described in U.S. Patent US8,491,895. An example of an anti-VISTA antibody is described in U.S. Patent Application US20130177557. Preferably, the ICI is selected from tremelimumab, ipilimumab, lirilumab, nivolumab, pembrolizumab, pidilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL3280A, AMP-224, durvalumab, atezolizumab, avelumab, rHIgM12B7, IMP321, BMS-986016, and PBF-509.

[0152] The present invention also relates to products containing modified myeloid cells, modified iPS cells, or modified HSC cells according to the present invention and immune checkpoint therapies associated with costimulatory antibodies that deliver positive signals through immunoregulatory receptors, including but not limited to ICOS, CD137, CD27, OX-40, and GITR, as a combined preparation for simultaneous, separate, or sequential use in the treatment of cancer, autoimmune diseases, or inflammatory diseases.

[0153] The present invention also relates to products containing modified myeloid cells, modified iPS cells, or modified HSCs according to the present invention and an additional cancer therapy as a combined preparation for simultaneous, separate, or sequential use in the treatment of cancer. In particular, products containing modified myeloid cells, modified iPS cells, or modified HSCs according to the present invention may be administered in combination with targeted therapy, immunotherapy such as immune checkpoint therapy and / or immune checkpoint inhibitors, costimulatory antibodies, chemotherapy, and / or radiation therapy.

[0154] In some embodiments, products containing modified myeloid cells, modified iPS cells, or modified HSCs according to the present invention can be used in combination with targeted therapy. As used herein, the term "targeted therapy" refers to targeted therapeutic agents, which are drugs designed to interfere with specific molecules necessary for tumor growth and progression. For example, targeted therapeutic agents, such as therapeutic monoclonal antibodies, target specific antigens found on the cell surface, such as transmembrane receptors or extracellular growth factors. Small molecules can penetrate the cell membrane and interact with intracellular targets. Small molecules are usually designed to interfere with the enzymatic activity of target proteins, such as proteasome inhibitors, tyrosine kinase or cyclin-dependent kinase inhibitors, and histone deacetylase inhibitors. Targeted therapy can also use cytokines.Examples of such targeted therapies include: Ado-trastuzumab emtansine (HER2), afatinib (EGFR (HER1 / ERBB1), HER2), aldesleukin (Proleukin), alectinib (ALK), alemtuzumab (CD52), axitinib (kit, PDGFR beta, VEGFR1 / 2 / 3), belimumab (BAFF), belinostat (HDAC), bevacizumab (VEGF ligand), blinatumomab (CD19 / CD3), bortezomib (proteasome), brentuximab Vedotin (CD30), bosutinib (ABL), brigatinib (ALK), cabozantinib (FLT3, KIT, MET, RET, VEGFR2), canakinumab (IL-1beta), carfilzomib (proteasome), ceritinib (ALK), cetuximab (EGFR), cobimetinib (MEK), crizotinib (ALK, MET, ROS1), dabrafenib (BRAF), daratumumab (CD38), dasatinib (ABL), denosumab (RANKL), dinutuximab (B4GALNT1) (GD2), elotuzumab (SLAMF7), enasidenib (IDH2), erlotinib (EGFR), everolimus (mTOR), gefitinib (EGFR), ibritumomab Tiuxetan (CD20), sonidegib (Smoothened), sipuleucel-T, siltuximab (IL-6), sorafenib (VEGFR, PDGFR, KIT, RAF), tocilizumab (IL-6R), temsirolimus (mTOR), tofacitinib (JAK3), trametinib (MEK), tositumomab (CD20), trastuzumab (HER2), vandetanib (EGFR), vemurafenib (BRAF), venetoclax (BCL2), vismodegib (PTCH, Smoothened), vorinostat (HDAC), Ziv-aflibercept (PIGF, VEGFA / B), olaparib (PARP inhibitor).

