Regulated expression in tumor microenvironment

Abstract

The present invention relates generally to the field of medicine. More particularly, it relates to an animal eukaryotic cell for use in the treatment of a solid tumor in a patient in need of treatment, said cell comprising a novel expression cassette allowing controlled expression of a first therapeutic transgene within and by said solid tumour.

Description

[0001] DESCRIPTION

[0002] TITLE: REGULATED EXPRESSION IN THE TUMOR MICROENVIRONMENT

[0003] FIELD OF INVENTION

[0004] The present invention relates generally to the field of medicine. More particularly, it relates to an animal eukaryotic cell for use in the treatment of a solid tumor in a patient in need of it, said cell comprising a novel expression cassette allowing the controlled expression of a first therapeutic transgene within and by said solid tumor.

[0005] PREVIOUS ART

[0006] In certain cancers, the therapeutic efficacy of chimeric antigen receptor (CAR-T) lymphocytes in eradicating tumor cells is undeniable. However, this treatment is rarely indicated as a first-line option due to the high frequency of associated adverse reactions. These secondary reactions, such as the "cytoquinolone storm" or the recognition of the tumor cell-targeted antigen in healthy tissues, are primarily due to the constitutive expression of CAR by genetically modified lymphocytes. This represents a significant obstacle to the development of CAR-T cell therapies. Various strategies aimed at eliminating these adverse effects and improving the efficacy of CAR-T cells are the subject of active research. To date, none have been successful.

[0007] BRIEF OVERVIEW OF THE INVENTION

[0008] Faced with this major challenge, the inventors developed a novel synthetic promoter enabling the controlled expression of a downstream cloned transgene and overcoming all obstacles. With this technology, a primary objective of the invention is to provide an animal eukaryotic cell for use in treating solid tumors in patients who require it. This cell comprises a novel expression cassette allowing the controlled expression of a first therapeutic transgene within and by said solid tumor. Another objective of the invention is to make available to the medical community the modified animal eukaryotic cell itself and the means to produce it.

[0009] DETAILED DESCRIPTION

[0010] In its most general aspect, the invention relates to an animal eukaryotic cell for use in the treatment of a solid tumor in a patient who needs it, wherein said animal eukaryotic cell:

[0011] ■ is chosen from: the T lymphocyte and the NK cell; and

[0012] ■ includes an expression cassette comprising, from the 5' end to the 3' end: - an inducible promoter whose sequence has at least 90%, in particular 95%, identity with the SEQ ID NO: 1 (2XAARE) sequence YB - TATA ), 2 (2XAARE TK ), 3 (miniCHACl YB TATA ), 4 (ULBP1), 6 (HSPA1B) or 7 (CHAC1);

[0013] - at least one initial therapeutic transgene,

[0014] in which the expression of said at least one first therapeutic transgene is reversible and is induced by the tumor microenvironment of said solid tumor to be treated,

[0015] said patient who needs it being free from deficiency in essential or non-essential amino acids (i.e. that said patient who needs it is in a situation of care and / or nutrition which does not cause any deficiency in glucose or essential amino acids, namely: histidine (His, H), isoleucine (He, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), threonine (Thr, T), tryptophan (Trp, W) and valine (Vai, V), or non-essential, alanine (Ala, A), glutamic acid (Glu, E), arginine (Arg, R), aspartic acid (Asp, D), asparagine (Asn, N), cysteine ​​(Cys, C), glutamine (Gin, Q), tyrosine (Tyr, T), glycine (Gly, G), proline (Pro, P) and serine (Ser, S)

[0016] and in which said expression cassette is specifically devoid of a coding or non-coding sequence having post-transcriptional regulatory properties. As previously mentioned, the inventors have developed a novel synthetic promoter enabling the expression of a downstream cloned transgene, which advantageously allows for the spatial regulation of the expression of said at least one first transgene, or even a second transgene, in T lymphocytes or NK cells genetically modified by conditions characteristic of the tumor microenvironment (TME), such as amino acid or glucose deficiencies. Thus, the use of this promoter makes it possible to confine the expression of at least one first therapeutic transgene to the pathological tissue only and to prevent adverse effects due to the constitutive expression of this transgene.In the context of solid tumors, the present invention constitutes a considerable therapeutic tool due to its safety, particularly with NK cells (e.g. NK-92 cell line), considered safer than T lymphocytes (e.g. CD3+ T lymphocytes) in an allogeneic context and clinically approved for CAR-NK therapies.

[0017] It is also understood that the invention relates to a method of treating a solid tumor in a patient who needs it, comprising the administration of an animal eukaryotic cell:

[0018] ■ chosen from: the T lymphocyte and the NK cell; and

[0019] ■ including an expression cassette comprising from end 5' to end 3':

[0020] - an inducible promoter whose sequence has at least 90%, in particular 95%, identity with the SEQ ID NO: 1 (2XAARE) sequence YB - TATA ), 2 (2XAARE TK ), 3 (miniCHACl YB TATA), 4 (ULBP1), 6 (HSPA1B) or 7 (CHAC1);

[0021] - at least one initial therapeutic transgene,

[0022] in which the expression of said at least one first therapeutic transgene is reversible and is induced by the tumor microenvironment of said solid tumor to be treated, said patient in need of it being free from deficiency in essential or non-essential amino acids (i.e., said patient in need of it is in a care and / or nutritional situation that does not result in any deficiency in glucose or essential amino acids, namely: histidine (His, H), isoleucine (He, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), threonine (Thr, T), tryptophan (Trp, W) and valine (Vai, V), or non-essential amino acids, alanine (Ala, A), glutamic acid (Glu, E), arginine (Arg, R), aspartic acid (Asp, D), asparagine (Asn, N), cysteine ​​(Cys, C), glutamine (Gin, Q), tyrosine (Tyr, T), glycine (Gly G), proline (Pro, P) and serine (Ser, S)

[0023] and wherein said expression cassette is specifically devoid of a coding or non-coding sequence having post-transcriptional regulatory properties. "Animal eukaryotic cell" here refers to an immune cell, that is, a cell involved in the functioning of the immune system. Numerous examples exist, such as lymphocytes (e.g., T, B, NK, and NKT cells), phagocytes (e.g., macrophages and dendritic cells), and granulocytes (e.g., neutrophils, eosinophils, and basophils), as well as their precursor cells. These cells can be of autologous, syngeneic, allogeneic, or xenogeneic origin, primary, cancerous, or immortalized. Finally, these cells can be genetically modified. It should be noted that the invention specifically utilizes a genetically modified T lymphocyte and an NK cell.The term "T lymphocyte" in the invention refers in particular to a naïve, effector, or memory T lymphocyte with helper or cytotoxic properties. The term "NK cell" in the invention refers in particular to a primary, cancerous, or immortalized NK cell.

