Myeloid cells modified by cytokine chimeric receptor and uses thereof

Abstract

The invention relates to a modified myeloid cell comprising a cytokine chimeric receptor (CCR), wherein said CCR comprises an extracellular domain comprising an extracellular domain of a cytokine receptor which binds soluble molecules present in the tumor microenvironment (TME); a transmembrane domain; and an intracellular signaling domain comprising CD40 cytotail, CD3zeta intracellular domain and / or STING or one of its fragments. The invention also relates to a modified cell comprising a CCR and a transgene coding for a cytokine. The invention also relates to therapeutic uses thereof and to methods and pharmaceutical compositions for the treatment of cancer.

Description

[0001] MYELOID CELLS MODIFIED BY CYTOKINE CHIMERIC RECEPTOR

[0002] AND USES THEREOF

[0003] FIELD OF THE INVENTION:

[0004] The invention relates to a modified myeloid cell comprising a cytokine chimeric receptor (CCR), wherein said CCR comprises an extracellular domain comprising an extracellular domain of a cytokine receptor which binds soluble molecules present in the tumor microenvironment (TME); a transmembrane domain; and an intracellular signaling domain comprising CD40 cytotail, CD3zeta intracellular domain and / or STING or one of its fragments or variants thereof or a combination thereof. The invention also relates to a modified cell comprising a CCR and a transgene coding for a cytokine. The invention also relates to therapeutic uses thereof and to methods and pharmaceutical compositions for the treatment of cancer.

[0005] BACKGROUND OF THE INVENTION:

[0006] Solid tumors and their metastases are the most common and therapeutically challenging types of cancer today. The tumor microenvironment (TME) is a complex, heterogeneous mix of cellular populations that interact with one another and with the tumor cells. The TME is immunosuppressive, both evading the immune system and preventing therapeutic intervention from efficiently eliminating malignant cells. Myeloid cells within the TME play an important role in contributing to immune evasion by exhibiting potent immunosuppressive as well as pro- turn origenic properties.

[0007] TAMs (tumor-associated macrophages) are a key cell component of the TME in a variety of cancers. The prevailing consensus is that tumor-derived cytokines direct myeloid cell recruitment at the monocyte stage, and then the TME influences their development 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 hinder antitumor CD8+ T cells from infiltrating the tumor and attract or induce regulatory T cells (Treg). TAMs secrete growth factors like VEGF or TGFP, which promote tumor growth and invasive behavior. They are generally associated with poor prognosis, though recent studies have shown that their impact on prognosis can vary depending on their localization and polarization.

[0008] Various strategies have been adopted to harness the anti -tumor capacity of myeloid cells by genetic engineering. One of the most promising strategies was to virally transduce macrophages with CAR constructs to mobilize their capacity to phagocytose tumor cells and to promote an inflammatory phenotype. One of the major difficulties is to identify an antigen uniquely expressed on tumor cells.

[0009] To overcome this limitation, the inventors design chimeric receptors specific for soluble molecules present in the TME. This kind of receptor, which the inventors have named CCR (cytokine chimeric receptor), is composed of an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain being the extracellular domain of a cytokine receptor, and the intracellular domain being a domain inducing an inflammatory phenotype.

[0010] The inventors develop a variety of CCR that are specific of secreted molecules present in the TME. The CCR can be made of two chains, such as for the IL10R, IL4R and TGFbR, or of one chain such as for CSF1R. Even if encoded by two separate chains, the inventors can engineer the extracellular part of the Receptor (R) as a single chain linked by a spacer peptide. The CCR can also be made of the two chains of the IL6 receptor, IL6R alpha and gpl30 or only the IL6R alpha extracellular domain or only the gpl30 fused to the transmembrane domain. The appropriate transmembrane domain(s) would be fused to it and then the inventors use as an intracellular domain a truncation of STING fused or not to the zebrafish sequence as described in the invention. The inventors also use instead the CD40 or CD40-CD3z.

[0011] There is no disclosure in the art of a modified myeloid cell comprising a cytokine chimeric receptor (CCR) and its uses thereof in the treatment of cancer.

[0012] SUMMARY OF THE INVENTION:

[0013] The invention relates to a modified myeloid cell comprising a cytokine chimeric receptor (CCR), or a modified induced pluripotent stem cell (iPS) or hematopoietic stem cell (HSC) comprising a CCR, wherein said CCR comprises an extracellular domain comprising an extracellular domain of a cytokine receptor which binds soluble molecules present in the TME; a transmembrane domain; and an intracellular signaling domain comprising CD40 cytotail, CD3zeta intracellular domain and / or STING or one of its fragments. The invention also relates to a modified cell comprising a CCR and a transgene coding for a cytokine under the control of an inducible or constitutive promoter, which notably enables a locally contained expression of the cytokine. The invention also relates to therapeutic uses thereof and to methods and pharmaceutical compositions for the treatment of cancer. In particular, the invention is defined by the claims. DETAILED DESCRIPTION OF THE INVENTION:

[0014] The inventors design chimeric receptors specific for soluble molecules present in the TME. This kind of receptor, which the inventors have named CCR (cytokine chimeric receptor), is composed of an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain being the extracellular domain of a cytokine receptor, and the intracellular domain being a domain inducing an inflammatory phenotype. The inventors develop a variety of CCR that are specific of secreted molecules present in the TME. The CCR can be made of two chains, such as for the IL10R, IL4R and TGFbR, or of one chain like for CSF1R. Even if encoded by two separate chains, the inventors can engineer the extracellular part of the Receptor as a single chain linked by a spacer peptide. The appropriate transmembrane domain(s) would be fused to it and then the inventors use as an intracellular domain our truncations of STING fused or not to the zebrafish sequence as described in our patent. The inventors could also use instead the CD40 or CD40-CD3z described in or patents.

[0015] Accordingly, the invention relates to a modified myeloid cell comprising a cytokine chimeric receptor (CCR), wherein said CCR comprises an extracellular domain comprising an extracellular domain of a cytokine receptor which binds soluble molecules present in the TME; a transmembrane domain; and an intracellular signaling domain comprising CD40 cytotail, CD3zeta intracellular domain and / or STING or one of its fragments. The invention also relates to a modified cell comprising a CCR and a transgene coding for a cytokine. The invention also relates to therapeutic uses thereof and to methods and pharmaceutical compositions for the treatment of cancer.

[0016] Immune cell and Myeloid cell comprising a chimeric soluble antigen receptor (CSAR)

[0017] Accordingly, in a first aspect, the invention relates to a modified immune cell comprising a chimeric soluble antigen receptor (CSAR), wherein said CSAR comprises: an extracellular domain comprising a domain of a receptor which binds soluble molecules present in the TME; a transmembrane domain; and an intracellular signaling domain comprising CD40 cytotail, CD3zeta intracellular domain and / or STING or one of its fragments or variants thereof. In some embodiments, the invention relates to a modified myeloid cell comprising a chimeric soluble antigen receptor (CSAR), wherein said CSAR comprises: an extracellular domain comprising a domain of a receptor which binds soluble molecules present in the TME; a transmembrane domain; and an intracellular signaling domain comprising CD40 cytotail, CD3zeta intracellular domain and / or STING or one of its fragments or variants thereof.

[0018] In a further aspect, the invention relates to a modified induced pluripotent stem cell (iPS) or hematopoietic stem cell (HSC) comprising a chimeric soluble antigen receptor (CSAR), wherein said CSAR comprises: an extracellular domain comprising a domain of a receptor which binds soluble molecules present in the TME; a transmembrane domain; and an intracellular signaling domain comprising CD40 cytotail, CD3zeta intracellular domain and / or STING or one of its fragments or variants thereof.

[0019] Said iPS or HSC modified by a CSAR is called “CSAR iPS” or “CSAR HSC”, respectively, in the present invention.

[0020] Immune cell and Myeloid cell comprising a cytokine chimeric receptor (CCR}

[0021] In a further aspect, the invention relates to a modified immune cell comprising a cytokine chimeric receptor (CCR), wherein said CCR comprises: an extracellular domain comprising a domain of a cytokine receptor; a transmembrane domain; and an intracellular signaling domain comprising CD40 cytotail, CD3zeta intracellular domain and / or STING or one of its fragments or variants thereof.

[0022] In some embodiments, the invention relates to a modified myeloid cell comprising a cytokine chimeric receptor (CCR), wherein said CCR comprises: an extracellular domain comprising a domain of a cytokine receptor; a transmembrane domain; and an intracellular signaling domain comprising CD40 cytotail, CD3zeta intracellular domain and / or STING or one of its fragments or variants thereof. In a further aspect, the invention relates to a modified induced pluripotent stem cell (iPS) or hematopoietic stem cell (HSC) comprising a cytokine chimeric receptor (CCR), wherein said CCR comprises: an extracellular domain comprising a domain of a cytokine receptor; a transmembrane domain; and an intracellular signaling domain comprising CD40 cytotail, CD3zeta intracellular domain and / or STING or one of its fragments or variants thereof.

[0023] Said iPS or HSC modified by a CCR is called “CCR iPS” or “CCR HSC”, respectively, in the present invention.

[0024] The cells according to the invention are typically eukaryotic cells, such as mammalian cells (also named in the present invention animal cells), e.g., human cells.

[0025] The term “immune cell” has its general meaning in the art and refers to cells derived from the blood, bone marrow, lymph, or lymphoid organs (notably the thymus) and are cells of the immune system (i.e., immune cells), such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including monocytes, macrophages, lymphocytes, T cells (CD4+ or CD8+ T cell), NK cells, lymphoid progenitors and / or T cell progenitors.

[0026] Preferably according to the invention, immune cells are notably monocytes, macrophages.

[0027] Typically also, the engineered immune cell especially the myeloid cell is isolated from a subject. Preferably, said subject is suffering from a cancer, an autoimmune disease or an inflammatory disease or is at risk of suffering from a cancer, an autoimmune disease or an inflammatory disease.

[0028] The cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen.

[0029] With reference to the subject to be treated, the cells of the invention may be allogeneic and / or autologous.

[0030] The cells and compositions containing the cells according to the invention are isolated from a sample, notably a biological sample, e.g., obtained from or derived from a subject. Typically, the subject needs a cell therapy (adoptive cell therapy) and / or will receive the cell therapy. Samples include, in the context of cell therapy (typically adoptive cell therapy) samples from autologous and allogeneic sources. In some embodiments, the cells are derived from cell lines, e.g., myeloid or monocyte cell lines. The cells can also be obtained from a xenogeneic source, such as a mouse, a rat, a non-human primate, or a pig. Preferably, the cells are human cells.

[0031] The term “myeloid cell” has its general meaning in the art and refers to any type of cells derived from the myeloid tissue (bone marrow), or resembling bone marrow. Preferably, it is a monocyte, a macrophage or a dendritic cell, more preferably a monocyte.