[0155] In some embodiments, products containing modified myeloid cells, modified iPS cells, or modified HSCs according to the present invention may be used in combination with chemotherapy. As used herein, the term "anti-tumor chemotherapy" or "chemotherapy" has its general meaning in the art and refers to cancer therapeutic treatment using chemical or biochemical agents, particularly one or several anti-neoplastic or chemotherapeutic agents. Chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including their synthetic analogs adozelesin, carzelesin, and biceresin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including their synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatins; chlorambucil, chlornaphazine, chorofosfamidis, Nitrogen mustards such as cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine;Antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma II and calicheamicin omega II); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin; as well as neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin n), carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potofilomycin, puromycin, chelamycin, rodolubicin antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; Androgens such as lusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziconazole; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate;Hydroxyurea; Lentinan; Lonidamine; Maytansinoids such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidamol; Nitraerin; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine; PSK polysaccharide complex; Razoxane; Rhizoxin; Sizofuran; Spirogermanium; Tenuazonic acid; Triazicone; 2,2',2"-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, verlaculin A, roridin A, and anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; gemcitabine; 6-thioguanine; mercaptopurine; platinum complex compounds such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunorubicin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; anthracyclines, nitrosoureas, antimetabolites, epipodophyllotoxins, enzymes such as L-asparaginase; anthracenediones; antagonists including hormone and adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone, and aminoglutethimide; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs, and leuprolide;and nonsteroidal antiandrogens such as flutamide; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0156] In some embodiments, products containing modified myeloid cells, modified iPS cells, or modified HSCs according to the present invention are administered to a patient in combination with radiation therapy for simultaneous, separate, or sequential use in the treatment of cancer, autoimmune diseases, or inflammatory diseases. Suitable examples of radiation therapy include external beam radiation therapy (e.g., superficial X-ray therapy, orthovoltage X-ray therapy, megavoltage X-ray therapy, radiosurgery, stereotactic radiotherapy, fractionated stereotactic radiotherapy, cobalt therapy, electron beam therapy, fast neutron therapy, neutron capture therapy, proton beam therapy, intensity-modulated radiation therapy (IMRT), three-dimensional conformal radiation therapy (3D-CRT), and the like); brachytherapy; non-brachytherapy; tomotherapy; and the like. Gamma rays are another form of photons used in radiation therapy. Gamma rays are spontaneously produced when certain elements (e.g., radium, uranium, and cobalt-60) emit radiation during decomposition or decay. In some embodiments, the radiation therapy can be proton radiation therapy or proton minibeam radiation therapy.Proton radiotherapy is an ultra-high precision form of radiation therapy that uses proton beams (Prezado Y, Jouvion G, Guardiola C, Gonzalez W, Juchaux M, Bergs J, Nauraye C, Labiod D, De Marzi L, Pouzoulet F, Patriarca A, Dendale R, Tumor Control in RG2 Glioma-Bearing Rats: A Comparison Between Proton Minibeam Therapy and Standard Proton Therapy. Int J Radiat Oncol Biol Phys. 2019 / 06 / 1;104(2):266~271, doi: 10.1016 / j.ijrobp.2019.01.080;Prezado Y, Jouvion G, Patriarca A, Nauraye C, Guardiola C, Juchaux M, Lamirault C, Labiod D, Jourdain L, Sebrie C, Dendale R, Gonzalez W, Pouzoulet F. Proton minibeam radiation therapy widens the therapeutic index for high-grade gliomas. Sci Rep. 2018 Nov 7;8(1):16479, doi: 10.1038 / s41598-018-34796-8). The radiation therapy can also be FLASH radiotherapy (FLASH-RT) or FLASH proton irradiation.FLASH radiotherapy involves the ultrafast delivery of radiation treatments at spatial doses orders of magnitude higher than those currently used in routine clinical practice (ultra-high dose rate) (Favaudon V, Fouillade C, Vozenin MC, The radiotherapy FLASH to save healthy tissues. Med Sci (Paris) 2015; 31: 121-123, DOI: 10.1051 / medsci / 20153102002); Patriarca A., Fouillade CM, Martin F., Pouzoulet F., Nauraye C. et al., Experimental setup for FLASH proton irradiation of small animals using a clinical system. Int J Radiat Oncol Biol Phys, 102 (2018), 619-626, doi: 10.1016 / j.ijrobp.2018.06.403). Electronic publication July 11, 2018).

[0157] Also described is a method for treating cancer, an autoimmune disease, or an inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of modified myeloid cells, modified iPS cells, or modified HSCs according to the present invention.

[0158] 1. A method for treating cancer, an autoimmune disease, or an inflammatory disease, comprising the steps of: - collecting myeloid cells from the patient; - modifying at least one of said myeloid cells by transducing said cells with a first vector comprising a sequence encoding IL-2 under the control of an inducible or constitutive promoter, and optionally a second vector, preferably a lentiviral vector, comprising a nucleic acid sequence encoding a CAR; and - reinfusing the modified myeloid cells into the patient. A method is also described, including:

[0159] By "cancer," the present invention refers to tumors. Tumors that can be treated include primary tumors and metastatic tumors, as well as refractory tumors. Refractory tumors include tumors that do not respond to or are resistant to treatment with chemotherapeutic agents alone, antibodies alone, radiation alone, or combinations thereof. Refractory tumors also include tumors that appear to be suppressed by treatment with such agents but recur up to five years, and sometimes up to ten years or more, after treatment is discontinued.

[0160] Examples of cancers that can be treated with the modified myeloid cells according to the present invention include, but are not limited to, cancer cells derived from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testes, tongue, or uterus. In addition, the cancer may specifically be of the following histological types, but is not limited to: neoplasm, malignant tumor; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; hair matrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; adenocarcinoma, familial polyposis; solid carcinoma; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma. Chromophobe carcinoma; acidophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid adenocarcinoma; skin adnexal carcinoma; apocrine adenocarcinoma Adenocarcinoma; papillary serous cystic adenocarcinoma; mucinous cystic adenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; Tumor, malignant; granulosa cell tumor, malignant; and loblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomus angiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar striated melanoma Sarcoma; Stromal sarcoma; Mixed tumor, malignant; Mixed Müllerian tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymoma, malignant; Brenner tumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma; Mesothelioma, malignant; Dysgerminoma; Embryonal carcinoma; Teratoma, malignant; Ovarian goiter, malignant; Choriocarcinoma; Mesonephroma, malignant; Angiosarcoma; Hemangioendothelioma, malignant; Kaposi's sarcoma; Hemangiopericytoma, malignant; Lymphangiosarcoma; Osteosarcoma; Paracortical osteosarcoma; Chondrosarcoma; Chondroblastoma, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor, malignant; Ameloblastic odontosarcoma;Ameloblastoma, malignant; Ameloblastic fibrosarcoma; Pinealoma, malignant; Chordoma; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrillary astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal tumor; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granular cell tumor, malignant; Malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; Paragranuloma Lymphoma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0161] Preferably, the cancer is a solid tumor or metastasis.

[0162] Examples of autoimmune diseases that can be treated with modified myeloid cells according to the present invention include, but are not limited to, rheumatoid arthritis, inflammatory bowel disease (Crohn's disease, ulcerative colitis), or multiple sclerosis.

[0163] "Treatment" or "treating" refers to both prophylactic or preventative treatment and curative or disease-modifying treatment, and includes treatment of subjects at risk of or suspected of having contracted a disease, as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, including the suppression of clinical recurrence. Treatment may be administered to a subject who has or is likely to eventually acquire a medical disorder in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurrent disorder, or to prolong the subject's survival beyond that which would be expected in the absence of such treatment.

[0164] "Therapeutic regimen" means a pattern of treatment for a disease, e.g., a pattern of medications used during treatment. A therapeutic regimen can include an induction regimen and a maintenance regimen.