[0024] According to another embodiment, the invention therefore relates to the animal eukaryotic cell for its use as described above, said animal eukaryotic cell being a T lymphocyte. In particular, it is a CD3+ T lymphocyte.

[0025] According to another embodiment, the invention also relates to the animal eukaryotic cell for its use as described above, said animal eukaryotic cell being an NK cell. In particular, it is an NK cell belonging to the NK-92 lineage.

[0026] "Solid tumor" refers to a solid cancerous tumor, such as carcinomas, sarcomas, or blastomas, identifiable as a localized cluster of cells, which is distinct from blood cell cancers, such as leukemias, in which the cancerous cells circulating in the blood or lymph are dispersed throughout the body. According to another embodiment, the invention therefore relates to the animal eukaryotic cell for its use as described above, said solid tumor being selected from: carcinoma, sarcoma, and blastoma.

[0027] "Expression cassette" refers to a nucleic acid comprising an inducible promoter upstream of at least one first therapeutic transgene. Notably, this cassette is "devoid of a coding or non-coding sequence having post-transcriptional regulatory properties." In other words, it specifically does not include a nucleic acid sequence that can be either coding (capable of coding for proteins) or non-coding (not coding for proteins) and that possesses regulatory properties acting after the transcription process. Furthermore, in another embodiment, the invention relates to the animal eukaryotic cell for its use as described above, wherein said expression cassette is devoid of a coding or non-coding sequence having post-transcriptional regulatory properties.

[0028] "Inducible promoter" refers to a nucleic acid located upstream (at the 5' end) of a gene (or transgene) that controls its expression, notably by regulating its transcription. In the invention, this promoter is described as inducible because the expression of the gene (or transgene) it controls is not constitutive and is only induced after its activation in response to the appropriate stimulus. In the invention, this appropriate stimulus corresponds to the tumor microenvironment. In other words, the expression of said at least one first therapeutic transgene is induced only when the genetically modified eukaryotic animal cell according to the invention has infiltrated said solid tumor or is in close proximity to it. Otherwise, the expression of said at least one first therapeutic transgene is not induced. This means:

[0029] - After being genetically modified, the animal eukaryotic cell according to the invention includes an expression cassette that does not express itself and this can be safely administered to a patient who needs it.

[0030] - Once in the body of the patient who needs it, as long as the genetically modified animal eukaryotic cell according to the invention does not approach or infiltrate said solid tumor, said expression cassette is (still) not expressed; and conversely, if it approaches or infiltrates it, the tumor microenvironment will then cause the induction of said inducible promoter according to the invention and therefore the expression of said at least one first therapeutic transgene so that the genetically modified animal eukaryotic cell according to the invention plays its therapeutic role of destroying said solid tumor.

[0031] - However, in the event that the genetically modified animal eukaryotic cell according to the invention has destroyed said solid tumor or moves away from it, the tumor microenvironment can no longer play its induction role and the expression of said at least one first therapeutic transgene is repressed until the genetically modified animal eukaryotic cell according to the invention encounters a new solid tumor or infiltrates again into the solid tumor from which it has moved away.

[0032] It should be noted that these notions of induction and reversibility of the expression of the expression cassette of the invention, which make it a tool of immense safety, are explicitly found in the characteristic: "the expression of said at least one first therapeutic transgene is reversible and is induced by the tumor microenvironment of said solid tumor to be treated".

[0033] It should also be noted that in the invention, the inducible promoter is chosen from specific sequences, namely the one having at least 90%, in particular 95%, identity with the sequence SEQ ID NO: 1 (2XAARE YB - TATA ), 2 (2xAARE TK ), 3 (miniCHACl YB - TATA), 4 (ULBP1), 6 (HSPA1B) or 7 (CHAC1), all inducible by the tumor microenvironment. "% identity" refers to the percentage determined by direct comparison of two oligonucleotide sequences (nucleic acid sequences), by determining the number of identical nucleotides between the two sequences, then dividing it by the number of nucleotides in the longer of the two sequences, and multiplying the result by 100. By "having at least 90% identity" we therefore mean that the aforementioned percentage of identity is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100%. In this regard, it should be noted that this definition applies to all embodiments of the invention, including when it involves a direct comparison of two polypeptide sequences (amino acid sequence).Furthermore, it is understood that sequences having at least 90% identity with a reference sequence retain the same properties and functions, or even that these are improved.

[0034] According to another embodiment, the invention relates to the animal eukaryotic cell for its use as described above, said inducible promoter being selected from sequences having at least 95% identity with the SEQ ID NO: 1 (2XAARE) sequences. YB - TATA ), 2 (2XAARE TK ), 3 (miniCHAC I YB TAIA ), 4(ULBP1), 6(HSPA1B), and 7(CHAC1). In particular, the invention relates to the animal eukaryotic cell for its use as described above, said inducible promoter being selected from the sequences SEQ ID NO: 1 (2XAARE YB TATA ), 2 (2XAARE TK ), 3 (miniCHAC I YB TAIA), 4(ULBP1), 6(HSPA1B), and 7(CHAC1). In particular, the invention also relates to the animal eukaryotic cell for its use as described above, said inducible promoter being selected from the sequences SEQ ID NO: 1 (2XAARE YB - TATA ), 2 (2XAARE TK ) and 3 (miniCHACI YB - TATA ), which are known to be activated by an inducer that induces the expression of the transcription factor ATF4. Advantageously, the invention also relates to the animal eukaryotic cell for its use as described above; said inducible promoter is the sequence SEQ ID NO: 1 (2XAARE YB TATA ), 4(ULBP1), 6 (HSPA1B) and 7 (CHAC1).

[0035] “At least one first therapeutic transgene” means that the expression cassette according to the invention comprises, downstream of said inducible promoter, at least one transgene. That is to say, there may be several, such as two, three, or even four. Advantageously, downstream of said inducible promoter are two transgenes. They are, or can be, described as therapeutic because this / these therapeutic transgene(s) designate(s) a sequence of nucleic acids carrying the information for a therapeutic protein, which, following the transcription and translation of this nucleic acid, contributes to, or leads to, the cure of a patient in need. In the invention, a therapeutic transgene can be selected from: oncogenes, anti-oncogenes, transcription factors (e.g., c-JUN, FOXO1, B ATF, TBET, or NRF2), trophic factors, cytokines (e.g., IL2, IFNγ, TNFα, IL-18, or IL-12), enzymes, hormones, receptors, transporters (e.g.GLUT1), ligands, human proteins, heterologous proteins, chimeric proteins, immunogenic proteins, chimeric antigen receptors (CARs) and antibodies.