[0032] The term “stem cell” has its general meaning in the art and refers to a cell that, by successive divisions can give rise to specialized cells. The term “pluripotent stem cell” refers to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues 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. A “pluripotent stem cell” may be identified by the expression of one or more of the cell markers Klf4, Sox2, Oct4, cMyc, Nanog and SSEA1. A cell is considered as a pluripotent stem cell when it is capable of generating cells from any of the three germ layers: endoderm, identified by the expression of alpha-fetoprotein; mesoderm (identified by the expression of desmin and / or alpha smooth muscle actin) and ectoderm (identified by the expression of beta-tubulin III = Tuj 1 and / or E to N-cadherin). Assays to assess the pluripotentiality of a cell are known in the art.

[0033] The term “induced pluripotent stem cell” or “iPS” has its general meaning in the art and refers to a pluripotent cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, by inducing a forced expression of certain genes. An "induced pluripotent stem cell" is defined by the expression of several transcription factors including one or more of Klf4, Sox2, Oct4 and cMyc. iPS cells are typically derived by transfection of certain stem cell- associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection is typically achieved through viral vectors, such as retroviruses, 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), Nanog and LIN28 have been identified to increase the induction efficiency. After 3-4 weeks, small numbers of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells, and are typically isolated through morphological selection, doubling time, or through a 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 are, without limitation, fibroblasts, keratinocytes and adipocytes. These cells can be obtained from an adult being by methods well- known in the state of the art (Mochiduki and Okita, 2012).

[0034] The term “hematopoietic stem cells” or “HSC” has its general meaning in the art and refers to cells which possess the ability to fully reconstitute the immune system of a lethally irradiated host from which the cells are obtained. The hematopoietic stem cells give rise to all blood and immune cells.

[0035] The CSAR of the invention comprises, from its N-terminal end to its C-terminal end :

[0036] - an extracellular domain comprising a domain of a receptor which binds soluble molecules present in the TME;

[0037] - optionally a hinge domain;

[0038] - a transmembrane domain; and

[0039] - a first intracellular signaling domain comprising the CD40 cytotail, CD3zeta intracellular domain and / or STING or one of its fragments or variants thereof.

[0040] The CCR of the invention comprises, from its N-terminal end to its C-terminal end :

[0041] - an extracellular domain comprising a domain of a cytokine receptor;

[0042] - optionally a hinge domain;

[0043] - a transmembrane domain; and

[0044] - a first intracellular signaling domain comprising the CD40 cytotail, CD3zeta intracellular domain and / or STING or one of its fragments or variants thereof.

[0045] Between each domain, a linker, identical or different, may be present. Preferably, the CSAR or the CCR does not comprise any linker between the different domains. In other words, the CSAR or the CCR is obtained by direct fusion of the different domains.

[0046] The CSAR or CCR myeloid cell according to the invention, or the CSAR or CCR iPS or CSAR or CCR HSC according to the invention, comprises, at the N-terminal end of the CSAR or CCR, an extracellular domain comprising a domain of a receptor which binds soluble molecules present in the TME such as cytokines. Preferably, said extracellular domain does not bind to an antigen expressed at the surface of cells, i.e. tumor cells or TME cells. The term “chimeric soluble antigen receptor” or “CSAR” refers to a receptor or a fragment or variant thereof that binds to a soluble antigen or soluble molecule present in the TME. The term “chimeric soluble antigen receptor” or “CSAR” also refers to receptor or a fragment or variant thereof which comprises, from its N-terminal end to its C-terminal end an extracellular domain comprising a domain of a receptor which binds soluble molecules present in the TME. The CSAR according to the invention is different from classical chimeric antigen receptors that binds to an antigen expressed at the surface of cells.

[0047] The term “soluble molecules present in the TME” has its general meaning in the art and refers to soluble molecules such as cytokines, chemokines and hormones. The term “soluble molecules present in the TME” also relates to soluble molecules such as any polypeptide, ligand or fragment thereof expressed and produced by cells specifically present in the TME such as cancer cells and immune cells. The term “soluble molecules present in the TME” refers to a soluble molecule such as a ligand present in the TME that binds to a receptor. The TME includes the tissues and cells around a tumor; it notably includes the surrounding blood vessels, immune cells such as Treg cells or immunosuppressive macrophages, fibroblasts, signaling molecules and the extracellular matrix. The term “soluble molecules present in the TME” also relates to soluble molecules of TME expressed and produced by activated CAF, soluble molecules of TME expressed by T regs and soluble molecules of TME expressed by protumoral myeloid cells such as TREM-2. Preferably, the soluble molecules of TME expressed and produced is chosen from cytokines, chemokines, hormones, growth factors and soluble molecules of TME expressed by Tregs and TREM-2. In some embodiments, soluble molecules of TME are different from antigens that are expressed at the surface of cells.

[0048] The term “cytokine” has its general meaning in the art and refers to polypeptides which exert their functions by interacting with specific cytokine receptors on the target cell surface and involved several biological functions such as in autocrine, paracrine and endocrine signaling as immunomodulating agents. Cytokines include chemokines, interleukins, interferons, lymphokines, growth factors and tumor necrosis factors. Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, and T lymphocytes, as well as endothelial cells, fibroblasts, and various stromal cells. Cytokines act through cell surface receptors and are especially important in the immune system; cytokines modulate the balance between humoral and cell-based immune responses, and they regulate the maturation, growth, and responsiveness of some cell populations. Cytokines are to be differentiated from antigens that are expressed at the surface of cells. The term “cytokine” also refers to cytokines selected from but not limited to cytokines described in Cameron MJ, Kelvin DJ. Cytokines, Chemokines and Their Receptors. In: Madame Curie Bioscience Database [Internet], Austin (TX): Landes Bioscience; 2000-2013; in Landskron G, De la Fuente M, Thuwajit P, Thuwajit C, Hermoso MA. Chronic inflammation and cytokines in the tumor microenvironment. J Immunol Res. 2014;2014: 149185; and Kartikasari AER, Huertas CS, Mitchell A, Piebanski M. Tumor-Induced Inflammatory Cytokines and the Emerging Diagnostic Devices for Cancer Detection and Prognosis. Front Oncol. 2021 Jul 7; 11 :692142.

[0049] In some embodiments, the cytokine is selected from but not limited to the group consisting of IL-1, IL2, IL3, IL4, IL5, IL6, IL7, IL-8, IL9, IL10, IL11, IL12, IL13, IL15, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL26, IL27, IL28, IL31, IL33, TSLP, gpl30, LIF, GCSF, GM-CSF, CNTF, EPO, GH, PRL, IFN-a, IFN- , IFN-co, IFN-s, IFN-K, IFNy, IFN 1, IFNZ2, IFNZ3, TGFp such as TGFpi and TGFP2, TREM1, TREM2, TNFa, CCL2, CCL-5, CCL21, CCL22, GM-CSF, CXCL-1, CXCL-2, CX3CL1, CXCL9, CXCL10 and CXCL16.

[0050] The term “hormone” has its general meaning in the art and refers to a substance or polypeptide produced by glands with internal secretion, which serve to carry signals through the blood to target organs (Starka L, Duskova M. What is a hormone? Physiol Res. 2020 Sep 30;69(Suppl 2): S 183-S 185).

[0051] The term “Chemokine” has its general meaning in the art and refers to a substance or polypeptide selected from but not limited to the group consisting of CXCL12 (SDF-1), IL-8 and CCL21 and chemokines described in Kartikasari et al., 2021 and Landskron et al., 2014.

[0052] The term “growth factor” has its general meaning in the art and refers to a substance or polypeptide selected from but not limited to the group consisting of VEGF and TGFp and growth factors described in Landskron et al., 2014 and Raman et al., 2007.

[0053] The term “cytokine chimeric receptor” or “CCR” refers to a chimeric receptor or a fragment or variant thereof that binds to a cytokine present in the TME. By “chimeric receptor”, it is meant a receptor comprising at least one extracellular domain from a first protein and at least one intracellular domain from a second protein (different from the first one). The term “cytokine chimeric receptor” or “CCR” also refers to receptor or a fragment or variant thereof which comprises, from its N-terminal end to its C-terminal end an extracellular domain comprising a domain of a cytokine receptor which binds cytokine present in the TME. In some embodiments, the term CCR refers to pro-inflammatory cytokine receptor, anti-inflammatory cytokine receptor or a fragment or variant thereof. The cytokine chimeric receptor according to the invention comprises an extracellular domain comprising a domain of a cytokine receptor and is thus different from chimeric antigen receptors comprising an extracellular antigenbinding domain which binds to a tumor antigen or an antigen present on cells of the TME.

[0054] The term “cytokine receptor” or “CR” has its general meaning in the art and refers to cytokine receptor or a fragment or variant thereof that binds to a cytokine. The term “cytokine receptor” or “CR” also refers to extracellular domain of a cytokine receptor or a variant thereof which binds cytokine present in the TME. In some embodiments, the term CR refers to pro- inflammatory cytokine receptor, anti-inflammatory cytokine receptor or a fragment or variant thereof. The term “cytokine receptor” or “CR” also refers to cytokine receptor selected from but not limited to cytokines described in Cameron MJ, Kelvin DJ. Cytokines, Chemokines and Their Receptors. In: Madame Curie Bioscience Database [Internet], Austin (TX): Landes Bioscience; 2000-2013. According to the invention, the cytokine receptor is different from an antigen receptor and does not bind a tumor antigen or an antigen present on cells of the TME.

[0055] In some embodiments, the cytokine receptor is selected from but not limited to the group consisting of IL-1R, IL10R, ILlORa, ILlORb, IL4R, IL4Ra, IL12R, IL13R, IL13Ral, IL7R, IL7Ra, IL8R, IL9R, IL9Ra, IL21R, IL21Ra, IL2R, IL2Rb, IL2rgc, TSLPR, IL6R, IL6Ra, ILl lRa, gpl30, GCSFR, IL3R, IL3Ra, IL5R, IL5Ra, GMCSFRa, CSF2Rb, LIFRb, IL31Ra, CNTFR, IL27R, IL27Ra, EPOR, GHR, PRLR, IFNAR, IFNAR2, IFNGR, IFNGR1, IL28R, IL20R, IL20Ra, IL22R, TGFbeta Receptor such as TGFbRl and TGFbR2, TREM1,TREM2, IL17R, IL17Ra, IL17Rc, IL18R, IL23R, IL33R, TFN-yRl and TNFR2.

[0056] By "hinge domain", it is meant any hinge domain present in immunoglobulins or in cluster of differentiation CD molecules.

[0057] Preferably, the hinge domain is the one of CD8. CD8 comprises an alpha chain (CD8a) and a beta chain (CD8b). Preferably, the hinge domain is the one of the CD8a chain.

[0058] The human version of CD8a may be found in Uniprot under accession number Q8TAW8. CD8a comprises 235 amino acids. The hinge domain is the fragment of amino acids 138 to 182 of said sequence, which corresponds to SEQ ID NO: 1.

[0059] Preferably, the hinge domain is the one of CD8a, preferably of human CD8a. Preferably, the hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:1).

[0060] By "transmembrane domain", it is meant a single-pass or a multipass transmembrane sequence.