[0165] The phrase "induction regimen" or "induction phase" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide a subject with high levels of a drug during the initial period of a treatment regimen. An induction regimen may use (in part or in whole) a "loading regimen," which may involve administering a higher dose of the drug than the physician would use during a maintenance regimen, administering the drug more frequently than the physician administers the drug during a maintenance regimen, or both.

[0166] The phrase "maintenance regimen" or "maintenance phase" refers to a therapeutic regimen (or portion of a therapeutic regimen) used to maintain a subject during disease treatment, e.g., to keep the subject in remission for an extended period of time (months or years). A maintenance regimen can use continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon arrival of certain predetermined criteria (e.g., disease symptoms)).

[0167] By "therapeutically effective amount" is meant a sufficient quantity of modified myeloid cells, modified iPS cells, or modified HSC cells according to the present invention to treat a disease (e.g., cancer) at a reasonable benefit / risk ratio applicable to any medical treatment. It is understood that the total daily usage of the products of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and excretion rate of the particular compound used; the duration of treatment; drugs used in combination with or concomitantly with the product; and similar factors well known in the medical arts. For example, it is well within the skill of one in the art to start administering a compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0168] "Pharmaceutical" or "pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when administered to mammals, particularly humans, as appropriate. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical, or rectal administration, the active ingredient, alone or in combination with another active ingredient, can be administered to animals and humans in unit dosage form in admixture with a conventional pharmaceutical carrier.

[0169] Typically, pharmaceutical compositions contain a pharmaceutically acceptable vehicle for injectable formulations. These may be in particular isotonic, sterile saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, and the like, or mixtures of such salts), or dry, especially lyophilized, compositions that can be reconstituted as injectable solutions by the addition of sterile water or saline, if desired. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions containing the compounds of the invention as free bases or pharmaceutically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The product can be formulated into a composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the protein's free amino groups), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the required amount of the active polypeptide in an appropriate solvent with some of the other ingredients listed above, followed by filtered sterilization, if necessary. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield powders from previously sterile-filtered solutions of the active ingredient(s) plus any additional desired ingredients. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the injectable solutions described above, although drug-release capsules and the like can also be used. For example, for parenteral administration in aqueous solution, the solution should be suitably buffered, if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art in light of the present disclosure. For example, one dosage can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the proposed infusion site. Some variation in dosage will necessarily occur depending on the condition of the subject to be treated, and the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0170] The first and / or second vector, or the CAR2 band, can be administered via at least one lipid nanoparticle (LNP), at least one liposome, or at least one virus-like particle (VLP). LNP refers to stable nucleic acid-lipid nanoparticles, particularly in the field of nucleic acid and mRNA drug delivery systems. Liposome refers to a spherical vesicle made of a lipid bilayer. LNP or liposome comprises at least one ionizable lipid and at least one nucleic acid molecule. Preferably, LNP or liposome further comprises at least one helper lipid. Preferably, the helper lipid is selected from phospholipids, cholesterol lipids, and polymers.

[0171] The phospholipid may typically be selected from dioleoyl-phosphatidylethanolamine (DOPE) or a derivative thereof, distearoylphosphatidylcholine (DSPC) or a derivative thereof, distearoyl-phosphatidylethanolamine (DSPE) or a derivative thereof, stearoyloleoylphosphatidylcholine (SOPC) or a derivative thereof, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) or a derivative thereof, N-(2,3-dioleoyl)propyl)-N,N,N-trimethylammonium chloride (DOTAP) or a derivative thereof, or any combination thereof.

[0172] The cholesterol lipid can be cholesterol or a derivative thereof.

[0173] The polymer may be polyethylene glycol (PEG) or a derivative thereof.

[0174] The nucleic acid molecule is a DNA molecule or an RNA molecule. In some embodiments, the nucleic acid molecule is a cDNA, mRNA, miRNA, siRNA, sgRNA, modified RNA, antagomir, antisense molecule, guide RNA molecule, CRISPR guide RNA molecule, peptide, therapeutic peptide, targeting nucleic acid, or any combination thereof.

[0175] In some embodiments, the liposome is a ligand-targeted liposome, the surface of which is functionalized with at least one targeting ligand. The targeting ligand may be selected from antibodies, agonist peptides, and aptamers, which enable precise delivery of the first and / or second vector to specific cells, such as myeloid cells, preferably monocytes or macrophages, by recognizing the corresponding receptor or antigen.

[0176] Virus-like particles (VLPs) are vesicles with a hollow core and an envelope that mimic a virus but are not infectious. They can be composed of viral structural proteins that self-assemble into virus-like structures, chemical compounds, or polymers arranged in multiple layers surrounding a hollow core. The VLP can package a nucleic acid sequence corresponding to a transcription unit encoding a CAR and, optionally, at least one cytokine, preferably at least one interleukin.

[0177] The present invention also relates to a method for delivering at least one mRNA molecule encoding a first and / or second vector, or a CAR2 band, to a subject in need thereof. In some embodiments, the LNP or a composition thereof delivers the first and / or second vector, or the mRNA molecule encoding the CAR2 band, to a target, preferably a myeloid cell. The method can include a single administration or multiple administrations of the LNP or a composition thereof. In some embodiments, the LNP or a composition thereof is administered by a delivery route selected from the group consisting of intradermal, subcutaneous, intramuscular, intracerebroventricular, intrathecal, oral delivery, intravenous, intratracheal, intraperitoneal, intrauterine delivery, or any combination thereof.