[0036] Advantageously, the first therapeutic transgene is a chimeric antigen receptor (CAR). That is, a laboratory-created protein that recognizes and targets proteins present on the surface of cells in the solid tumor being treated. For example, the following CARs can be listed: CAR anti-A-folate receptor, CAR anti-Carbonic anhydrase IX, CAR anti-CD171, CAR anti-CD 19, CAR anti-CD20, CAR anti-CD276, CAR anti-CD319, CAR anti-CEA, CAR anti-cMet, CAR anti-EGFR, CAR anti-EGFRIII, CAR anti-EGFRn, CAR anti-FAP, CAR anti-GD2, CAR anti-GPL 00, CAR anti-GPC3, CAR anti-HER2, CAR anti-IL13Ra2, CAR anti-MAGE, CAR anti-MART-1, CAR anti-MSLN, CAR anti-Mucl, CAR anti-Mucl 6, CAR anti-NKG2D, CAR anti-PSMA, CAR anti-TRP-1, CAR anti-TRP2, CAR anti-VEGFR, CAR anti-HLA-G, CAR anti-CLDN18.2, CAR anti-EPCAM, CAR anti-FAP, CAR anti-RORl, CAR anti-ROR2, CAR anti-PDl, and CAR anti-PSCA (Marie-Thérèse Rubio et al. Biology, concepts and principles of CAR-T cells.Bulletin du Cancer, 2018, 105 (Suppl. 2), pp.S135-S146; Belovezhets, T. et al. Comparative Pre-Clinical Analysis of CD20-Specific CAR T Cells Encompassing IF 5-, Leu 16-, and 2F2-Based Antigen-Recognition Moieties. Int. J. Mol. Sci. 2023, 24, 3698.).

[0037] According to another embodiment, the invention therefore relates to the animal eukaryotic cell for its use as described above, wherein said at least one first therapeutic transgene is a chimeric antigen receptor (CAR),

[0038] in particular a CAR chosen from: an anti-A-folate receptor CAR, an anti-Carbonic anhydrase IX CAR, an anti-CD171 CAR, an anti-CD 19 CAR, an anti-CD20 CAR, an anti-CD276 CAR, an anti-CD319 CAR, an anti-CEA CAR, an anti-cMet CAR, an anti-EGFR CAR, an anti-EGFRIII CAR, an anti-EGFRn CAR, an anti-FAP CAR, an anti-GD2 CAR, an antigpl 00 CAR, an anti-GPC3 CAR, an anti-HER2 CAR, an anti-IL13Ra2 CAR, an anti-MAGE CAR, an anti-MART-1 CAR, an anti-MSLN CAR, an anti-Mucl CAR, an anti-Mucl6 CAR, an anti-NKG2D CAR, an anti-PSMA CAR, an anti-TRP-1 CAR, an anti-TRP2 CAR, an anti-VEGFR CAR, an anti-HLA-G CAR, an anti-HLA-G CAR, an anti-HLA-G CAR, an anti-HLA-G CAR, an anti-HLA-G CAR, an anti-HLA-G CAR, an anti-HLA-M anti-CLDN18.2, an anti-EPCAM CAR, an anti-FAP CAR, an anti-ROR1 CAR, an anti-ROR2 CAR, an anti-PD1 CAR and an anti-PSCA CAR.According to another embodiment, the invention relates to the animal eukaryotic cell for its use as described above, wherein said at least one first therapeutic transgene is a chimeric antigen receptor (CAR) selected from: an anti-A-folate receptor CAR, an anti-Carbonic anhydrase IX CAR, an anti-CD 171 CAR, an anti-CD 19 CAR, an anti-CD20 CAR, an anti-CD276 CAR, an anti-CD319 CAR, an anti-CEA CAR, an anti-cMet CAR, an anti-EGFR CAR, an anti-EGFRIII CAR, an anti-EGFRn CAR, an anti-FAP CAR, an anti-GD2 CAR, an anti-gpl O0 CAR, an anti-GPC3 CAR, an anti-HER2 CAR, an anti-IL13Ra2 CAR, an anti-MAGE CAR, an anti-MART-1 CAR, an anti-MSLN CAR, an anti-Mucl CAR, a CAR anti-Mucl6, an anti-NKG2D CAR, an anti-PSMA CAR, an anti-TRP-1 CAR, an anti-TRP2 CAR, an anti-VEGFR CAR, an anti-HLA-G CAR, an anti-CLDN18.2 CAR, an anti-EPCAM CAR, an anti-FAP CAR, an anti-RORl CAR, an anti-ROR2 CAR, an anti-PDl CAR and an anti-PSCA CAR.

[0039] As mentioned above, a second transgene may be present in the expression cassette according to the invention. In this case, it is located downstream of at least one first transgene and is subject to the regulation of said inducible promoter upstream of these two transgenes. According to another embodiment, the invention therefore relates to the animal eukaryotic cell for its use as described above, wherein said expression cassette further comprises a second transgene downstream of said first transgene.

[0040] the expression of said second transgene being reversible and being induced by the tumor microenvironment of said solid tumor to be treated,

[0041] said second transgene being in particular: ■ a transcription factor, in particular a transcription factor chosen from: c-JUN, FOXO1, B ATF, TBET and NRF2;

[0042] ■ a cytokine, in particular a cytokine chosen from: IL2, IFNγ, TNFα, IL18, and IL12; or

[0043] ■ a carrier, in particular the GLUT1 carrier.

[0044] According to another embodiment, the invention relates to the animal eukaryotic cell for its use as described above, said second transgene being:

[0045] ■ a transcription factor, in particular a transcription factor chosen from:

[0046] c-JUN, FOXO1, B ATF, TBET and NRF2;

[0047] ■ a cytokine, in particular a cytokine chosen from: IL2, IFNγ, TNFα, IL18, and IL12; or

[0048] ■ a carrier, in particular the GLUT1 carrier.

[0049] According to another embodiment, the invention relates to the animal eukaryotic cell for its use as described above, said second transgene being a transcription factor, in particular a transcription factor selected from: c-JUN, FOXO1, BATF, TBET, and NRF2. Advantageously, the invention relates to the animal eukaryotic cell for its use as described above, wherein said second transgene is c-JUN, the nucleic acid of which has a sequence having at least 90% identity with the nucleic acid of sequence SEQ ID NO: 9, or whose nucleic acid codes for an amino acid sequence having at least 90% identity with sequence SEQ ID NO: 10.

[0050] According to another embodiment, the invention relates to the animal eukaryotic cell for its use as described above, said second transgene being a cytokine, in particular a cytokine selected from: IL2, IFNγ, TNFα, IL18, and IL12. In particular, the invention therefore relates to the animal eukaryotic cell for its use as described above, said second transgene being a cytokine selected from: IL2, IFNγ, TNFα, IL18, and IL12.