[0061] Single-pass transmembrane regions are found in certain CD molecules, tyrosine kinase receptors, serine / threonine kinase receptors, TGF, BMP, activin and phosphatases. Single-pass transmembrane regions often include a signal peptide region and a transmembrane region of about 20 to about 25 amino acids, many of which are hydrophobic amino acids and can form an alpha helix. A short track of positively charged amino acids often follows the transmembrane span to anchor the protein in the membrane.

[0062] Multipass transmembrane domains are present in proteins such as ion pumps, ion channels and transporters, and include two or more helices that span the membrane multiple times.

[0063] Sequences for single-pass and multipass transmembrane domains are known and can be selected for incorporation into the CSAR or the CCR.

[0064] The transmembrane domain can be chosen from wild-type transmembrane domains and mutated transmembrane domains. Mutated transmembrane domains may be modified by a mutation, such as an amino acid substitution (for example, an amino acid which is typically charged is substituted by a hydrophobic residue). Preferably, the transmembrane domain is the one of the alpha, beta or zeta chain of the T cell receptor, CD3-8, CD3zeta, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD28, CD33, CD38, CD64, CD80, CD86, CD134, CD137 or CD 154. Preferably, the transmembrane domain is a CD8 transmembrane domain.

[0065] The transmembrane domain may also be chosen from transmembrane domain of a cytokine receptor. In some embodiments, the transmembrane domain may be chosen from the transmembrane domain of the cytokine receptor used in the extracellular domain or from another cytokine receptor.

[0066] The transmembrane domain may also be synthesized de novo, comprising mostly hydrophobic residues, such as, for example, leucine and valine.

[0067] According to the invention, the transmembrane domain is fused at its N-terminal end to the extracellular domain comprising a domain of a receptor which binds soluble molecules present in the TME of the CSAR or the CCR, and at its C-terminal end to the intracellular signaling domain. In certain embodiments, a short polypeptide linker may form the linkage between the transmembrane domain and the intracellular signaling domain of the CSAR or the CCR.

[0068] The CSAR or the CCR may further comprise a stalk, that is, an extracellular region of amino acids between the extracellular domain and the transmembrane domain. For example, the stalk may be a sequence of amino acids naturally associated with the selected transmembrane domain.

[0069] Preferably, the CSAR or the CCR comprises a CD8 transmembrane domain. Preferably, the CSAR or the CCR comprises a CD8 transmembrane domain, and a CD8 hinge domain. Said hinge domain is preferably fused (preferably directly), at its C-terminal end, to the N-terminal end of the transmembrane domain.

[0070] Preferably, the transmembrane domain comprises the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:2). This transmembrane domain is the one of human CD8.

[0071] Preferably, the hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 1).

[0072] By "intracellular domain", it is meant an intracellular signaling domain at the C-terminal end of the CSAR or the CCR. This intracellular domain can be chosen depending on the targeted effector activity aimed by the CSAR or the CCR. The intracellular domain can be derived from other receptors and can be designed to elicit a given cell function, such as phagocytosis, inflammatory activation or TME modulation.

[0073] The intracellular domain can comprise one or more intracellular signaling domains derived from a phagocytic receptor, a scavenger receptor or an integrin receptor. For example, the intracellular domain can comprise one or more intracellular signaling domains that promote phagocytic activity, an inflammatory response or integrin activation.

[0074] The intracellular signaling domain may be derived from a phagocytic or tethering receptor or may comprise a phagocytosis activation domain. Preferably, the intracellular signaling domain that promote phagocytic activity (i.e. also called phagocytosis activation domain) comprises an intracellular signaling domain derived from FcyR, FcaR or FcsR. In some embodiments, the intracellular signaling domain is derived from a receptor other than a phagocytic receptor selected from Megfl 0, MerTk, FcR-alpha or Bail. In some embodiments, the intracellular signaling domain is derived from a phagocytic receptor chosen from lectin, dectin 1, CD206, scavenger receptor Al (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. In some embodiments, the intracellular signaling domain of the invention is not derived from a cytokine receptor.

[0075] Preferably, the intracellular signaling domains that promote an inflammatory response (i.e. also called proinflammatory signaling domain) comprises aPI3-kinase (PI3K) recruitment domain. Preferably, it comprises 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, NODI, NOD2, Pyrin, AIM2, NLRC4 and / or CD40.

[0076] Preferably, the intracellular domain comprises at least two intracellular signaling domains, which comprise: either (i) a first intracellular signaling domain derived from FcyR or FcsR, and (ii) a second intracellular signaling domain: (A) comprising a PI3K recruitment domain, or (B) derived from CD40; or (i) a first intracellular signaling domain derived from a phagocytic receptor, and (ii) a second intracellular signaling domain: (A) comprising a PI3K recruitment domain, or (B) derived from CD40.

[0077] Examples of the intracellular domain include a fragment or domain from one or more molecules or receptors including, but are 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), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 127, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD 1 id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD 150, 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.

[0078] In some embodiments, the intracellular signaling domain of the invention comprises CD40 cytotail, CD3zeta intracellular domain and / or STING or one of its fragments or variants thereof.

[0079] In some embodiments, the intracellular signaling domain of the invention comprises CD40 cytotail.

[0080] In some embodiments, the intracellular signaling domain of the invention comprises CD3zeta intracellular domain.

[0081] In some embodiments, the intracellular signaling domain of the invention comprises STING or one of its fragments or variants thereof.

[0082] In some embodiments, the intracellular signaling domain of the invention comprises from N-terminal to C-terminal, a first intracellular signaling domain comprising the CD40 cytotail (cytoplasmic tail), which is fused to a second intracellular signaling domain comprising the CD3zeta intracellular domain.

[0083] By “CD40 cytotail”, it is meant 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) may be found in Uniprot under accession number P25942. It comprises 277 amino acids. The fragment comprising amino acids 216-277 of said sequence is the cytosolic part. Said fragment corresponds to SEQ ID NO:3.

[0084] In some embodiments, the intracellular signaling domain comprises a CD40 cytotail which is a fragment of human CD40.

[0085] In some embodiments, the intracellular signaling domain comprises the amino acid sequence: KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISV QERQ (SEQ ID NO:3).

[0086] Said first intracellular signaling domain is fused, at its C-terminal end, to a second intracellular signaling domain comprising the CD3zeta intracellular domain. Preferably said fusion is directly performed, i.e. without any linker.

[0087] CD3zeta, also called 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, whereas in 5% of T cells the TCR consists of gamma and delta chains. In the plasma membrane, the TCR chains alpha and beta associate with six additional adaptor proteins to form an octameric complex. Said complex comprises both alpha and beta chains, forming the ligandbinding site, but also one CD3gamma chain, one CD3delta chain, two CD3epsilon chains and two CD3zeta chains.

[0088] The sequence of human CD3zeta chain (hCD3zeta) may be found in Uniprot under accession number P20963. It comprises 164 amino acids. The fragment comprising amino acids 52-164 of said sequence is the cytosolic part. Said fragment corresponds to SEQ ID NO:4.

[0089] In some embodiments, the second intracellular signaling domain comprises the human CD3zeta intracellular domain.

[0090] In some embodiments, the second intracellular signaling domain comprises the amino sequence: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR (SEQ ID NO:4).

[0091] In some embodiments, the intracellular signaling domain is either a first intracellular signaling domain comprising the CD40 cytoplasmic tail, preferably of sequence SEQ ID NO:3, or a first intracellular signaling domain comprising the CD40 cytoplasmic tail, preferably of sequence SEQ ID NO:3, which is fused to a second intracellular signaling domain comprising the CD3zeta intracellular domain, preferably of sequence SEQ ID NO:4. The above embodiments and definitions for the modified myeloid cell expressing a CSAR or a CCR, except the intracellular signaling domain, also apply to the modified myeloid cell with a CSAR or a CCR including STING or one of its fragments or variants thereof.

[0092] In some embodiments, the intracellular signaling domain of the modified myeloid cell comprises STING or one of its fragments or variants theroef.

[0093] The STimulator of INterferon Genes (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 transcriptional activation of type I interferons and inflammatory cytokines in the presence of pathological nucleic acid species. STING activation relies 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 lead to cGAS -dependent synthesis of cyclic GMP-AMP (cGAMP). Interaction of cGAMP with STING activates a pathway that finally leads to the transcription of pro-inflammatory cytokines and type I interferons.

[0094] The sequence of human STING may be found in Uniprot under accession number A0A2R3XZB7. It comprises 379 amino acids. Preferably, a sequence with some amino acid deletions in the N-terminal end is used. STING may be used in its wild-type version, or in a mutated form to attenuate its activity.

[0095] In some embodiments, a fragment of STING is used, preferably comprising deletions of the N-terminal end. Preferably, the fragment corresponds to amino acids 137 to 379 of A0A2R3XZB7.

[0096] In some embodiments, the intracellular signaling domain comprises the amino sequence KGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGA VSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDRAGIKDRVYSNSIYELLENG QRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNN CRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISG MEKPLPLRTDFS (SEQ ID NO:5).

[0097] Preferably, the intracellular signaling domain is encoded by the nucleic sequence SEQ

[0098] ID N0:6.

[0099] >STINGt_nt SEQ ID NO:6. aagggcctggccccagctgagatctctgcagtgtgtgaaaaagggaatttcaacgtggcccatgggctggcatggtcatatt acatcggatatctgcggctgatcctgccagagctccaggcccggattcgaacttacaatcagcattacaacaacctgctacggggtgca gtgagccagcggctgtatattctcctcccattggactgtggggtgcctgataacctgagtatggctgaccccaacattcgcttcctggata aactgccccagcagaccggtgaccgtgctggcatcaaggatcgggtttacagcaacagcatctatgagcttctggagaacgggcagc gggcgggcacctgtgtcctggagtacgccacccccttgcagactttgtttgccatgtcacaatacagtcaagctggctttagccgggag gataggcttgagcaggccaaactcttctgccggacacttgaggacatcctggcagatgcccctgagtctcagaacaactgccgcctcat tgcctaccaggaacctgcagatgacagcagcttctcgctgtcccaggaggttctccggcacctgcggcaggaggaaaaggaagagg ttactgtgggcagcttgaagacctcagcggtgcccagtacctccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaa gcccctccctctccgcacggatttctcttga

[0100] In some embodiments, the present invention further relates to a modified cell comprising a CSAR, wherein said CSAR comprises:

[0101] - an extracellular domain comprising a domain of a receptor which binds soluble molecules present in the TME;

[0102] - a transmembrane domain; and

[0103] - an intracellular signaling domain comprising a chimeric fragment of STING; and wherein said modified cell is a myeloid cell.

[0104] The above embodiments and above definitions for the CSAR myeloid cell, except the intracellular signaling domain, are also valid for such a modified myeloid cell with a CSAR including a chimeric fragment of STING.

[0105] In some embodiments, the present invention further relates to a modified cell comprising a CCR, wherein said CCR comprises:

[0106] - an extracellular domain comprising a domain of a cytokine receptor;

[0107] - a transmembrane domain; and

[0108] - an intracellular signaling domain comprising a chimeric fragment of STING; and wherein said modified cell is a myeloid cell.