[0178] Preparation method The present invention also provides a method for producing modified myeloid cells, modified iPS cells, or modified HSCs, comprising the steps of: - providing at least one cell selected from isolated myeloid cells, iPS cells, and HSCs; - transducing the cells with at least a first vector, preferably a lentiviral vector, comprising a nucleic acid sequence encoding IL-2 or at least one interleukin selected from IL-10, IL-15, IL-13, IL-7A, IFN alpha, IFN beta, IFN lambda, IFN gamma, IL-1A, IL-1B, IL-12, and IL-21, under the control of an inducible or constitutive promoter; and optionally a second vector, preferably a lentiviral vector, comprising a sequence encoding a CAR. The present invention relates to a method, comprising:

[0179] The CAR is - an extracellular antigen-binding domain that binds to a tumor antigen or a TME antigen; - optionally, a hinge domain; - a transmembrane domain; and - First intracellular signaling domain Includes: A first intracellular signaling domain comprising a CD40 cytotail, preferably fused to a second intracellular signaling domain comprising a CD3 zeta intracellular domain. Includes:

[0180] The first step in the preparation method is the provision of at least one cell selected from isolated myeloid cells, iPS cells, or HSCs.

[0181] The cells are then transduced with at least a first vector, preferably a lentiviral vector, containing a sequence encoding IL-2 under the control of an inducible or constitutive promoter. The first vector can be used to introduce IL-2 into isolated myeloid cells, preferably monocytes.

[0182] Preferably, the cells are transduced with two vectors, preferably lentiviral vectors, a first vector comprising a sequence encoding IL-2 and a second vector comprising a nucleic acid sequence encoding a CAR, under the control of an inducible or constitutive promoter. The vectors can be used to introduce CAR and IL-2 into isolated myeloid cells, preferably monocytes.

[0183] Alternatively, the cells can be transduced with a single vector (i.e., a first vector) comprising a sequence encoding IL-2 under the control of an inducible or constitutive promoter and a nucleic acid sequence encoding a CAR under the control of a promoter.

[0184] In one embodiment, the vector is a plasmid vector, a viral vector, a retrotransposon (e.g., piggyback, sleeping beauty) or a site-specific insertion vector (e.g., CRISPR, zinc finger nuclease, TALEN). Preferably, the vector is a viral vector, preferably a lentiviral vector. Vectors, including vectors derived from retroviruses such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have the additional advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells. They also have the additional advantage of causing low immunogenicity in subjects into which they are introduced.

[0185] Expression of natural or synthetic nucleic acids is typically achieved by operably linking the nucleic acid to a promoter and incorporating the construct into an expression vector. The vector is generally capable of replication in mammalian cells and / or integration into the mammalian cell genome. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for controlling the expression of the desired nucleic acid sequence.

[0186] The nucleic acid sequence (nucleic acid) encoding the CAR or the IL-2 can be cloned into any number of different types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, or a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0187] The expression vector can be administered to cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Vol. 1-4, Cold Spring Harbor Press, NY. Viruses useful as vectors include retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. Additional promoter elements, such as enhancers, control the frequency of transcription initiation. Depending on the promoter, individual elements appear to function cooperatively or independently to activate transcription.

[0188] In one embodiment, the vector is a recombinant lentiviral vector with altered tropism, comprising (i) a mutant glycoprotein (G protein) that eliminates the natural receptor tropism and (ii) an insertion of an antibody, receptor ligand, or peptide that specifically targets cells, preferably myeloid cells, preferably monocytes or macrophages. The G protein plays an important role during the initial step of viral infection because it is involved in the attachment of the virus to a specific receptor. After binding, the G protein triggers fusion between the viral membrane and the endosomal membrane, and the endosome releases the viral genome into the cytosol for the next step of infection. Preferably, the recombinant lentiviral vector containing the mutant G protein enables antigen-specific infection of myeloid cells, preferably monocytes or macrophages.

[0189] The expression vector that can be introduced into cells to assess the expression of a polypeptide or portion thereof can also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells desired to be transfected or infected via the viral vector. In other embodiments, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like. Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Generally, reporter genes are genes that are not present or expressed in the recipient organism or tissue and encode a polypeptide whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assessed at an appropriate time after the DNA is introduced into the recipient cells. Suitable reporter genes may include luciferase, beta-galactosidase, chloramphenicol acetyltransferase, genes encoding secreted alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, a construct with a minimal 5' flanking region that exhibits the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be linked to reporter genes and used to evaluate agents for their ability to modulate promoter-driven transcription.

[0190] The method comprises the step of introducing into said cell a first vector, said vector comprising a sequence encoding IL-2 under the control of a cytokine-specific promoter, preferably selected from the interleukin-6 promoter or the interleukin-8 promoter.

[0191] Preferably, the gene for IL-2 is the human gene of the nucleic acid sequence SEQ ID NO:1.

[0192] Preferably, the method comprises introducing into said cell a second vector, said vector comprising a CAR. Preferably, the CAR comprises, from its N-terminus to its C-terminus: - an extracellular antigen-binding domain of sequence SEQ ID NO: 3, - optionally a hinge domain of sequence SEQ ID NO: 4, - a transmembrane domain of sequence SEQ ID NO: 5, - a first intracellular signaling domain of sequence SEQ ID NO: 6 fused, preferably directly, with a second intracellular signaling domain of sequence SEQ ID NO: 7; Includes:

[0193] The sequences of this application can be summarized as follows: array

[0194] [Table 1A]

[0195] [Table 1B]

[0196] [Table 1C]

[0197] [Table 1D]

[0198] [Table 1E]

[0199] [Table 1F]

[0200] [Table 1G]

[0201] [Table 1H]

[0202] [Table 1I]

[0203] [Table 1J]

[0204] [Table 1K] [Brief explanation of the drawings]