[0051] According to another embodiment, the invention relates to the animal eukaryotic cell for its use as described above, said second transgene being a transporter, in particular the GLUT1 transporter. In particular, the invention therefore relates to the animal eukaryotic cell for its use as described above, said second transgene being the GLUT1 transporter.

[0052] According to another embodiment, the invention relates to the animal eukaryotic cell for its use as described above, wherein said expression cassette comprises a nucleic acid sequence encoding the p2A peptide, which is located downstream of said first therapeutic transgene and upstream of said second transgene (i.e., between said first therapeutic transgene and upstream of said second transgene). Advantageously, the nucleic acid encoding the p2A peptide corresponds to the sequence SEQ ID NO: 11. According to another embodiment, the invention relates to the animal eukaryotic cell for its use as described above, wherein said expression cassette comprises at its 3' end (downstream of said first therapeutic transgene or downstream of said second transgene when present) a nucleic acid sequence allowing the transcription of a polyA tail.Advantageously, the nucleic acid enabling the transcription of a polyA tail corresponds to the sequence SEQ ID NO: 12.

[0053] According to another embodiment, the invention relates to the animal eukaryotic cell for its use as described above, the animal eukaryotic cell being in a form suitable for administration to the patient in need. This may take the form of a pharmaceutical composition comprising (at least) one animal eukaryotic cell according to the invention and at least one pharmaceutically acceptable excipient, said pharmaceutical composition being in a form suitable for administration by one of the following routes: parenteral, injectable, intratumoral, subcutaneous, intraperitoneal, intravenous, intrathecal, intraventricular, intraparenchymal, intrapleural, or pulmonary.

[0054] According to another embodiment, the invention therefore relates to a pharmaceutical composition for use in the treatment of a solid tumor in a patient who needs it, said pharmaceutical composition comprising (at least) one animal eukaryotic cell according to the invention and at least one pharmaceutically acceptable excipient.

[0055] In view of the foregoing, it is understood that another aspect of the invention relates to an animal eukaryotic cell comprising an expression cassette including from the 5' end to the 3' end:

[0056] ■ an inducible promoter whose sequence has at least 90%, in particular 95%, identity with the SEQ ID NO: 1 (2XAARE) sequence YB - TATA ), 2 (2xAARE TK ), 3 (miniCHACl YB TATA), 4(ULBP1), 6 (HSPA1B) or 7 (CHAC1); and ■ at least one first therapeutic transgene, said at least one first therapeutic transgene being a chimeric antigen receptor (CAR), said expression cassette being in particular devoid of a coding or non-coding sequence having post-transcriptional regulatory properties and said animal eukaryotic cell being selected from: the T lymphocyte and the NK cell.

[0057] It is also understood that the invention relates to a pharmaceutical composition comprising (at least) one animal eukaryotic cell and at least one pharmaceutically acceptable excipient, said animal eukaryotic cell comprising an expression cassette comprising from the 5' end to the 3' end:

[0058] ■ an inducible promoter whose sequence has at least 90%, in particular 95%, identity with the SEQ ID NO: 1 (2XAARE) sequence YB - TATA ), 2 (2xAARE TK), 3 (miniCHACl YB TATA ), 4(ULBP1), 6 (HSPA1B) or 7 (CHAC1); and ■ at least one first therapeutic transgene, said at least one first therapeutic transgene being a chimeric antigen receptor (CAR), said expression cassette being in particular devoid of a coding or non-coding sequence having post-transcriptional regulatory properties and said animal eukaryotic cell being selected from: the T lymphocyte and the NK cell.

[0059] According to another embodiment, the invention relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), said animal eukaryotic cell being a T lymphocyte. In particular, it is a CD3+ T lymphocyte. According to another embodiment, the invention also relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), said animal eukaryotic cell being an NK cell. In particular, it is an NK cell belonging to the NK-92 cell line.

[0060] According to another embodiment, the invention relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), said expression cassette being devoid of a coding or non-coding sequence having regulatory properties at the post-transcriptional level.

[0061] According to another embodiment, the invention relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), said inducible promoter being selected from sequences having at least 95% identity with the SEQ ID NO: 1 (2XAARE) sequences. YB - TATA ), 2 (2xAARE TK ), 3 (miniCHACl YB - TATA ), 4 (ULBP1), 6 (HSPA1B), and 7 (CHAC1). In particular, the invention relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), said inducible promoter being selected from the sequences SEQ ID NO: 1 (2XAARE YB - TATA ), 2 (2XAARE TK ), 3 (miniCHACl YB - TATA), 4 (ULBP1), 6 (HSPA1B), and 7 (CHAC1). In particular, the invention also relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), said inducible promoter being selected from the sequences SEQ ID NO: 1 (2XAARE YB TATA ), 2 (2XAARE TK ) and 3 (miniCHACl YB - TATA Advantageously, the invention also relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), said inducible promoter being the sequence SEQ ID NO: 1 (2xAARE YB TATA ), 4 (ULBP1), 6 (HSPA1B) and 7 (CHAC1).

[0062] According to another embodiment, the invention relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), in which said at least one first therapeutic transgene is a chimeric antigen receptor (CAR),

[0063] in particular a CAR chosen from: an anti-A-folate receptor CAR, an anti-Carbonic anhydrase IX CAR, an anti-CD171 CAR, an anti-CD 19 CAR, an anti-CD20 CAR, an anti-CD276 CAR, an anti-CD319 CAR, an anti-CEA CAR, an anti-cMet CAR, an anti-EGFR CAR, an anti-EGFRIII CAR, an anti-EGFRn CAR, an anti-FAP CAR, an anti-GD2 CAR, an antigpl 00 CAR, an anti-GPC3 CAR, an anti-HER2 CAR, an anti-IL13Ra2 CAR, an anti-MAGE CAR, an anti-MART-1 CAR, an anti-MSLN CAR, an anti-Mucl CAR, an anti-Muclô CAR, an anti-NKG2D CAR, an anti-PSMA CAR, an anti-TRP-1 CAR, an anti-TRP2 CAR, an anti-VEGFR CAR, an anti-HLA-G CAR, an anti-CLDN18.2, an anti-EPC AM CAR, an anti-FAP CAR, an anti-ROR1 CAR, an anti-ROR2 CAR, an anti-PDI CAR and an anti-PSCA CAR.According to another embodiment, the invention relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), wherein said at least one first therapeutic transgene is a chimeric antigen receptor (CAR) selected from: an anti-A-folate receptor CAR, an anti-Carbonic anhydrase IX CAR, an anti-CD171 CAR, an anti-CD19 CAR, an anti-CD20 CAR, an anti-CD276 CAR, an anti-CD319 CAR, an anti-CEA CAR, an anti-cMet CAR, an anti-EGFR CAR, an anti-EGFRIII CAR, an anti-EGFRn CAR, an anti-FAP CAR, an anti-GD2 CAR, an anti-gpl O0 CAR, an anti-GPC3 CAR, an anti-HER2 CAR, an anti-IL13Ra2 CAR, an anti-MAGE CAR, an anti-MART-1 CAR, an anti-MSLN CAR, an anti-Mucl CAR, an anti-Muclô CAR, an anti-NKG2D CAR, an anti-PSMA CAR, an anti-TRP-1 CAR, an anti-TRP2 CAR, an anti-VEGFR CAR, an anti-HLA-G CAR, an anti-CLDN18 CAR.2, an anti-EPCAM CAR, an anti-FAP CAR, an anti-RORl CAR, an anti-ROR2 CAR, an anti-PDI CAR and an anti-PSCA CAR.