[0109] The above embodiments and above definitions for the CCR myeloid cell, except the intracellular signaling domain, are also valid for such a modified myeloid cell with a CCR including a chimeric fragment of STING.

[0110] By “a chimeric fragment of STING”, it is meant a modified STING C-terminal tail (CTT). Said modified STING CTT contains two sequence motifs known as IRF3 and TBK1 and, optionally, an additional fish-specific NF-KB motif. Preferably, said modified STING CTT is a chimera construct where IRF3 motif is from human STING, and TBK1 and NF-KB motifs are from human STING or fish STING. Preferably, the fish STING is a zebrafish STING. Preferably, said modified STING CTT is a chimera construct where IRF3 and TBK1 motifs are from human STING and the NF-KB is from zebrafish STING (said CCR is called “CCR- STINGtz”). Preferably, CCR-STINGtz comprises the amino sequence:

[0111] KGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNL LRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDRAGIKDRVYSNSIYEL LENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPE SQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPE LLISGMEKPLPLRTDPVETTDYFNPSSAMKQN (SEQ ID NO:7).

[0112] Preferably, CCR-STINGtz is encoded by the following nucleic sequence:

[0113] AAGGGCCTGGCCCCAGCTGAGATCTCTGCAGTGTGTGAAAAAGGGAATTT CAACGTGGCCCATGGGCTGGCATGGTCATATTACATCGGATATCTGCGGCTGATC CTGCCAGAGCTCCAGGCCCGGATTCGAACTTACAATCAGCATTACAACAACCTGC TACGGGGTGCAGTGAGCCAGCGGCTGTATATTCTCCTCCCATTGGACTGTGGGGT GCCTGATAACCTGAGTATGGCTGACCCCAACATTCGCTTCCTGGATAAACTGCCC CAGCAGACCGGTGACCGTGCTGGCATCAAGGATCGGGTTTACAGCAACAGCATCT ATGAGCTTCTGGAGAACGGGCAGCGGGCGGGCACCTGTGTCCTGGAGTACGCCA CCCCCTTGCAGACTTTGTTTGCCATGTCACAATACAGTCAAGCTGGCTTTAGCCGG GAGGATAGGCTTGAGCAGGCCAAACTCTTCTGCCGGACACTTGAGGACATCCTGG CAGATGCCCCTGAGTCTCAGAACAACTGCCGCCTCATTGCCTACCAGGAACCTGC AGATGACAGCAGCTTCTCGCTGTCCCAGGAGGTTCTCCGGCACCTGCGGCAGGAG GAAAAGGAAGAGGTTACTGTGGGCAGCTTGAAGACCTCAGCGGTGCCCAGTACC TCCACGATGTCCCAAGAGCCTGAGCTCCTCATCAGTGGAATGGAAAAGCCCCTCC CTCTCCGCACGGATCCTGTGGAGACCACCGATTATTTTAACCCATCTAGCGCAAT GAAACAAAACTAA (SEQ ID NO:8).

[0114] Said intracellular signaling domain comprising STING or one of its fragments or variants thereof may further comprise the CD40 cytotail, preferably as described above, and / or the CD3zeta intracellular domain, preferably as described above.

[0115] In some embodiments, the CSAR or the CCR comprises, from its N-terminal end to its C -terminal end: an extracellular domain, optionally a hinge domain of sequence SEQ ID NO: 1, a transmembrane domain of sequence SEQ ID NO:2, either a first intracellular signaling domain of sequence SEQ ID NO: 3, or a first intracellular signaling domain of sequence SEQ ID NO:3 which is fused, preferably directly, to a second intracellular signaling domain of sequence SEQ ID NON.

[0116] Preferably, the CSAR or the CCR comprises, from its N-terminal end to its C-terminal end: an extracellular domain, optionally a hinge domain of sequence SEQ ID NO: 1, a transmembrane domain of sequence SEQ ID NO:2, a first intracellular signaling domain of sequence SEQ ID NON, fused, preferably directly, to a second intracellular signaling domain of sequence SEQ ID NON.

[0117] Preferably, the CSAR or the CCR comprises, from its N-terminal end to its C-terminal end: an extracellular domain, optionally a hinge domain of sequence SEQ ID NO: 1, a transmembrane domain of sequence SEQ ID NON, and an intracellular signaling domain of sequence SEQ ID NO:7.

[0118] The present invention also relates to the nucleic acid sequence coding for a CSAR or a CCR according to the invention. Said nucleic acid sequence may be a DNA or RNA sequence. Said nucleic acid sequence may be used in therapy, especially for treating a cancer, an autoimmune disease or an inflammatory disease. Preferably, said nucleic acid sequence is administered to a subject, preferably by injection. Accordingly, the macrophages of said subject receive said nucleic acid sequence, and subsequently express the CSAR or a CCR, notably the CSAR or a CCR including STING or one of its fragments or variants theroef, preferably the CCR-STINGtz.

[0119] Immune cell and Myeloid cell further comprising a vector coding for at least one cytokine, preferably at least one interleukin, preferably with IL-2 The inventors investigate the transduction of monocytes with the gene coding for at least one cytokine such as interleukin-2 (IL-2) under the control of a specific promoter and with specific CSAR or CCR of the invention which may be used as treatment notably in cancer therapy.

[0120] Indeed, said monocytes are able to penetrate tumor and differentiate as IL-2 expressing macrophages able to bind a given antigen, activating thus their great capacity to phagocytose tumor cells, but also to further mobilize the antigen presentation and co-stimulatory capacities of macrophages upon their encounter of tumor cells, and thus to stimulate anti-tumor immunity.

[0121] Accordingly, in a further aspect of the invention, the modified immune cell, the modified myeloid cell, the modified iPS or the modified HSC of the invention further comprises a vector comprising a sequence coding for at least one cytokine, preferably at least one interleukin, preferably with IL-2.

[0122] Said cell is also called the “modified cell” in the present application.

[0123] The presence of both the first vector comprising a sequence coding for at least one cytokine, preferably at least one interleukin, preferably IL-2 and the second vector comprising a sequence coding for the CSAR or the CCR of the invention is called “CSAR+2bands” or “CCR+2bands”. The first and second vector may be combined in one vector comprising a sequence coding for both the at least one interleukin and the CSAR or the CCR of the invention.

[0124] Accordingly, in a further aspect, the invention relates to modified cell, wherein the cell is chosen from immune cell, myeloid cells, induced pluripotent stem cells (iPS) and hematopoietic stem cells (HSC), and wherein the cell comprises:

[0125] (i) either a first vector comprising a sequence coding for 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 coding for a chimeric soluble antigen receptor (CSAR); or

[0126] (ii) only comprises the first vector, which encodes for both the at least one cytokine, preferably at least one interleukin, and for the CSAR.

[0127] Accordingly, in a further aspect, the invention relates to modified cell, wherein the cell is chosen from immune cell, myeloid cells, induced pluripotent stem cells (iPS) and hematopoietic stem cells (HSC), and wherein the cell comprises: (i) either a first vector comprising a sequence coding for 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 coding for a cytokine chimeric receptor (CCR); or

[0128] (ii) only comprises the first vector, which encodes for both the at least one cytokine, preferably at least one interleukin, and for the CCR.

[0129] First vector coding for at least one cytokine, preferably at least one interleukin.

[0130] Interleukin-2 (IL-2) is the interleukin that induces the proliferation of responsive 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 the initial T cell is also stimulated by an antigen, thus helping the body fight off infections. Together with other polarizing cytokines, IL-2 stimulates naive CD4+ T cell differentiation into Thl and Th2 lymphocytes while it impedes differentiation into Thl7 and folicular Th lymphocytes. IL-2 increases also the cell killing activity of both natural killer cells and cytotoxic T cells.

[0131] Preferably, IL-2 is human IL-2 (hIL-2).

[0132] Preferably, IL-2 is encoded by the following nucleic sequence: (SEQ ID NO:9)

[0133] Atggggatccttcccagccctgggatgcctgcgctgctctccctcgtgagccttctctccgtgctgctgatgggttgcgtagc tgaaaccggtgcccccaccagctccacaaagaagacccagctgcagctggagcacctgctgctggacctgcagatgatcctgaacg gcatcaacaattacaagaatccaaagctgacacggatgctgaccttcaagttttatatgcccaagaaggccacagagctgaagcacctg cagtgcctggaggaggagctgaagcctctggaggaggtgctgaacctggcccagtccaagaatttccacctgcggccaagagacct gatctctaacatcaatgtgatcgtgctggagctgaagggcagcgagaccaccttcatgtgcgagtacgccgatgagaccgccacaatc gtggagttcctgaacaggtggatcaccttttgtcagtccatcatctctaccctgacatga

[0134] Preferably, IL-2 is encoded by the following amino acid sequence: (SEQ ID NO: 10)

[0135] MGILPSPGMPALLSLVSLLSVLLMGCVAETGAPTSSTKKTQLQLEHLLLDLQM ILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHL RPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0136] The first vector of the modified myeloid cell, the modified iPS or the modified HSC according to the invention comprises a sequence coding for IL-2 under the control of an inducible or constitutive promoter. Preferably, the sequence coding for IL-2 is inserted in a lentivector, preferably pCDHl, optionally containing a resistance gene, and under control of an inducible or constitutive promoter. Preferably, the promoter is inducible.

[0137] Constitutive or inducible promoters are usually associated with constitutive or inducible genes, respectively. A constitutive gene is a gene that is permanently transcribed, as opposed to a facultative gene. An inducible gene is a gene whose expression responds to a stimulus, such as an environmental change or the position in the cell cycle. An inducible gene is inactive unless there is the presence of an inducer that allows the gene to be expressed. The use of such constitutive or inducible promoters in genetic engineering allows the regulation of the transduced genes of interest to be controlled. Inducible promoters are generally preferred over constitutive promoters for their reversibility and flexibility. In addition, compared to constitutive promoters, inducible promoters are generally more efficient and have fewer side effects, such as cell death and delayed growth or development.

[0138] Preferably, the constitutive promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may 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, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, the actin promoter, the myosin promoter, the hemoglobin promoter and the creatine kinase promoter.

[0139] Preferably, the first vector comprises an inducible promoter. Preferably said inducible promoter is specific, i.e. it activates transcription mainly in response to CSAR or CCR activation. An inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Inducible promoters are well- known in the art.

[0140] Preferably, the inducible promoter is chosen from positive and negative inducible promoters. In the case of a positive inducible promoter, the activator protein binds to the promoter to initiate transcription. In contrast, in the case of a negative inducible promoter, the promoter is inactive, because a bound repressor protein actively prevents transcription. Once an inducer binds to the repressor protein, said repressor protein is removed from the DNA, and transcription can be activated.

[0141] The inducible promoter can be activated in response to a stimulus, such as the presence or absence and / or the amount of a chemical agent, or a change of temperature and / or light.