[0205] [Figure 1] Lentiviral transduction of monocytes to obtain IL-2-producing macrophages. (A) Untransduced macrophages and macrophages transduced with control or IL-2-encoding lentiviral vectors were collected on day 10, permeabilized, stained with anti-IL-2-PE antibody, and analyzed by FACS. Left: Representative histogram of PE fluorescence. Right: Summary of results for multiple donors. MFI ratios are shown relative to untransduced macrophages. (B) Quantification of IL-2 in supernatants of approximately 300,000 macrophages / mL cultured for 72 hours. Each dot represents one donor. Bars represent mean values ​​+ / - SD. (C) IL-2 concentration present in the supernatant (SN) of macrophages as a function of time after transduction. [Figure 2]Transduced macrophages secrete functional IL-2. STAT5 phosphorylation detected by flow cytometry in various T cell subsets after 10 min of contact with (A) supernatant (SN) or (B) recombinant human IL-2 (hIL-2). Mean values ​​+ / - SD. N=3 donors. (C) IL-2-macrophages (IL-2-MΦ) were seeded 24 h before adding the indicated T cell subsets at various ratios for 10 min. Mean values ​​+ / - SD of two technical replicates. Tconv: CD3+ CD4+ CD25- FoxP3-. Treg: CD3+ CD4+ CD25+ FoxP3+. CD8 T cells: CD3+ CD4-. [Figure 3] Figure 1 shows that IL-2-monocytes induce tumor regression in vivo. (A) NSG mice were injected subcutaneously with 5x106 MDA-MB-231 CD19+ GFP+ cells. Half of the mice were injected intravenously with CD14- PBMCs (equivalent to 7x106 cells). Mice were left untreated or injected intratumorally (it) with IL-2-Mono or control monocytes. (B) Body weight measured over 35 days. (C) Tumor size measured with a vernier caliper over 35 days. [Figure 4] Figure 3 shows flow cytometry analysis of spleens and tumors from mice that underwent cell therapy. Mice from the experiment depicted in Figure 3 were sacrificed 35 days after tumor injection. Their spleens and remaining tumors were harvested, stained for the indicated markers, and analyzed by flow cytometry to determine the percentage of human and tumor cells present in these preparations. For mice with less than 1% CD45+ cells, the % of CD3+ cells was not presented. [Figure 5]Figure 1 shows the tumor growth-regulating ability of CAR-MΦ in 3D. Purified CD14+ cells were transduced with lentivectors encoding the indicated CAR constructs and cultured for 6 days in the presence of M-CSF without any antibiotic selection. On day 3, 10 MDA-MB-231 cells (MDA-GFP-CD19) were seeded into ultra-low attachment 96-well plates, resulting in the formation of tumor spheroids grown in 3D. Three days later, 2 × 10 untransduced macrophages (UTD) or CAR-macrophages (CAR-STOP, CAR-CD3z, or CAR-STINGtz) were added to the established spheroids. GFP intensity, representing spheroid growth of MDA-GFP-CD19 tumor cells, was tracked every 3 hours for 168 hours by time-lapse microscopy. Mean + / - SD values ​​of GFP intensity calculated from triplicate spheroids are shown. [Figure 6] Figure 1 shows that antigen stimulation of CAR macrophages induces the secretion of pro-inflammatory cytokines and interferons. Purified CD14+ cells were transduced with lentivectors encoding the indicated CAR constructs and cultured for 6 days in the presence of M-CSF without any antibiotic selection. Macrophages were harvested and plated alone or with A549 tumor cells expressing or not expressing CD19+ for 24 hours. Cytokine quantification was performed by Legendplex on the supernatants of the indicated cocultures. Untransduced (UTD) macrophages or macrophages transduced with CAR-STOP, CAR-CD3z, or CAR-STINGtz were cultured with vehicle alone (0), A549 cells, or A549-CD19+ cells at a 1:1 E:T ratio for 24 hours. Each dot represents one donor. [Figure 7] FIG. 1 is a schematic diagram of the CAR2 band provirus. [Figure 8]Figure 1 shows that antigen stimulation of CAR2-banded macrophages induces a type I interferon response and production of the payload IL-2. Purified CD14+ cells were transduced with a lentivector encoding the indicated CAR2-banded construct and cultured for 3 days in the presence of M-CSF without any antibiotic selection. Macrophages were harvested and seeded alone or with A549 tumor cells expressing or not expressing CD19+ for 24 hours. Quantification of the indicated cytokines was performed by CBA on the supernatants of the indicated cocultures. Untransduced (UTD) macrophages or macrophages transduced with IL2-STOP or IL2-STING were cultured with vehicle alone (no tumor), A549 cells, or A549-CD19+ cells. CAR macrophages were cultured at an E:T ratio of 1:1 for 24 hours. Each dot represents one donor. [Example]

[0206] Example 1 Engineering autologous monocytes to express IL-2 and / or CAR-STING and their effects on anti-tumor immunity Materials and Methods cell line The MDA-MB-231 human breast adenocarcinoma cell line was maintained in RPMI complete medium (Gibco™ Roswell Park Memorial Institute 1640 supplemented with 10% fetal bovine serum and 1% Gibco™ penicillin-streptomycin (Thermofischer)). HEK 293 FT cells were maintained in DMEM complete medium (Gibco™ Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin).

[0207] MDA-MB-231-GFP and MDA-MB-231-GFP-CD19 were obtained by lentiviral transduction with pWPXLd-GFP encoding GFP and pCDH1-CD19 encoding hCD19. Transduced cell lines were FACS-sorted to obtain homogeneous cell populations.

[0208] primary cells Peripheral blood mononuclear cells (PBMCs) were isolated from the plasmapheresis residue using Ficoll-Paque (GE Healthcare). Informed consent was obtained from all donors, and samples were anonymized before use in the study. Monocytes were purified by CD14 magnetic microbeads (Miltenyi 130-050-201). + Isolate by positive selection and CD3 + T cells were isolated by negative selection using (Miltenyi 130-096-535).

[0209] Plasmid construction and virus production The CAR construct and human IL-2 (hIL-2) were cloned into the pCDH1 lentiviral vector, which contains a puromycin resistance gene under the control of the EF1α promoter.

[0210] Lentivirus was produced in HEK293FT cells. The lentiviral vector was co-transfected with psPAX2 (a second-generation lentiviral packaging plasmid) and pMD2.G (encoding VSV-G) using PEI MAX® (Polysciences). Vpx-VLPs were produced in HEK293FT cells by transfection with pSIV3 and pMD2.G (S. Bobadilla et al., 2013).