[0064] According to another embodiment, the invention relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), in which said expression cassette further comprises a second transgene downstream of said first transgene, said second transgene being in particular:

[0065] ■ a transcription factor, in particular a transcription factor chosen from:

[0066] c-JUN, FOXO1, B ATF, TBET and NRF2;

[0067] ■ a cytokine, in particular a cytokine chosen from: IL2, IFNγ, TNFα, IL18, and IL12; or

[0068] ■ a carrier, in particular the GLUT1 carrier.

[0069] According to another embodiment, the invention therefore relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), said second transgene being:

[0070] ■ a transcription factor, in particular a transcription factor chosen from:

[0071] c-JUN, FOXO1, B ATF, TBET and NRF2;

[0072] ■ a cytokine, in particular a cytokine chosen from: IL2, IFNγ, TNFα, IL18, and IL12; or

[0073] ■ a carrier, in particular the GLUT1 carrier.

[0074] According to another embodiment, the invention relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), said second transgene being a transcription factor, in particular a transcription factor selected from: c-JUN, FOXO1, BATF, TBET and NRF2. In particular, the invention relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), said second transgene being a transcription factor, selected from: c-JUN, FOXO1, BATF, TBET and NRF2.Advantageously, the invention relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), in which said second transgene is c-JUN, the nucleic acid of which has a sequence having at least 90% identity with the nucleic acid of sequence SEQ ID NO: 9 or the nucleic acid of which codes a sequence of amino acids having at least 90% identity with the sequence SEQ ID NO: 10.

[0075] According to another embodiment, the invention therefore relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), said second transgene being a cytokine, in particular a cytokine selected from: IL2, IFNγ, TNFα, IL18, and IL12. In particular, the invention therefore relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), said second transgene being a cytokine selected from: IL2, IFNγ, TNFα, IL18, and IL12.

[0076] According to another embodiment, the invention relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), said second transgene being a transporter, in particular the GLUT1 transporter. In particular, the invention relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), said second transgene being the GLUT1 transporter.

[0077] According to another embodiment, the invention relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), wherein said expression cassette comprises a nucleic acid sequence encoding the p2A peptide, which is located downstream of said first therapeutic transgene and upstream of said second transgene (i.e., between said first therapeutic transgene and upstream of said second transgene). Advantageously, the nucleic acid encoding the p2A peptide corresponds to the sequence SEQ ID NO: 11.

[0078] According to another embodiment, the invention relates to the animal eukaryotic cell as described above (or the pharmaceutical composition as described above), wherein said expression cassette comprises at its 3' end (downstream of said first therapeutic transgene or downstream of said second transgene when present) a nucleic acid sequence allowing the transcription of a polyA tail. Advantageously, the nucleic acid allowing the transcription of a polyA tail corresponds to the sequence SEQ ID NO: 12.

[0079] According to another aspect, the invention relates to the use of a lentiviral vector to transduce an animal eukaryotic cell to obtain (manufacture) the animal eukaryotic cell of the invention.

[0080] "Lentiviral vector" means a laboratory-produced recombinant lentiviral particle comprising an encapsulated and / or enveloped modified lentiviral genome, said genome including an expression cassette according to the invention. This lentiviral vector, which is obtained using conventional laboratory techniques, is efficient at entering the animal eukaryotic cell, is non-replicative, and leads either to the integration (targeted or untargeted) of said genome and therefore of said expression cassette according to the invention into the genome of an infected (transduced) animal eukaryotic cell, or to the presence of a non-integrated episome in the cellular genome.Classically, the structure of this lentiviral genome after reverse transcription comprises: the cis sequences of the HIV lentiviral genome, namely two Long Terminal Repeats (LTRs) flanking the ends of the vector genome with the U3, R, and U5 regions, but where the U3 region is mutant (AU3), lacking its enhancer sequence and therefore without promoter activity. Lentiviral vectors with AU3 in the LTRs are called "self-inactivating" since they cannot be transcribed by wild-type HIV. The 5' LTR of the genome is followed by a "psi" sequence for the encapsidation of the vector's RNA genome, a Rev Responsive Element (RRE) sequence for the export of the vector's RNA genome, the Central Polypurine Tract "cppt" and Central Termination Sequence "cts" sequences for the formation of the central DNA triplex during reverse transcription, and a 3' LTR. In addition to the cis sequences of the virus, these vectors contain said expression cassette according to the invention, placed between the two LTRs.By "expression cassette according to the invention", which is also an object of the invention, is meant, as mentioned above, a nucleic acid comprising from the 5' end to the 3' end:.

[0081] ■ an inducible promoter whose sequence has at least 90%, in particular 95%, identity with the SEQ ID NO: 1 (2XAARE) sequence YB - TATA ), 2 (2xAARE TK ), 3 (miniCHACl YB TATA ), 4(ULBP1), 6 (HSPA1B) or 7 (CHAC1); and ■ at least one first therapeutic transgene, said at least one first therapeutic transgene being a chimeric antigen receptor (CAR), said expression cassette being in particular devoid of a coding or non-coding sequence having post-transcriptional regulatory properties. It should be noted that this expression cassette according to the invention may further comprise a second transgene downstream of said first transgene, said second transgene being in particular:

[0082] ■ a transcription factor, in particular a transcription factor chosen from:

[0083] c-JUN, FOXO1, B ATF, TBET and NRF2;

[0084] ■ a cytokine, in particular a cytokine chosen from: IL2, IFNγ, TNFα, IL18, and IL12; or

[0085] ■ a carrier, in particular the GLUT1 carrier.