[0142] Chemically regulated promoters are among the most common inducible promoters. The inducible positive tetracycline ON (Tet-On) system, for example, works by direct activation. In this system, the tetracycline-controlled reverse transactivator (rtTA) is normally inactive and cannot bind to tetracycline response elements (TREs) in a promoter. Tetracycline and its derivatives are used as inducing agents to enable promoter activation. The inducible promoter may also be a promoter inducible by a chemical agent whose administration would be controlled over time or even locally. The administered chemical agent can be chosen from a wide possibility of molecules, such as tetracycline, biotin or the combination Tet-Off / Tet-On. The possibility of using photoimmunotherapy would also allow a localized and limited time action.

[0143] Cytokine specific promoters are also known; they are activated once the cytokine binds to the promoter. Preferably, the cytokine specific promoter is the promoter of interleukin-6 or the promoter of interleukin-8.

[0144] Other examples include the negative inducible promoter pLac or the negative inducible promoter pBad.

[0145] Some promoters are temperature inducible. They show almost no expression at normal temperatures but can be induced by exposure to heat or cold. For example, the Hsp70 promoter is inducible by heat shock.

[0146] Light is another means of activating gene expression, and two-component systems used in synthetic biology use light to regulate transcription. This requires a light-sensitive protein, such as YF 1 (known as Histidine Kinase), that is capable of inducing a transcriptional response, including acting on the synthesis of a repressor or an inducer.

[0147] Thus, preferably, the inducible promoter is chosen from chemically regulated promoters, temperature inducible promoters and light inducible promoters. Preferably, the inducible promoter is chosen from cytokine specific promoters (more preferably from the promoter of interleukin-6 and the promoter of interleukin-8), metallothionine promoters, glucocorticoid promoters, progesterone promoters, tetracycline promoters (such as Tet-On promoter), pLac, pBad, heat shock protein promoters (more preferably Hsp70 promoter) and YF1. The inducible promoter may also be chosen from artificial promoters containing response elements that are activated via the activation of the CSAR or the CCR. Artificial promoters may be designed from minimal promoters complemented with multiple binding sites for transcription factors such as NF-KB, API or ISRE (IFN-sensitive response element). Preferably, the artificial promoter comprises at least ISRE. Preferably, the artificial promoter is a mouse INFbeta promoter (that comprises ISRE), a promoter complemented with NF-KB responsive elements, or a combination thereof. The promoter may also be promX, of SEQ ID NO: 12. A preferred inducible promoter is a cytokine specific promoter (more preferably the promoter of interleukin-6 or the promoter of interleukin-8 or the promoter of interferon beta) which comprises response elements (i.e. binding sequences) to NF-KB or ISRE, or both. Preferably, the inducible promoter is promX of SEQ ID NO: 11 or a cytokine-specific promoter which comprises response elements to NF-KB or ISRE, preferably the inducible promoter is promX of SEQ ID NO: 12 or the promoter NF-KB of SEQ ID NO: 13.

[0148] The inducible promoter promX of SEQ ID NO: 11 : aagggcctggccccagctgagatctctgcagtgtgtgaaaaagggaatttcaacgtggcccatgggctggcatggtcatatt acatcggatatctgcggctgatcctgccagagctccaggcccggattcgaacttacaatcagcattacaacaacctgctacggggtgca gtgagccagcggctgtatattctcctcccattggactgtggggtgcctgataacctgagtatggctgaccccaacattcgcttcctggata aactgccccagcagaccggtgaccgtgctggcatcaaggatcgggtttacagcaacagcatctatgagcttctggagaacgggcagc gggcgggcacctgtgtcctggagtacgccacccccttgcagactttgtttgccatgtcacaatacagtcaagctggctttagccgggag gataggcttgagcaggccaaactcttctgccggacacttgaggacatcctggcagatgcccctgagtctcagaacaactgccgcctcat tgcctaccaggaacctgcagatgacagcagcttctcgctgtcccaggaggttctccggcacctgcggcaggaggaaaaggaagagg ttactgtgggcagcttgaagacctcagcggtgcccagtacctccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaa gcccctccctctccgcacggatcctgtggagaccaccgattattttaacccatctagcgcaatgaaacaaaactaa

[0149] >promX_nt (The inducible promoter promX of SEQ ID NO: 12) tgaattagtttcactttccagtttcagtttccagtttcattttccagtttcattttccagtttcattttcctgatatcctgcaggagcttga ataaaatgaatattagaagctgttagaataagagaaaatgacagaggaaaactgaaagggagaactgaaagtgggaaattcctctgag gcagaaaggaccatcccttataaatagcacaggccatgaaggaagatcattctcactgcagcctttgacagcctttgcctcatcttg

[0150] >Promotor NFKB RE (The inducible promoter NF-KB of SEQ ID NO: 13) ggggactttccgggaatttccggggactttccgggaatttccgggaatttccggggactttccgggaatttccggggactttc cgggaatttccagatctggcctcggcggccaagcttgctagcggggggctataaaagggggtgggggcgttcgt Said inducible promoter is preferably activated by the tumor environment around the IL-2 expressing cell. Preferably, the inducible promoter is a cytokine specific promoter. Preferably, the cytokine specific promoter is the promoter of interleukin-6 or the promoter of interleukin-8 or a synthetic promoter containing various elements able to bind transcription factors.

[0151] The inducible promoter is typically activated once the modified cell reaches the tumor.

[0152] Preferably, according to a first alternative, the sequence coding for IL-2 is inserted in a lentivector, preferably pCDHl, under control of a constitutive promoter. This alternative does not allow the control of IL-2 expression.

[0153] Preferably, according to a second alternative, the sequence coding for IL-2 is inserted in a lentivector, preferably pCDHl, under control of an inducible promoter.

[0154] The invention also relates to a modified cell expressing at least one cytokine, preferably at least one interleukin chosen from IL-10, IL-15, IL-13, IL-7A, IFNalpha, IFNbeta, IFNlambda, IFNgamma, IL-1A, IL-1B, IL-12 and IL-21, wherein the cell is chosen from myeloid cells, induced pluripotent stem cells (iPS) and hematopoietic stem cells (HSC), and wherein the cell comprises a first vector comprising a sequence coding for said interleukin under the control of an inducible or constitutive promoter, and a second vector comprising a sequence coding for a chimeric soluble antigen receptor (CSAR), wherein said CSAR comprises: an extracellular domain comprising a domain of a receptor which binds soluble molecules present in the TME; optionally a hinge domain; a transmembrane domain, and an intracellular signaling domain.

[0155] All the above embodiments for the first vector are also applicable to said modified cell expressing a cytokine. In this case, the sequence of the cytokine to be expressed is introduced into the first vector instead of the sequence coding for IL-2.

[0156] Preferably, all the above embodiments for the first vector are also applicable to said modified cell expressing an interleukin (different from IL-2). In this case, the sequence of the interleukin to be expressed is introduced into the first vector instead of the sequence coding for IL-2.

[0157] In some embodiments, the modified immune cell, the modified myeloid cell, iPS or HSC according to the invention comprises a first vector comprising a sequence coding for IL-2 under the control of an inducible or constitutive promoter, and further a second vector comprising a sequence coding for a chimeric soluble antigen receptor (CSAR), wherein said CSAR comprises: an extracellular domain comprising a domain of a receptor which binds soluble molecules present in the TME; optionally a hinge domain; a transmembrane domain, and an intracellular signaling domain.

[0158] In some embodiments, the modified immune cell, the modified myeloid cell, iPS or HSC according to the invention comprises a first vector comprising a sequence coding for IL-2 under the control of an inducible or constitutive promoter, and further a second vector comprising a sequence coding for a cytokine chimeric receptor (CCR), wherein said CCR comprises: an extracellular domain comprising a domain of a cytokine receptor; optionally a hinge domain; a transmembrane domain, and an intracellular signaling domain.

[0159] Such a cell is able to bind to soluble molecule present in the tumor microenvironment and typically presents a targeted effector activity when activated by the appropriate chemokine / cytokine be able to perform phagocytosis of tumor cells.

[0160] Second vector with chimeric soluble antigen receptor (CSAR) or cytokine chimeric

[0161] The modified immune cell, the modified myeloid cell, the modified iPS or the modified HSC of the invention further comprises a second vector comprising a sequence coding for the chimeric soluble antigen receptor (CSAR) or the cytokine chimeric receptor (CCR) of the invention. The presence of both said first vector and said second vector is called respectively “CSAR+2bands” or “CCR+2bands”. In another embodiment, the CSAR or the CCR with its promoter can be included in the first vector (i.e. the vector comprising at least one interleukin sequence). In such a case, the modified myeloid cell, the modified iPS or the modified HSC of the invention only comprises the first vector, which encodes both for the interleukin and for the CSAR or the CCR; said vector is called “CSAR+2bands” or “CCR+2bands”. The CSAR or the CCR with its promoter can be upstream or downstream the at least one interleukin sequence. Preferably, the CSAR and the CCR with its promoter is downstream the at least one interleukin sequence. Preferably, the CSAR or the CCR with its promoter is downstream the IL2 sequence. Preferably, the CSAR2bands or CCR2bands vector comprises the nucleic acid sequence SEQ ID NO:9.

[0162] Thus, according to this embodiment, preferably the modified myeloid cell, iPS or HSC according to the invention comprises a first vector (“CSAR+2bands”) comprising a sequence coding for IL-2 under the control of an inducible or constitutive promoter, and a sequence coding for a chimeric soluble antigen receptor (CSAR) under the control of an inducible or constitutive promoter, wherein said CSAR comprises: an extracellular domain comprising a domain of a receptor which binds soluble molecules present in the TME; optionally a hinge domain; a transmembrane domain, and an intracellular signaling domain.

[0163] Preferably, the CSAR2bands comprises the CSAR with its promoter downstream the sequence coding for IL-2 under the control of an inducible or constitutive promoter. In other words, preferably, the CSAR2bands comprises the CSAR with its promoter in 3’ of the sequence coding for IL-2 under the control of an inducible or constitutive promoter.

[0164] In some embodiments, the modified myeloid cell, iPS or HSC according to the invention comprises a first vector (“CCR+2bands”) comprising a sequence coding for IL-2 under the control of an inducible or constitutive promoter, and a sequence coding for a cytokine chimeric receptor (CCR) under the control of an inducible or constitutive promoter, wherein said CCR comprises: an extracellular domain comprising a domain of a cytokine receptor; optionally a hinge domain; a transmembrane domain, and an intracellular signaling domain.

[0165] Preferably, the CCR2bands comprises the CCR with its promoter downstream the sequence coding for IL-2 under the control of an inducible or constitutive promoter. In other words, preferably, the CCR2bands comprises the CCR with its promoter in 3’ of the sequence coding for IL-2 under the control of an inducible or constitutive promoter.

[0166] Preferably, the modified cell of the invention is a myeloid cell and comprises a first vector comprising a sequence coding for IL-2 under the control of an inducible or constitutive promoter, and a second vector comprising a sequence coding for a chimeric soluble antigen receptor (CSAR), wherein said CSAR comprises:

[0167] - an extracellular domain comprising a domain of a receptor which binds soluble molecules present in the TME;

[0168] - optionally a hinge domain,

[0169] - a transmembrane domain; and

[0170] - an intracellular signaling domain comprising STING or one of its fragments or variants thereof.