[0211] After 18 hours, the medium was replaced with fresh medium to remove the transfection reagent. The supernatant containing the lentivector was collected 24 hours after the medium change and filtered through a 0.45 μm filter.

[0212] Monocyte transduction and differentiation CD14 +Cells were transduced with the lentivector in the presence of Vpx-VLP and 4 μg / mL protamine. Monocytes were then differentiated into macrophages in macrophage medium (RPMI + 5% fetal bovine serum + 5% human serum + 1% penicillin-streptomycin) containing 50 ng / mL M-CSF for 10 days in Corning® 100 mm TC untreated culture dishes.

[0213] Intracellular FACS Human primary macrophages were treated with 1 / 1000 GolgiPlug™ (BD Biosciences) for 4 hours, detached with Accutase, fixed, permeabilized with BD Cytofix / Cytoperm™, labeled with anti-IL-2-PE antibody, and analyzed on a BD FACSVerse™ flow cytometer.

[0214] FACS-based phagocytosis assay 1×10 5 1 x 10 CAR macrophages 5 The cells were co-cultured with A549-GFP or A549-GFP-CD19 cells for 3 hours at 37°C. The cells were harvested with Accutase, stained with anti-CD45-Alexa700 (Biolegend) antibody in the presence of FcBlock™ (BD Biosciences), and analyzed by FACS using a Bio-Rad ZE5. + The percent of GFP+ events within the population was plotted as a percentage of phagocytosis.

[0215] STAT5 phosphorylation assay Human primary T cells were isolated from PBMCs using the Pan T Cell Isolation Kit Human (Miltenyi Biotec). 200,000 T cells were contacted with cells, supernatant, or recombinant human IL-2 for 10 minutes and then fixed with 2% paraformaldehyde. Cells were permeabilized with methanol, labeled with antibodies (anti-CD3-PECy7, anti-CD4-APCCy7, anti-CD25-PE, anti-FoxP3-Alexa488, and anti-pSTAT5-Alexa647), and analyzed on a BD FACSVerse™ flow cytometer.

[0216] In vivo studies A schematic of the xenograft model used is shown in the relevant figure panel. Cells were injected in 100 μl PBS for IV, IT, and SC injections. Tumor size was measured twice weekly with a caliper. Mice were weighed weekly and routine veterinary evaluation for overt signs of disease was performed. Animals were sacrificed at the end of the experiment.

[0217] result Generation of IL-2-expressing macrophages Human primary monocytes purified from PBMCs were transduced with a lentiviral vector encoding IL-2 under the control of a constitutive CMV promoter. After 7 days of culture in the presence of monocyte colony-stimulating factor (M-CSF), the supernatant was replaced with fresh medium. After 3 days, macrophages were harvested. IL-2 cellular expression was assayed by intracellular flow cytometry with an anti-IL-2-PE antibody after cell permeabilization. Untransduced or control vector-transduced macrophages did not express IL-2, whereas the majority (>90%) of IL-2-transduced macrophages (IL-2-MΦ) expressed IL-2 (Figure 1A). The supernatant of IL-2-MΦ cultured for 3 days contained 300 ng / ml + / - 150 ng / ml of IL-2, as measured by CBA (Figure 1B). IL-2 concentration was measured in the monocyte supernatant as a function of time after transduction. IL-2-transduced monocytes secreted detectable levels of IL-2 as early as 3 days after transduction (Fig. 1C). We concluded that monocytes transduced with our IL-2 lentivector gave rise to a high percentage of macrophages (estimated 1 fg IL-2 / MΦ) that rapidly produced significant amounts of IL-2.

[0218] IL-2 produced by IL-2-MΦ is functionally active on primary T lymphocytes We assayed the functionality of IL-2 produced by IL-2-MΦ by measuring IL-2-induced phosphorylation of STAT5, which initiates a signaling cascade leading to T cell survival and proliferation. IL-2-MΦ supernatants were incubated with human primary T cells for 10 min, after which the cells were fixed, permeabilized, and stained with an antibody specific for phosphorylated STAT5. Freshly purified total CD3+ cells from PBMCs isolated from healthy donors were used. + T cells were used in this assay. Because they express IL-2R at different levels, and the high-affinity IL-2R is predominantly expressed by Tregs in the steady state, STAT5 phosphorylation was measured in three T cell subsets: CD4 + T conv (CD3 + CD4 +CD25 - Foxp3 - conventional T cells), T reg (CD3 + CD4 + CD25 + Foxp3 + regulatory T cells, defined as CD8 + T cells (CD3 + CD4 - Only supernatants of IL-2-MΦ induced STAT5 phosphorylation in all T cell populations to varying degrees consistent with their respective expression of IL-2R chains: Treg > Tconv > CD8 T cells (Fig. 2A).

[0219] In parallel, we titrated purified recombinant human IL-2 (hIL-2) and different dilutions of IL-2-MΦ supernatants in the same assay (Fig. 2B). The curves obtained for the three T cell populations were similar in both situations, demonstrating T conv and CD8 + Compared with T cells, T reg In IL-2-MΦ, the rate of STAT5 phosphorylation was higher due to their expression of the high-affinity IL-2R. Thus, IL-2 secreted by IL-2-MΦ was functionally active on all T cell populations.

[0220] We also more directly assessed the potency of IL-2-MΦs by plating them for 24 hours before adding them directly to primary T cells for 10 minutes (Figure 2C). Various ratios of macrophages to T cells were tested, resulting in precise dose-effect curves. The presence of macrophages did not impair the ability of secreted IL-2 to phosphorylate STAT5 in a dose-dependent manner, similar to hIL-2. Collectively, our data demonstrate that IL-2-MΦs secrete substantial amounts of IL-2 capable of activating all primary T cell populations.