[0086] In other words, the invention relates to the use of a lentiviral vector as described above, said lentiviral vector comprising a lentiviral genome comprising an expression cassette comprising from the 5' end to the 3' end:

[0087] ■ an inducible promoter whose sequence has at least 90%, in particular 95%, identity with the SEQ ID NO: 1 (2XAARE) sequence YB - TATA ), 2 (2xAARE TK ), 3 (miniCHACl YB TATA), 4(ULBP1), 6 (HSPA1B) and 7 (CHAC1); and ■ at least one first therapeutic transgene, said at least one first therapeutic transgene being a chimeric antigen receptor (CAR), said expression cassette being in particular devoid of a coding or non-coding sequence having post-transcriptional regulatory properties and possibly further comprising a second transgene downstream of said first transgene, said second transgene being in particular:

[0088] ■ a transcription factor, in particular a transcription factor chosen from:

[0089] c-JUN, FOXO1, B ATF, TBET and NRF2; ■ a cytokine, in particular a cytokine selected from: IL2, IFNγ, TNFα, IL18, and IL12; or

[0090] ■ a carrier, in particular the GLUT1 carrier.

[0091] In connection with this use, it is understood that another aspect of the invention relates to a method of transducing an animal eukaryotic cell using the lentiviral vector as described above to obtain (manufacture) an animal eukaryotic cell comprising an expression cassette including from the 5' end to the 3' end:

[0092] ■ an inducible promoter whose sequence has at least 90%, in particular 95%, identity with the SEQ ID NO: 1 (2XAARE) sequence YB - TATA ), 2 (2xAARE TK ), 3 (miniCHACl YB TATA), 4(ULBP1), 6 (HSPA1B) and 7 (CHAC1); and ■ at least one first therapeutic transgene, said at least one first therapeutic transgene being a chimeric antigen receptor (CAR), said expression cassette being in particular devoid of a coding or non-coding sequence having post-transcriptional regulatory properties and said animal eukaryotic cell being selected from: the T lymphocyte and the NK cell.

[0093] "Method for transducing an animal eukaryotic cell" refers to a conventional and standard laboratory method for transducing an animal eukaryotic cell using a lentiviral vector to modify its genome. Specifically, it involves introducing the expression cassette according to the invention into an animal eukaryotic cell (T lymphocyte or NK cell) in order to produce a powerful and safe therapeutic tool, which is then made available to the medical community to treat a solid tumor in a patient who needs it. For example, and to achieve this, the method of the invention comprises at least:

[0094] 1. A step of contacting (infecting) an animal eukaryotic cell with a lentiviral vector at an infection multiplicity (IMO) of 10 for 24 hours to obtain a transduced animal eukaryotic cell; and

[0095] 2. a washing step of said transduced animal eukaryotic cell to obtain the animal eukaryotic cell according to the invention.

[0096] In all respects, it should be noted that the various aspects of the invention, as well as its various embodiments, are interdependent. They can therefore be combined as many times as necessary to obtain preferred aspects and / or embodiments of the invention not explicitly described. This also applies to all the definitions provided in this description, which apply to all aspects of the invention and its embodiments.

[0097] Furthermore, the present invention is illustrated, but not limited to, by the following figures and examples. LIST OF FIGURES

[0098] Figure 1. Diagram illustrating the structure of the expression cassettes used.

[0099] 1) EFla (SEQ ID NO: 13), promoter of the EEF1A1 gene controlling the constitutive expression of a chimeric antigen receptor (CAR) anti-CD19 (SEQ ID NO: 14). 2) 2XAARE YB TATA (SEQ ID NO: 1), Link sequences to ATF4 and TATA YB box controlling the regulated expression of CAR anti-CD 19 (SEQ ID NO: 14) or GFP (SEQ ID NO: 15). 3) 2XAARE YB TATA (SEQ ID NO: 1) controlling the regulated expression of the anti-CD19 CAR (SEQ ID NO: 14) and the JUN gene (SEQ ID NO: 9). These expression cassettes were cloned into a lentiviral vector to transduce T lymphocytes or NK-92 cells.

[0100] Figure 2. Regulated CAR expression by amino acid deficiency, in CD3+ lymphocytes transduced with a 2XAARE lentiviral vector YB - TATA -CARCD19.

[0101] (A) Bar plot illustrating CAR expression in CD3 lymphocytes +after 48 hours of culture in complete (Ctl), arginine-deficient (Arg), leucine-deficient (Leu), lysine-deficient (Lys), glutamine-deficient (Glt), methionine-deficient (Met), or tryptophan-deficient (Trp) medium. (B) Graph illustrating the expression of anti-CD19 CAR in CD3 lymphocytes + During induction at T0 with a medium deficient in Arg (•), Glt (A), or Trp (■), CAR expression was performed by adding the deficient medium, removing it after 48 hours for 48 hours by culture in complete medium, and then re-inducing it for 48 hours in the initial induction medium. CAR expression was assessed by CAR immunostaining and flow cytometry every 24 hours until T0+144h. Maximum CAR expression was observed after 48 hours of culture in a highly amino acid-deficient medium, which was normalized to 100% for each donor (n=5).

[0102] Figure 3. Regulated CAR expression by glucose deprivation in NK-92 cells transduced with a 2XAARE lentiviral vector YB - TATA -CARCD19.

[0103] (A) Bar plot illustrating CAR expression in NK-92 cells after 48 h of culture in complete or glucose-poor medium. NK-92 cells (n=3 per experiment) were transduced with the lentiviral vector 2XAARE YB TATA -CARCD19. (B) Graph illustrating CAR expression in NK-92 cells during induction at T0 with low-glucose medium. Induction was performed at T0, reversed at 48 h, cells were rested for 48 h in complete medium, and then re-induced for 48 h in the same initial low-glucose induction medium. CAR expression was assessed by flow cytometry every 24 h until T0+144 h. Maximum CAR expression was observed after 48 h of culture in highly glucose-restricted medium, which was normalized to 100% for each donor.

[0104] Figure 4. Spontaneous induction of GFP in lymphocytes or genetically modified NK-92 cells in a mass of cultured tumor cells.

[0105] Bar plot illustrating GFP expression in T lymphocytes (A) and NK-92 cells (B) infiltrating a melanoma spheroid (EST-109) or breast cancer (MDA-MB 231) after 72 h of co-culture, or alone in a control medium without a spheroid. T lymphocytes (n=5 donors) and NK-92 cells (n=3 experiments) were transduced with a 2XAARE lentiviral vector YB TATA -GFP. Figure 5. Regulated expression of JUN by an amino acid deficiency, in genetically modified CD3+ lymphocytes.