[0171] Said cell is called “modified myeloid cell with a CSAR including STING or one of its fragments or variants thereof’ in the present application.

[0172] In some embodiments, the modified cell of the invention is a myeloid cell and comprises a first vector comprising a sequence coding for IL-2 under the control of an inducible or constitutive promoter, and a second vector comprising a sequence coding for a cytokine chimeric receptor (CCR), wherein said CCR comprises:

[0173] - an extracellular domain comprising a domain of a cytokine receptor;

[0174] - optionally a hinge domain,

[0175] - a transmembrane domain; and

[0176] - an intracellular signaling domain comprising STING or one of its fragments or variants thereof.

[0177] Said cell is called “modified myeloid cell with a CCR including STING or one of its fragments” in the present application. The above embodiments and definitions for the modified myeloid cell expressing a CSAR or a CCR, except the intracellular signaling domain, also apply to the modified myeloid cell with a CSAR or a CCR including STING or one of its fragments or variants thereof.

[0178] The modified immune cell, myeloid cell, iPS or HSC according to the invention, comprising the first and second vectors, preferably presents targeted effector activity. By “targeted effector activity”, it is meant at least one effector activity chosen from phagocytosis, targeted cellular cytotoxicity, production of cytokines, production of reactive oxygen species (ROS), myeloid activation, antigen processing and presentation to T cells, and in vivo capacity in NSG mice complemented with human T cells to induce human antigen-dependent tumor regression. Preferably, the targeted effector activity is selected from antigen-dependent phagocytosis of tumor cells, antigen-dependent tumor cell cytokine secretion, and in vivo capacity in NSG mice complemented with human T cells to induce human antigen-dependent tumor regression. Said human antigen-dependent tumor regression is probably 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 may be measured according to methods well-known in the art, which are illustrated in the examples. In vivo capacity in NSG mice complemented with human T cells to induce human antigen-dependent tumor regression is evaluated according to the protocol described in the examples.

[0179] All the above embodiments for the second vector are also applicable to the modified cell expressing a cytokine.

[0180] All the above embodiments for the second vector are also applicable to the modified cell expressing an interleukin (different from IL-2). In such a case, the sequence of the interleukin to be expressed is introduced into the first vector instead of the sequence coding for IL-2.

[0181] In another embodiment, the invention also relates to a modified cell expressing at least one cytokine, preferably an interleukin, wherein the cell is chosen from myeloid cells, induced pluripotent stem cells (iPS) and hematopoietic stem cells (HSC), and wherein the cell comprises a first vector comprising a sequence coding for said cytokine under the control of an inducible or constitutive promoter, and optionally a second vector comprising a sequence coding for a CSAR or a CCR, wherein said CSAR or CCR comprises at least the CD40 cytotail and / or CD3zeta intracellular domain and / or STING or one of its fragments or variants thereof as intracellular signaling domain. In this embodiment, said modified cell is preferably a macrophage.

[0182] In another embodiment, the invention also relates to a modified cell expressing at least one interleukin chosen from IL-2, IL-10, IL-15, IL-13, IL-7A, IFNalpha, IFNbeta, IFNlambda, IFNgamma, IL-1A, IL-1B, IL-12 and IL-21, wherein the cell is chosen from myeloid cells, induced pluripotent stem cells (iPS) and hematopoietic stem cells (HSC), and wherein the cell comprises a first vector comprising a sequence coding for said interleukin under the control of an inducible or constitutive promoter, and optionally a second vector comprising a sequence coding for a CSAR or a CCR, wherein said CSAR or CCR comprises at least the CD40 cytotail and / or CD3zeta intracellular domain and / or STING or one of its fragments or variants thereof as intracellular signaling domain. In this embodiment, said modified cell is preferably a macrophage.

[0183] The present invention also relates to the nucleic acid sequence coding for a CSAR or CCR according to the invention. Said nucleic acid sequence may be a DNA or RNA sequence.

[0184] In some embodiments, the invention relates to nucleic acid sequence coding for the first and / or second vector, or the CSAR2bands or the CCR2bands of invention. Said nucleic acid sequence may be used in therapy, especially for treating a cancer, an autoimmune disease or an inflammatory disease. Preferably, said nucleic acid sequence is administered to a subject, preferably by injection. Accordingly, the macrophages of said subject receive said nucleic acid sequence, and subsequently express the CSAR or CCR, notably the CSAR or CCR including STING or one of its fragments or variants thereof, preferably the CCR-STINGtz.

[0185] In some embodiments, the invention relates to at least one lipid-based nanoparticle (LNP), at least one liposome or at least one virus-like particle (VLP) comprising a nucleic acid encoding for the first and / or second vector, or the CSAR2bands or the CCR2bands of invention.

[0186] Therapeutic uses

[0187] The present invention also relates to the use of a modified immune cell, a modified myeloid cell, a modified iPS or a modified HSC according to the invention, as a medicament.

[0188] The present invention also relates to a pharmaceutical composition comprising a modified immune cell, a modified myeloid cell, a modified iPS or a modified HSC according to the invention, and a pharmaceutical acceptable carrier. The present invention also relates to the use of the modified immune cell, the modified myeloid cell, the modified iPS or the modified HSC according to the invention, or of 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.

[0189] All these embodiments also apply to the modified cell expressing a cytokine.

[0190] All these embodiments also apply to the modified cell expressing an interleukin (different from IL-2). The sequence of the interleukin to be expressed in introduced into the first vector instead of the sequence coding for IL-2.

[0191] Preferably, the myeloid cells are obtained from a blood sample of the treated patient (treated donor). Preferably, they are modified and reinjected to the patient (donor), i.e. they are autologous.

[0192] By “autologous”, the present invention means that the modified immune cells, the modified myeloid cells or the therapeutic composition comprising the modified immune cells, the modified myeloid cells are used as a treatment for the patient from which the immune cells or myeloid cells are originated.

[0193] The present invention also relates to products containing a modified immune cell, a modified myeloid cell, a modified iPS or a modified HSC according to the invention, and a CAR-T cell, as a combined preparation for simultaneous, separate or sequential use in treatment of cancer, an autoimmune disease or an inflammatory disease.

[0194] CAR-T cells are well-known in the art. Preferably, CAR-T cells are chosen from tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel and idecabtagene vicleucel.

[0195] The present invention also relates to products containing a modified immunes cell, a modified myeloid cell, a modified iPS or a modified HSC according to the invention, and an Immune Checkpoint Inhibitor (ICI) as a combined preparation for simultaneous, separate or sequential use in treatment of cancer, an autoimmune disease or an inflammatory disease. An "immune checkpoint inhibitor" refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. 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.

[0196] Immune checkpoint proteins that may be quoted are CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR (such as KIR3DL2, KIR2DL1 / 2 / 3, KIR2L3), TIGIT, VISTA, IDO, CEACAM-1 or A2aR.

[0197] The immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligand or receptors, or preferably antibodies, such as human antibodies. Known inhibitors of the immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies.

[0198] Preferably the ICI is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG- 3, BTLA, B7H3, B7H4, TIM3, KIR (such as 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 aspects, 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 chosen from 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 antibodies described in International patent applications W02004004771, W02004056875, W02006121168, WO2008156712, W02009014708, W02009114335, WO2013043569 and W02014047350. Preferably, the inhibitor of PD-L1 is durvalumab, atezolizumab, LY3300054 or avelumab. Preferably, the inhibitor of PD-L2 is rHIgM12B7. Preferably, the LAG3 inhibitor is IMP321, BMS-986016 or inhibitors of the LAG3 receptor described in US patent US5,773,578. Preferably, the inhibitor of A2aR is PBF-509. Preferably, the inhibitor of CTLA-4 is an anti-CTLA-4 antibodies including, but not limited to, ipilimumab (see, e.g., US patents US6,984,720 and US8,017,114), tremelimumab (see, e.g., US 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 US patent US8,491,895. Example of anti-VISTA antibodies are described in US patent application US20130177557. Preferably, the ICI is chosen 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.

[0199] The present invention also relates to products containing the modified immunes cell, the modified myeloid cell, the modified iPS or the modified HSC according to the invention, and an immune checkpoint therapy related to co-stimulatory antibodies delivering positive signals through immune-regulatory receptors including but not limited to ICOS, CD137, CD27, OX- 40 and GITR, as a combined preparation for simultaneous, separate or sequential use in treatment of cancer, an autoimmune disease or an inflammatory disease.

[0200] The present invention also relates to products containing the modified immunes cell, the modified myeloid cell, the modified iPS or the modified HSC according to the invention, and additional cancer therapies as a combined preparation for simultaneous, separate or sequential use in treatment of cancer. In particular, products containing the modified immunes cell, the modified myeloid cell, the modified iPS or the modified HSC according to the invention may be administered in combination with targeted therapy, immunotherapy such as immune checkpoint therapy and / or immune checkpoint inhibitor, co-stimulatory antibodies, chemotherapy and / or radiotherapy.

[0201] In some embodiments, the products containing the modified immunes cell, the modified myeloid cell, the modified iPS or the modified HSC according to the invention may be used in combination with targeted therapy. As used herein, the term “targeted therapy” refers to targeted therapy agents, drugs designed to interfere with specific molecules necessary for tumor growth and progression. For example, targeted therapy 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 to interact with targets inside a cell. Small molecules are usually designed to interfere with the enzymatic activity of the target protein such as for example proteasome inhibitor, tyrosine kinase or cyclin- dependent kinase inhibitor, histone deacetylase inhibitor. Targeted therapy may also use cytokines. Examples of such targeted therapy include: Ado-trastuzumab emtansine (HER2), Afatinib (EGFR (HER1 / ERBB1), HER2), Aldesleukin (Proleukin), alectinib (ALK), Alemtuzumab (CD52), axitinib (kit, PDGFRbeta, VEGFR1 / 2 / 3), Belimumab (BAFF), Belinostat (HD AC), Bevacizumab (VEGF ligand), Blinatumomab (CD19 / CD3), bortezomib (proteasome), Brentuximab vedotin (CD30), bosutinib (ABL), brigatinib (ALK), cabozantinib (FLT3, KIT, MET, RET, VEGFR2), Canakinumab (IL-1 beta), carfilzomib (proteasome), ceritinib (ALK), Cetuximab (EGFR), cofimetinib (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).