[0221] In vivo antitumor activity of IL-2-MΦ To evaluate the antitumor activity of IL-2-MΦ in vivo, we used the human breast cancer MDA-MB-231 cell line to generate cells expressing CD19 and GFP by lentiviral transduction. + CD19 + Cells were injected subcutaneously into immunodeficient NSG mice. Half of the mice received monocyte-depleted PBMCs (CD14 - The CD3 cells were injected intravenously (iv). + The percentage of T cells was estimated by flow cytometry, and the number of CD14 cells injected was determined to be 7×10 6 CD3 + On day 11, mice were inoculated with 10 x 10 6 Each mouse received an intratumoral (i.t.) injection of either IL-2-transduced monocytes (Mono-IL-2) or the same number of monocytes transduced with an empty vector (control monocytes). Of note, monocytes were transduced in vitro on day 10, left overnight in an incubator, and injected on day 11, i.e., less than 24 hours after purification and transduction.

[0222] Mouse weight and sc tumor growth were monitored periodically over 35 days (Fig. 3A). Treatment did not affect mouse weight compared to untreated control mice, suggesting a lack of widespread toxic effects (Fig. 3B). We found that Mono-IL-2 cells were unable to control tumor growth by themselves and expressed CD14 - We observed that CD14 cells mediated limited control of tumor growth. - Five of six mice that received both cells and Mono-IL-2 showed a significant reduction in tumor burden. These data suggest that injected Mono-IL-2 plays a key role in the regulation of tumor growth by CD14. - This suggests that cooperation with cells (presumably T cells) is required (Figure 3C). n=2 experiments.

[0223] We conclude that transduction of monocytes with an IL-2-encoding vector generates cells capable of inducing tumor regression in immunodeficient mice partially reconstituted with T cells. Thus, Mono-IL-2 and CD14 - Injection of the cells induces an efficient graft-versus-tumor response in NSG mice bearing human tumors.

[0224] Flow cytometric analysis of cell populations present in treated mice Mice from the experiment described in Figure 3 were sacrificed 35 days after tumor injection, and their spleens and remaining tumors were harvested, stained, and analyzed by flow cytometry (Figure 4). Human myeloid cells remained undetectable with a CD64-specific antibody at both sites. Nevertheless, mice injected with Mono-IL-2 showed a significant increase in CD3 + High levels of human CD45 on the majority of T cells + Notably, the purity of our monocyte preparations was uniformly greater than 98% (not shown), indicating that in the group of mice receiving only Mono-IL-2, the very few contaminating T cells were efficiently consumed, presumably with the aid of IL-2 produced by Mono-IL-2 (CD3 T cells in tumors). + See panel A showing % of T cells).

[0225] MDA-MB-231 GFP co-cultured with various CAR-MΦs + CD19 + Cell spheroid growth assay We assayed the ability of CAR-macrophages (CAR-MΦ) to control tumor growth using tumor spheroids of MDA-MB-231 cells expressing GFP and CD19 (MDA-GFP-CD19). After 3 days, untransduced macrophages (UTD) or CAR-MΦ (CAR-STOP, CAR-CD3z, or CAR-STINGtz) were added to the established spheroids. Spheroid growth was measured by live-cell imaging using GFP fluorescence (Figure 5). We concluded that adding 2,000 CAR-STINGtz macrophages to established tumor spheroids (3 days old) induced highly efficient tumor regression, which was not observed with untransduced, CAR-STOP, or CAR-CD3z-transduced macrophages.

[0226] Antigen stimulation of CAR macrophages induces secretion of pro-inflammatory cytokines and interferons To assess the ability of CAR-macrophages (CAR-MΦ) to produce cytokines in an antigen-dependent manner, CAR-MΦ were incubated alone or in combination with CD19 + CAR-STINGtz-expressing macrophages were seeded with A549 tumor cells expressing or not expressing CAR-STINGtz for 24 hours. Cytokine quantification was performed in the indicated co-cultures (Figure 6). We concluded that macrophages expressing CAR-STINGtz are able to produce substantial amounts of a panel of cytokines of interest in a strictly antigen-dependent manner compared to CAR-STOP or CAR-CD3z. Notably, IL12 (IL-12p70) is essential for T cell priming, GM-CSF promotes myeloid cell survival and activates macrophages, and IFN alpha (IFN-a2) and beta (IFN-b) are essential for locally eliciting robust antitumor responses. IL-1 (IL-1b) and IL-6 activate T cells, but not T reg IL-10 production reflects macrophage activation. TNF-alpha (TNF-a) enhances phagocytosis. Therefore, CAR-STINGtz macrophages according to the present invention are capable of expressing CD19 +Antigen binding activates both the IRF3 and NF-KB pathways, allowing for the stimulation of the production of several cytokines. Importantly, in the absence of tumor antigen, very few cytokines are released by CAR macrophages.

[0227] Antigen stimulation of CAR2-banded macrophages induces a type I interferon response and payload IL-2 production We evaluated the ability of CAR2-banded macrophages to induce a type I interferon response (type I IFN) and to produce IL-2 in an antigen-dependent manner. The structure of the CAR2-banded vector is shown in Figure 7. Untransduced (UTD) macrophages or CAR2-banded macrophages transduced with IL2-STOP or IL2-STING were incubated with vehicle alone (no tumor), A549 cells, or A549-CD19 cells. + We cultured macrophages expressing the CAR2 band IL2-STING and quantified cytokines in the supernatant (Figure 8). + We conclude that activation of these CARs containing truncated STING leads to type I IFN production, which in turn induces the expression of interferon-stimulated genes, such as IP10, when contacted with expressing cells. Furthermore, activation of STING also leads to activation of a minimal promoter that drives the expression of the payload IL2. Thus, significant amounts of IL2 are produced in response to antigen exposure.