[0106] (A) Comparison of JUN transgene expression in lymphocytes (n=4 donors) transduced with a 2xAARE lentiviral vector YB TATA -CARCD19 (abbreviated 2xAARE-CAR) or 2xAARE YB TATA-CARCD19_p2A_c-Jun (abbreviated 2xAARE-CAR Jun) graph illustrating the expression of the JUN transgene in genetically modified T lymphocytes after 48 hours of culture in an environment low in arginine (Arg), glutamine (Glt), or tryptophan (Trp). (B) Graph illustrating the expression of the JUN transgene in CD3 lymphocytes + During induction at T0 with a medium low in arginine or tryptophan, the cells were induced. Induction was performed at T0, withdrawn at 48 hours, and then re-induced for 48 hours in the initial induction medium. JUN expression was assessed every 24 hours until T0+144h. CD3 lymphocytes + (n=5 donors) were transduced with 2xAARE lentiviral vectors YB TATA -CARCD19_p2A_c-Jun (abbreviated 2xAARE-CAR Jun). JUN expression was represented by fluorescence intensity.

[0107] Figure 6. Effect of the promoter controlling CAR expression on the phenotype of genetically modified lymphocytes.

[0108] Graphs illustrating (A) the proportion of terminal effector memory (TEM), central stem memory (SCM), central memory (CM), and effector memory (EM) cells, and (B) the expression of markers associated with lymphocyte depletion, in T lymphocytes after 10 days of culture in expansion medium with CD3 and CD28 receptor activation. T lymphocytes (n=5 donors) were transduced with the lentiviral vector 2XAARE YB - TATA -CARCD19 (abbreviated 2xAARE-CAR) or EFla-CARCD19 (abbreviated EFla-CAR).

[0109] Figure 7. Measurement of the cytotoxic activity of CAR-T lymphocytes under amino acid deficiency conditions.

[0110] (A) Graph illustrating the cytotoxicity of anti-CD19 CAR-T lymphocytes on CD19 melanoma cells +(EST-109) after 3 ohms of co-culture in complete (ctl), arginine-low (Arg), glutamine-low (Glt), or tryptophan-low (Trp) medium. CD19+ T cells and EST-109 cells were co-cultured with an effector-to-target (E:T) ratio of 2:1. Anti-CD19 CAR-T cells (n=5 donors) were transduced with the 2xAARE lentiviral vectors YB TATA -CARCD19 (abbreviated 2xAARE-CAR) or 2xAARE YB - TATA -CARCD19_p2A_c-Jun (abbreviated 2xAARE-CAR Jun). (B) Graph illustrating the cytotoxicity of modified T lymphocytes on CD19 melanoma spheroids + (EST-109), and (C) breast cancer CD19 + (MDA-MB 231) after 72h or 80h of co-culture respectively in complete medium (ctl), deficient in arginine, glutamine, or tryptophan. T lymphocytes (n=5 donors) were either untransduced or transduced with VSVg 2XAARE vectors YB - TATA -CARCD19, VSVg 2xAARE YB TATA-CARCD19_p2A_c-Jun or VSVg lentivirals EFla-CARCD19. (B and C) CAR cell cytotoxicity is proportional to the reduction in spheroid size (•, control cells; ■, 2xAAREC-CARCD19; A, 2xAARE) YB - TATA -CARCD19_p2A_c-Jun ; ♦, EFla-CARCD19). Figure 8. Measurement of the cytotoxic activity of NK92-CAR-T under glucose deficiency conditions.

[0111] (A) Graph illustrating the cytotoxicity of NK-92-2xAARE cells YB TATA -CARCD19 modified on CD19 melanoma cells + (EST-109) after 48 h of co-culture in complete (ctl) or glucose-deficient medium. Cells were co-cultured with an effector-to-target (E:T) ratio of 2:1. NK-92 cells (n=3 experiments) were transduced with VSVg 2xAARE lentiviral vectors YB TATA -CARCD19. (B) Graph illustrating the cytotoxicity of modified NK-92 cells on CD19 melanoma spheroids + (EST-109), and (C) breast cancer CD19 +(MDA-MB 231) after 74 h of co-culture in complete or glucose-deficient medium. NK-92 cells (n=3 experiments) were either untransduced (•) or transduced with the 2XAARE lentiviral vectors YB - TATA -CARCD19 (A), or EFla-CARCD19 (■).

[0112] Figure 9. CAR expression in different tissues after CAR-T lymphocyte transplantation in mice.

[0113] Graph illustrating CAR expression by genetically modified T lymphocytes in the blood and different tissues of NXG mice bearing MDA-MB 231 CD19 subcutaneous tumors + (n=6 mice). T lymphocytes transduced with the 2XAARE lentiviral vector YB TATA -CARCD19 were injected intravenously into mice 96h before euthanasia.

[0114] Figure 10. Tumor growth after CAR-T lymphocyte transplantation in mice.

[0115] Graph illustrating the growth curves of EST-109 CD19+ tumors, with an arrow marking the injection point of non-transduced T lymphocytes (•), 2xAARE YB TATA -CAR-Jun (abbreviated 2xAARE-YB-CAR Juri) (A) or EFla-CAR (abbreviated EFla-CAR) (■).

[0116] Figure 11. Spontaneous induction of GFP in genetically modified lymphocytes in a mass of cultured tumor cells.

[0117] Graph illustrating GFP expression in T lymphocytes infiltrating a melanoma spheroid (EST-109) between 0 and 96 h of co-culture, or alone in a control medium without spheroid. T lymphocytes were transduced with a 2XAARE lentiviral vector YB - TATA-GFP or a ULBP1-GFP, HSPAlb-GFP, or CHAC1-GFP vector. The graph represents the increase in GFP fluorescence intensity measured in lymphocytes co-cultured with a spheroid compared to the GFP fluorescence intensity measured in the same lymphocytes cultured alone. Imaging and measurements were performed on Incucyte. EXAMPLES

[0118] Regulated expression of the CAR (chimeric antigen receptor) in the microenvironment of solid tumors: materials and methods

[0119] Human primary T lymphocytes

[0120] Peripheral blood mononuclear cells (PBMCs) were isolated from donor whole blood using the Ficoll-Hypaque method. Primary human CD3+ T cells were then isolated from PBMCs by negative selection using the Pan T cell Isolation Kit, human (Miltenyi Biotec). After isolation from PBMCs, the CD3+ T cells were recultured at a concentration of 500,000 T cells per ml in medium (CTStm OpTmizer (Thermo Fisher Scientific), supplemented with IX GlutaMAX (Gibco), IX penicillin-streptomycin (Gibco), and 100 U / ml of recombinant human IL-2 (Miltenyi Biotec)). Human CD3 / CD28 T-cell activator (Thermo Fisher Scientific; 2:1 bead / cell ratio) or ImmunoCult CD3 / CD28 / CD2 T-cell activator (STEMCELL technologies; 12.5 µl / ml) were added to the culture medium. After 48 hours, the T lymphocytes were transduced (see "lentiviral transduction").Next, 72h after activation, the beads were removed by magnetic separation (if the T cells were activated with the human CD3 / CD28 T activator). The modified T cells were cultured with fresh T cell expansion medium (CTStm OpTmizer supplemented with 2.5% human serum (Gibco), IX GlutaMAX (Gibco), IX penicillin-streptomycin (Gibco) and 100U / ml of recombinant human IL-2 (Miltenyi Biotec)) and were maintained at a density of 500,000 cells per ml.