[0202] In some embodiments, the products containing the modified immunes cell, the modified myeloid cell, the modified iPS or the modified HSC according to the invention may be used in combination with chemotherapy. As used herein, the term “antitumor chemotherapy” or “chemotherapy” has its general meaning in the art and refers to a cancer therapeutic treatment using chemical or biochemical substances, in particular using one or several antineoplastic agents or chemotherapeutic agents. Chemotherapeutic agents include, but are not limited to alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g. , calicheamicin, especially calicheamicin gammall and calicheamicin omegall) ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; methylhydrazine derivatives including N-m ethylhydrazine (MIH) and procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT- 11); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; anthracyclines, nitrosoureas, antimetabolites, epipodophylotoxins, enzymes such as L-asparaginase; anthracenediones; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0203] In some embodiments, the products containing the modified immunes cell, the modified myeloid cell, the modified iPS or the modified HSC according to the invention is administered to the patient in combination with radiotherapy for simultaneous, separate or sequential use in treatment of cancer, an autoimmune disease or an inflammatory disease. Suitable examples of radiation therapies include external beam radiotherapy (such as superficial X-rays therapy, orthovoltage X-rays therapy, megavoltage X-rays therapy, radiosurgery, stereotactic radiation therapy, Fractionated stereotactic radiation therapy, cobalt therapy, electron therapy, fast neutron therapy, neutron-capture therapy, proton therapy, intensity modulated radiation therapy (IMRT), 3 -dimensional conformal radiation therapy (3D-CRT) and the like); brachytherapy; unsealed source radiotherapy; tomotherapy; and the like. Gamma rays are another form of photons used in radiotherapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay. In some embodiments, radiotherapy may be proton radiotherapy or proton minibeam radiation therapy. Proton radiotherapy is an ultra-precise form of radiotherapy 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 Jun 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). Radiotherapy may also be FLASH radiotherapy (FLASH-RT) or FLASH proton irradiation. FLASH radiotherapy involves the ultra-fast delivery of radiation treatment at dose rates several orders of magnitude greater than those currently 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 C. M., Martin F., PouzouletF., Nauraye C., et al. Experimental set-up for FLASH proton irradiation of small animals using a clinical system. Int J Radiat Oncol Biol Phys, 102 (2018), pp. 619-626. doi: 10.1016 / j ijrobp.2018.06.403. Epub 2018 Jul 11). It is also described a method for treating cancer, an autoimmune disease or an inflammatory disease in a subject in need thereof, comprising a step of administering to said subject a therapeutically effective amount of the modified immunes cell, the modified myeloid cell, the modified iPS or the modified HSC according to the invention.

[0204] It is also described a method for treating cancer, an autoimmune disease or an inflammatory disease, which comprises: collecting immune cells or myeloid cells from a patient; modifying at least one of said immune cells or myeloid cells by transducing said cell with a first vector comprising a sequence coding for IL-2 under the control of an inducible or constitutive promoter, and with a second vector comprising a nucleic sequence coding for the CSAR or the CCR of the invention, preferably a lentiviral vector; and re-injecting said modified immune cells or myeloid cells into the patient.

[0205] By "cancer", the invention means tumors. The tumors to be treated include primary tumors and metastatic tumors, as well as refractory tumors. Refractory tumors include tumors that fail to respond or are resistant to treatment with chemotherapeutic agents alone, antibodies alone, radiation alone or combinations thereof. Refractory tumors also encompass tumors that appear to be inhibited by treatment with such agents, but recur up to five years, sometimes up to ten years or longer after treatment is discontinued.

[0206] Examples of cancers that may be treated by the modified myeloid cell according to the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomangio sarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangio sarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical 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; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified nonHodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid 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.

[0207] Preferably, cancer is a solid tumor or a metastasis.

[0208] Examples of autoimmune diseases that may be treated by the modified immunes cell or the modified myeloid cell according to the invention include, but are not limited to, rheumatoid arthritis, inflammatory bowel disease (Crohn disease, ulcerative colitis) or multiple sclerosis.

[0209] By "treatment” or “treat", it is meant both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subj ect beyond that expected in the absence of such treatment.

[0210] By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen.

[0211] The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.

[0212] The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

[0213] By a "therapeutically effective amount", it is meant a sufficient amount of the modified myeloid cell, the modified iPS or the modified HSC according to the invention, to treat the disease (e.g. cancer) at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the product of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the product; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0214] By "pharmaceutical" or “pharmaceutically acceptable”, it is meant that molecular entities and compositions do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.

[0215] Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including 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 must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and 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 a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be 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 type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or inj ected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0216] The first and / or second vector, or the CSAR2bands or the CCR2bands, may be administered via at least one lipid nanoparticle (LNP) or at least one liposome or at least one virus-like particle (VLP). LNPs refer to stable nucleic acid-lipid nanoparticles, particularly in the field of nucleic acid and mRNA drug delivery systems. Liposomes refer to spherical vesicles made of a lipid bilayer. The LNP or liposome comprise at least one ionizable lipid and at least one nucleic acid molecule. Preferably, the LNP or liposome further comprise at least one helper lipid. Preferably the helper lipid is chosen from phospholipids, cholesterol lipids, and polymers.

[0217] The phospholipid may typically be chosen from dioleoyl-phosphatidylethanolamine (DOPE) or a derivative thereof, distearoylphosphatidylcholine (DSPC) or a derivative thereof, distearoyl-phosphatidylethanolamine (DSPE) or a derivative thereof, stearoyl oleoylphosphatidyl choline (SOPC) or a derivative thereof, l-stearioyl-2-oleoyl- phosphatidyethanol amine (SOPE) or a derivative thereof, N-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP) or a derivative thereof, or any combination thereof.

[0218] The cholesterol lipid may be cholesterol or a derivative thereof.

[0219] The polymer may be polyethylene glycol (PEG) or a derivative thereof.

[0220] The nucleic acid molecule is a DNA molecule or an RNA molecule. In some embodiments, the nucleic acid molecule is cDNA, mRNA, miRNA, siRNA, sgRNA, modified RNA, antagomir, antisense molecule, guide RNA molecule, CRISPR guide RNA molecule, peptide, therapeutic peptide, targeted nucleic acid, or any combination thereof.

[0221] In some embodiments, the liposome is a ligand-targeted liposome which surface is functionalized with at least one target ligand. Said target ligand may be chosen from antibodies, agonist peptides and aptamers. It allows a precise delivery of the first and / or second vector(s) to specific cells such as myeloid cells, preferably monocytes or macrophages, by recognizing corresponding receptors or antigens.

[0222] Virus-like particles (VLP) are vesicles having a hollow core and an envelope, that mimic viruses but that are not infectious. They can be made up of viral structural protein(s) that selfassemble into the virus-like structure, of chemical compound(s), or of polymers that are arranged in multiple layers surrounding a hollow core. Said VLP can package nucleic acid sequences corresponding to transcription units encoding the CSAR or the CCR of the invention and, optionally, transcription units encoding the at least one cytokine, preferably at least one interleukin.

[0223] The present invention also relates to a method of delivering at least one mRNA molecule encoding the first and / or second vector, or the CSAR2bands or the CCR2bands to a subject in need thereof. In some embodiments, the LNP or the composition thereof delivers the mRNA molecule encoding the first and / or second vector, or the CSAR2bands or the CCR2bands to a target, preferably myeloid cells. Said method may comprise a single administration or multiple administrations of the LNP or the composition thereof. In some embodiments, the LNP or the composition thereof is administered by a delivery route selected from the group consisting of intradermal, subcutaneous, intramuscular, intraventricular, intrathecal, oral delivery, intravenous, intratracheal, intraperitoneal, in utero delivery, or any combination thereof.

[0224] The invention also provides kits comprising the compound of the invention. Kits containing the compound of the invention find use in therapeutic methods.

[0225] Preparation method

[0226] The present invention also relates to a method for manufacturing a modified immune cell, a modified myeloid cell, a modified iPS or a modified HSC, wherein the method comprises: providing at least one cell chosen from isolated immune cell, myeloid cells, iPS or HSC; transducing said cell with at least a vector, preferably a lentiviral vector, comprising a sequence coding for the CSAR or the CCR of the invention. The present invention also relates to a method for manufacturing a modified immune cell, a modified myeloid cell, a modified iPS or a modified HSC, wherein the method comprises: providing at least one cell chosen from isolated immune cell, myeloid cells, iPS or HSC; transducing said cell with at least a first vector, preferably a lentiviral vector, comprising a nucleic sequence coding for IL-2 or a nucleic sequence coding for at least one interleukin chosen from IL-10, IL-15, IL-13, IL-7A, IFNalpha, IFNbeta, IFNlambda, IFNgamma, IL-1A, IL- IB, IL- 12 and IL-21, under the control of an inducible or constitutive promoter; and a second vector, preferably a lentiviral vector, comprising a sequence coding for the CSAR or the CCR of the invention.

[0227] The first step of the preparation method is the provision of at least one cell chosen from isolated immune cells, myeloid cells, iPS or HSC.

[0228] Then, said cells are transduced with at least a vector, preferably lentiviral vectors, comprising a sequence coding for the CSAR or the CCR of the invention.

[0229] Preferably, said cells are transduced with two vectors, preferably lentiviral vectors, the first one comprising a sequence coding for IL-2 under the control of an inducible or constitutive promoter, and the second one comprising a nucleic sequence coding for a CSAR or a CCR. The vectors may be used to introduce the CSAR or the CCR and IL-2 into an isolated immune or myeloid cell, preferably a monocyte.

[0230] Alternatively, said cells may be transduced with a single vector (i.e. first vector) comprising a sequence coding for IL-2 under the control of an inducible or constitutive promoter, and a nucleic sequence coding for a CSAR or CCR under the control of a promoter.

[0231] In one embodiment, the vector is a plasmid vector, a viral vector, a retrotransposon (e.g. piggyback, sleeping beauty) or a site directed insertion vector (e.g. CRISPR, Zn finger nucleases, TALEN). Preferably, the vector is a viral vector, preferably a lentiviral vector. Vectors, including those derived from retroviruses such as lentivirus, are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells. They also have the added advantage of resulting in low immunogenicity in the subject into which they are introduced. The expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid to a promoter, and incorporating the construct into an expression vector. The vector is one generally capable of replication in a mammalian cell, and / or also capable of integration into the cellular genome of the mammal. Typical vectors contain transcription and translation terminators, initiation sequences and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0232] The nucleic sequence (nucleic acid) coding for said CSAR, said CCR or said 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.

[0233] The expression vector may be provided to a cell 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, volumes 1-4, Cold Spring Harbor Press, NY). Viruses which are useful as vectors include retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and lentiviruses. In general, a suitable vector contains 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, e.g., enhancers, regulate the frequency of transcriptional initiation. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0234] In one embodiment, the vector is a recombinant lentiviral vector with modified tropism comprising (i) a mutant glycoprotein (G protein) which ablates the natural receptor tropism and (ii) an insertion of an antibody, a receptor ligand or a peptide targeting specifically a cell, preferably a myeloid cell, preferably a monocyte or a macrophage. Said G protein plays a critical role during the initial steps of virus infection as it is responsible for virus attachment to specific receptors. After binding, G triggers the fusion between the viral and endosomal membranes, which releases the viral genome in the cytosol for the subsequent steps of infection. Preferably, said recombinant lentiviral vector comprising a mutant G protein allows for an antigen-specific infection of myeloid cells, preferably of monocytes or macrophages.

[0235] In order to assess expression of a polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibioticresistance genes, such as neo and the like. Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assessed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene. Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.

[0236] The method comprises introducing into said cell a first vector, said vector comprising a sequence coding for IL-2 under the control of a cytokine specific promoter. Preferably, the cytokine specific promoter is chosen from promoter of interleukin-6 or the promoter of interleukin-8.

[0237] Preferably, the gene of IL-2 is a human gene of nucleic sequence SEQ ID NO:9.