Claims

1. selected from myeloid cells, induced pluripotent stem cells (iPS), and hematopoietic stem cells (HSC); (i) a first vector comprising a sequence encoding at least one cytokine, preferably at least one interleukin, under the control of an inducible or constitutive promoter, and a second vector comprising a sequence encoding a chimeric antigen receptor (CAR); or (ii) A modified cell comprising only a first vector encoding both at least one cytokine, preferably at least one interleukin, and a CAR.

2. 2. The modified cell of claim 1, which is a modified myeloid cell, preferably a monocyte, macrophage, or dendritic cell, more preferably a monocyte.

3. 3. The modified cell of claim 1 or 2, wherein the cytokine is IL-2 and the sequence encoding IL-2 is the sequence of SEQ ID NO:

2.

4. 4. The modified cell of any one of claims 1 to 3, wherein the promoter is inducible.

5. The CAR is - an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells of the tumor microenvironment (TME); - optionally, a hinge domain; - a transmembrane domain, and - Intracellular signaling domains 5. The modified cell of any one of claims 1 to 4, comprising:

6. The extracellular antigen-binding domains are CD19, MUC16, MUC1, CA1X, carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD20, CD22, CD30, 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, GD2Ac, GD3, ITER-2, hTERT, IL-13R-a2, K-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, ERBB2, MAGEA3, p53, MARTl, GPl00, and proteinase 3. (PR1), tyrosinase, survivin, EphA2, NKG2D ligand, NY-ES0-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, integrin B7, ICAM-1, CD70, Tim3, CLEC12A, ER, human telomerase reverse transcriptase (hTERT), mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 6. The modified cell of claim 5, wherein the extracellular antigen-binding domain is selected from the group consisting of CYP1B, HER2 / neu, p95HER2, Wilms' tumor gene 1 (WT1), livin, alpha-fetoprotein (AFP), prostate-specific membrane antigen (PSMA), cyclin (DI), mesothelin, B-cell maturation antigen (BCMA), and tumor-associated calcium transducer and activator of 2 (TROP2), and preferably the extracellular antigen-binding domain is an anti-CD19 binding domain, preferably an anti-CD19 scFv.

7. 7. The modified cell of Claim 5 or 6, wherein the intracellular signaling domain is a first intracellular signaling domain comprising the CD40 cytoplasmic tail, preferably of sequence SEQ ID NO: 6, or a first intracellular signaling domain comprising the CD40 cytoplasmic tail, preferably of sequence SEQ ID NO: 6, fused to a second intracellular signaling domain comprising the CD3 zeta intracellular domain, preferably of sequence SEQ ID NO:

7.

8. CAR is, from its N-terminus to its C-terminus, - an extracellular antigen-binding domain of sequence SEQ ID NO: 3, - optionally a hinge domain of sequence SEQ ID NO: 4, - a transmembrane domain of sequence SEQ ID NO: 5, and - either a first intracellular signalling domain of sequence SEQ ID NO: 6, or a first intracellular signalling domain of sequence SEQ ID NO: 6 fused, preferably directly, with a second intracellular signalling domain of sequence SEQ ID NO: 7; 8. The modified cell of any one of claims 1 to 7, comprising:

9. The CAR is - an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells of the TME; - optionally, a hinge domain; - a transmembrane domain; and - an intracellular signaling domain comprising STING or one of its fragments 7. The modified cell of any one of claims 1 to 6, wherein the modified cell is a myeloid cell.

10. The modified cell according to any one of claims 1 to 9, wherein the promoter of the first vector is a cytokine-specific promoter, preferably an interleukin-6 promoter or an interleukin-8 promoter.

11. 10. The modified cell of any one of claims 5 to 9, wherein the promoter of the first vector is an artificial promoter containing a response element that is activated via activation of the CAR.

12. 1. A modified cell that expresses at least one cytokine, preferably an interleukin selected from IL-10, IL-15, IL-13, IL-7A, IFN alpha, IFN beta, IFN lambda, IFN gamma, IL-1A, IL-1B, IL-12, and IL-21, wherein the cell is selected from a myeloid cell, an induced pluripotent stem cell (iPS), and a hematopoietic stem cell (HSC), the cell comprising a first vector comprising a sequence encoding the cytokine or interleukin under the control of an inducible or constitutive promoter, and optionally a second vector comprising a sequence encoding a chimeric antigen receptor (CAR); The CAR is - an extracellular antigen-binding domain that binds to a tumor antigen or an antigen present on cells of the tumor microenvironment (TME); - optionally, a hinge domain; - a transmembrane domain, and - Intracellular signaling domains A modified cell comprising:

13. A method for producing the modified myeloid cells, modified iPS cells, or modified HSCs of any one of claims 1 to 12, comprising: - providing at least one cell selected from isolated myeloid cells, iPS cells, and HSCs; - transducing said cells with a first vector, preferably a lentiviral vector, comprising a sequence encoding IL-2 or a sequence encoding at least one cytokine, preferably an interleukin selected from IL-10, IL-15, IL-13, IL-7A, IFN alpha, IFN beta, IFN lambda, IFN gamma, IL-1A, IL-1B, IL-12, and IL-21, under the control of an inducible or constitutive promoter, preferably under the control of an interleukin-6 promoter or an interleukin-8 promoter; and - optionally transducing said cells with a second vector, preferably a lentiviral vector, comprising a nucleic acid sequence encoding a CAR. A method comprising:

14. A pharmaceutical composition comprising the modified cells of any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

15. 15. The modified cell of any one of claims 1 to 12 or the pharmaceutical composition of claim 14 for use in the treatment of cancer, an autoimmune disease, or an inflammatory disease, preferably wherein the cancer is a solid tumor.

16. 16. The modified cells of claim 15, which are myeloid cells and are autologous to the donor.

17. 13. A product comprising the modified cells of any one of claims 1 to 12 and a CAR-T cell or immune checkpoint inhibitor as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer, an autoimmune disease, or an inflammatory disease.

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