[0238] The method also comprises introducing into said cell a second vector, said vector comprising the CSAR or the CCR of the invention.

[0239] The invention will be further illustrated by the following examples. However, these examples should not be interpreted in any way as limiting the scope of the present invention.

[0240] EXAMPLE:

[0241] Myeloid cell comprising a cytokine chimeric receptor (CCR) Materials and methods Primary cells

[0242] Peripheral blood mononuclear cells (PBMC) were separated from plasmapheresis residues using Ficoll-Paque (GE Healthcare). Informed consent was obtained from all donors, and samples were deidentified prior to use in the study. Monocytes were isolated by CD14+ positive selection using CD14magnetic microbeads (Miltenyi 130-050-201).

[0243] Plasmid construction and virus production

[0244] CCR constructs were cloned under a SSFV promoter into a pTRIP lentiviral vector.

[0245] Lenti virus were produced in HEK293 LTV cells. Lentiviral vectors were co transfected with psPAX2 (2nd generation lentiviral packaging plasmid), pMD2.G (encoding VSV-G) and a plasmid encoding a Vpr-Vpx fusion protein, using PEI MAX® (Polysciences).

[0246] After 18h the media was replaced with fresh media to remove transfection reagent. Supernatants containing lentivector were collected 24h after medium change and filtered with a 0.45pm filter.

[0247] Monocyte transduction and differentiation

[0248] CD14+ cells were transduced with lentivectors in presence of 4pg / mL protamine. Monocytes were then allowed to differentiate in macrophages for 10 days in macrophage medium (RPMI + 5% fetal calf serum + 5% human serum + 1% Penicillin-Streptomycin) with 50ng / mL M-CSF in Corning® 100 mm Not TC-treated Culture Dish.

[0249] Detection of CCR expression

[0250] Cells were harvested with StemPro Accutase Cell Dissociation Reagent (ThermoFischer) and washed with PBS then stained with antibodies directed against the CCR extracellular domain for 30 min at 4°C. Human FcBlockTM (BD Biosciences) was added during the staining. Cells were analyzed with FACS using a NocoCyte.

[0251] Cell Cyte-based spheroid growth assay

[0252] 1x103 tumor cells were seeded with 1.6x104 macrophages in Corning® Costar® UltraLow Attachment 96-Well Plates (Merck). GFP fluorescence was then followed and measured on several days with a CellCyte analysis system. GFP intensity analysis was performed with CellCyte software. After background removing, green objects were defined with a threshold. Total GFP+ integrated intensity is the sum of pixels belonging to all green objects.

[0253] Cytokine secretion assay

[0254] CCR-M were cultured in media, media supplemented with the target cytokine or cocultured with cytokines+ tumor cells for 24h. Supernatant was collected and clarified by centrifugation. Cytokines secreted were measured by Legendplex (Anti Viral response panel, Biolegend) following the manufacturer instructions.

[0255] Cytokine secretion assay

[0256] CCR-M were cultured in media, media supplemented with the target cytokine or cocultured with cytokines+ tumor cells for 24h. Cells were detached by accutase treatment and labelled with anti-CD86, anti-CD4, anti-HLADR, anti-CD169, anti-CD206 and and anti- CD163 conjugated antibodies and analyzed with NocoCyte flow cytometer.

[0257] Results

[0258] Generation of CCR expressing macrophages (CCR-MQ).

[0259] Monocytes were transduced with lentivectors encoding the various CCR constructs and allow to differentiate for 6 days into CCR-M . The CCR-M displayed high surface expression of their CCR as assayed by FACS using an antibody directed against the extracellular domain.

[0260] CCR-MQ acquired a pro-inflammatory phenotype.

[0261] The inventors tested the polarization of CCR-M upon stimulation with the target cytokine. CCR-M harboring a STING or STINGtz domain secreted high levels of pro- inflammatory cytokines and up-regulated inflammatory surface markers.

[0262] Next, the inventors tested the polarization of CCR-M upon stimulation with tumor cells expressing the target cytokine and similar results were obtained.

[0263] CCR-MQ impede the growth of tumor spheroids.

[0264] The inventors tested the capacity of CCR-M to impact the growth in 3D of tumor spheroid secreting the target cytokines over a 7-day period. CCR-M slowed down the growth of cytokine+ tumor spheroids.

[0265] CCR-MQ activated by their specific cytokine / chemokine become phagocytic.

[0266] The inventors tested the capacity of CCR-M to perform phagocytosis. CCR-M exposed to their specific cytokine / chemokine were assayed for their capacity to phagocytose fluorescently labeled tumor cells. Indeed CCR-M when activated by the appropriate chemokine / cytokine were able to perform phagocytosis of tumor cells. CCR-MQ can perform antigen cross-presentation

[0267] Since CCR-M were able to phagocytose tumor cells, the inventors tested their ability to perform Ag cross presentation. Monocytes from an HLA-A2 donor ware co-cultured with a melanA+ melanoma cell line HAL-A2 negative. CCR-M were then recovered and co-cultured with a melanaA-HLA-A2 specific CD8+ T cell clone. Measurement of T cell activation and killing of an HLA-A2 MelanA+ melanoma cell line revealed that our CCR-M were efficient at performing Ag cross presentation to the T clone.

[0268] The inventors develop a variety of CCR that are specific of secreted molecules present in the TME. The CCR can be made of two chains, such as for the IL10R, IL4R and TGFbR, or of one chain such as for CSF1R. Even if encoded by two separate chains, the inventors can engineer the extracellular part of the Receptor (R) as a single chain linked by a spacer peptide. The CCR can also be made of the two chains of the IL6 receptor, IL6R alpha and gpl30 or only the IL6R alpha extracellular domain or only the gpl30 fused to the transmembrane domain. The appropriate transmembrane domain(s) would be fused to it and then the inventors use as an intracellular domain a truncation of STING fused or not to the zebrafish sequence as described in the invention. The inventors also use instead the CD40 or CD40-CD3z.

[0269] REFERENCES:

[0270] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0271] Cameron MJ, Kelvin DJ. Cytokines, Chemokines and Their Receptors. In: Madame Curie Bioscience Database [Internet], Austin (TX): Landes Bioscience; 2000-2013. Available from: https: / / www.ncbi.nlm.nih.gov / books / NBK6294 / .

[0272] Kartikasari AER, Huertas CS, Mitchell A, Piebanski M. Tumor-Induced Inflammatory Cytokines and the Emerging Diagnostic Devices for Cancer Detection and Prognosis. Front Oncol. 2021 Jul 7; 11 :692142.

[0273] Landskron G, De la Fuente M, Thuwajit P, Thuwajit C, Hermoso MA. Chronic inflammation and cytokines in the tumor microenvironment. J Immunol Res. 2014;2014: 149185. doi: 10.1155 / 2014 / 149185. Epub 2014 May 13. PMID: 24901008; PMCID: PMC4036716. Raman D, Baugher PJ, Thu YM, Richmond A. Role of chemokines in tumor growth. Cancer Lett. 2007 Oct 28;256(2): 137-65. doi: 10.1016 / j.canlet.2007.05.013. Epub 2007 Jul 12. PMID: 17629396; PMCID: PMC2065851.

[0274] Starka L, Duskova M. What is a hormone? Physiol Res. 2020 Sep 30;69(Suppl 2):S183- S185. doi: 10.33549 / physiolres.934509. PMID: 33094616; PMCID: PMC8603735.

Claims

CLAIMS:

1. A modified cell comprising a cytokine chimeric receptor (CCR), wherein said CCR comprises: an extracellular domain comprising a domain of a cytokine receptor; a transmembrane domain; and an intracellular signaling domain comprising CD40 cytotail, CD3zeta intracellular domain and / or STING or one of its fragments or variants thereof.

2. The modified cell according to claim 1, wherein the cell is a modified immune cell, a modified myeloid cell, preferably a monocyte, a macrophage or a dendritic cell, and more preferably a monocyte.

3. The modified cell according to claim 1, wherein the cell is an induced pluripotent stem cells (iPS) or a hematopoietic stem cells (HSC).

4. The modified cell according to any one of claims 1 to 3, wherein the CCR comprises from its N-terminal end to its C-terminal end :- an extracellular domain comprising a domain of a cytokine receptor;- optionally a hinge domain;- a transmembrane domain; and- a first intracellular signaling domain comprising the CD40 cytotail, CD3zeta intracellular domain and / or STING or one of its fragments or variants thereof.

5. The modified cell according to any one of claims 1 to 3, wherein the CCR comprises, from its N-terminal end to its C-terminal end: an extracellular domain, optionally a hinge domain of sequence SEQ ID NO: 1, a transmembrane domain of sequence SEQ ID NO:2, an intracellular signaling domain of sequence SEQ ID NO:3.

6. The modified cell according to any one of claims 1 to 3, wherein the CCR comprises, from its N-terminal end to its C-terminal end: an extracellular domain, optionally a hinge domain of sequence SEQ ID NO: 1, a transmembrane domain of sequence SEQ ID NO:2,a first intracellular signaling domain of sequence SEQ ID NO:3, fused, preferably directly, to a second intracellular signaling domain of sequence SEQ ID NO:4.

7. The modified cell according to any one of claims 1 to 3, wherein the CCR comprises, from its N-terminal end to its C-terminal end: an extracellular domain, optionally a hinge domain of sequence SEQ ID NO: 1, a transmembrane domain of sequence SEQ ID NO:2, and an intracellular signaling domain of sequence SEQ ID NO:7.

8. The modified cell according to any one of claim 1-7, wherein the cell comprises an additional vector, said vector comprising a sequence coding for a gene of interest under the control of a cytokine specific promoter.

9. The modified cell according to any one of claim 1-8, wherein the cell comprises:(i) either a first vector comprising a sequence coding for 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 coding for a cytokine chimeric receptor (CCR); or(ii) only comprises the first vector, which encodes for both the at least one cytokine, preferably at least one interleukin, and for the CCR.

10. The modified cell according to claim 9, wherein the cytokine is IL-2, and the sequence coding for IL-2 is the sequence SEQ ID NO: 9.

11. The modified cell according to any one of claims 9 or 10, wherein the promoter is inducible.

12. A lipid-based nanoparticle (LNP), a liposome or virus-like particle (VLP) comprising a nucleic acid encoding for the cytokine chimeric receptor (CCR) of any one of claims 1 to 8 or the first and / or second vector of any one of claims 9-11.

13. A pharmaceutical composition comprising the modified cell of any one of claims 1-11, or the lipid-based nanoparticle (LNP), the liposome or the virus-like particle (VLP) of claim 12 and a pharmaceutical acceptable carrier.

14. The modified cell according to any one of claims 1-11, the lipid-based nanoparticle (LNP), the liposome or the virus-like particle (VLP) of claim 12 or the pharmaceutical composition of claim 13 for use in the treatment of cancer or an inflammatory disease.

15. The modified cell, the lipid-based nanoparticle (LNP), the liposome or the virus-like particle (VLP) or the pharmaceutical composition for use of claim 14, wherein the cancer is a solid tumor.

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