[0121] Cell lines

[0122] MDA-MB 231 (HTB-26) cells were purchased from the ATCC, and ESTDAB-109 or URKV-Mel-2 (EST-109) melanoma cells were obtained from Dr. Federico Garrido Torres-Puchol. Cell lines were maintained in culture in Roswell Park Memorial Institute (RPMI, Gibco) medium supplemented with 10% fetal bovine serum (FBS) (Gibco), GlutaMAX IX (Gibco), and penicillin-streptomycin IX (Gibco). Cell lines were routinely tested for mycoplasma using the MycoBlue Mycoplasma Detector (Vazyme). To obtain EST-109 and HTB-26 cells expressing the CD 19 antigen, these cells were transduced with a lentiviral vector "TRAP-CD19" (SEQ ID NO: 5) carrying the fusion gene containing the sequences, splice acceptor site -T2A (autocatalytic peptide) - CD 19 (cDNA of the CD 19 gene) - P2A (autocatalytic peptide) -bsd (Blasticidine-S deaminase).This vector allows the selection of blasticidin-resistant cells expressing the CD19 antigen. Following transduction with this vector and blasticidin selection, the resistant cells were labeled with an anti-CD19 antibody, and the cells expressing this antigen most strongly were sorted and amplified. The stability of CD19 expression by these cells was regularly monitored. EST-109 and HTB-26 cells expressing the CD19 antigen were designated EST-109, respectively. CD19+ and MDA CD19+ Lentiviral transduction

[0123] CD3+ T cells were transduced with lentiviral vectors 48 hours after activation. Briefly, lentivirus concentrates were added to the medium with a multiplicity of infection (MOI) of 10. After 24 hours, the cells were washed with fresh and expanded T cell expansion medium (see "Activation and Culture of Human T Cells").

[0124] Vector construction and lentiviral production

[0125] For these experiments, self-inactivating lentiviral vectors were used, lacking a promoter region in the U3 region of the promoter and featuring a central flap. The cDNA sequences of the CAR anti-CD19 transgene (SEQ ID NO: 14) and c-Jun (NM_002228.4; SEQ ID NO: 9) were cloned under the control of a promoter of the EF la gene (SEQ ID NO: 13) or 2XAARE. YB TATA(SEQ ID NO: 1). Recombinant particles were obtained by the transcomplementary plasmid transfection method in HEK-293T cells. Briefly, HIV-1 SIN-derived lentiviral vector particles were produced by transient co-transfection using the calcium chloride method. Human embryonic kidney 293T cells (ATCC-CRL-11268) were transfected with a mixture of DNA constructs comprising i) the plasmid encoding vesicular stomatitis virus G glycoprotein (pVSV; SEQ ID NO: 16), ii) the plasmid expressing HIV structural proteins and enzymes (p8.9; SEQ ID NO: 17), and the plasmid encoding the vector genome (pLV; SEQ ID NO: 18). Vector particles were collected 48 hours after transfection, filtered, and concentrated by ultracentrifugation at 19,000 rpm for 90 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended in PBS-BSA and then frozen at -80°C until use.The recombinant particles were titrated by measuring the viral protein p24 (HIV-1 p24 Antigen ELISA 2.0 kit (ref. 0801008, Zeptometrix); Subsequently, Ipg of p24 was considered to be equivalent to 10. 2 transduction units.

[0126] The expression cassettes produced were:

[0127] - EF 1 a-CARCD 19 (SEQ ID NO: 19);

[0128] - 2XAARE YB TATA -CARCD 19 (SEQ ID NO: 20);

[0129] - 2XAARE YB TATA -GFP (SEQ ID NO: 21); and

[0130] - 2XAARE YB TATA -CARCD19_p2A_c-Jun (SEQ ID NO: 22);

[0131] - ULBP 1 -GFP (SEQ ID NO: 8);

[0132] - HSPAlb-GFP (SEQ ID NO: 27);

[0133] - CHAC1-GFP (SEQ ID NO: 28).

[0134] and the lentiviral vectors produced were:

[0135] - pLV-EF 1 a-CARCD 19 (SEQ ID NO: 23);

[0136] - pLV-2xAARE YB TATA -CARCD19 (SEQ ID NO: 24);

[0137] - pLV-2xAARE YB TATA -GFP (SEQ ID NO : 25) ; et

[0138] - pLV-2xAARE YB TATA -CARCD 19_p2 A c- Jun (SEQ ID NO : 26),

[0139] - pLV-ULBP 1 -GFP (SEQ ID NO : 29);

[0140] - pLV-HSPAlb-GFP (SEQ ID NO : 30);

[0141] - pLV-CHAC 1 -GFP (SEQ ID NO : 31 ). Cytométrie en flux

[0142] T lymphocytes were washed in FACS buffer (PBS + 0.5% FBS) and stained with fluorophore-conjugated cell surface antibodies for 15 minutes at room temperature. The cells were then washed twice with FACS buffer before flow cytometry analysis. CAR-expressing cells were incubated with CD19 CAR Detection Reagent (Miltenyi Biotec) at a 1 / 500 dilution for 15 minutes at room temperature and then washed twice with FACS buffer before the previously described cell surface staining, supplemented with the anti-biotin antibody REAfinity (clone REA746, Miltenyi Biotec), to detect CAR expression. Cell surface antibodies were used at a 1 / 100 dilution, except for the anti-biotin antibody, which was used at a 1 / 200 dilution.Intracellular staining was performed using the same cell surface staining protocol, after which the cells were fixed, permeabilized, and stained using the BD Cytofix / Cytoperm Fixation / Permeabilization Kit (BD Biosciences) according to the manufacturer's instructions. Intracellular antibodies were used at a 1:50 dilution, and the LIVE / DEAD solution was used at a 1:500 dilution. For experiments involving CellTrace CFSE (Thermofisher Scientific), T cells were stained with 2.5 pM dye in RPMI supplemented with 10% S VF for 20 minutes at 37°C. After staining, the cells were analyzed using a BD LSR Fortessa flow cytometer and then the FACS Diva software. Downstream analysis was then performed using FlowJo v.10.9 software.

[0143] In vitro studies under tumor microenvironment conditions

[0144] After activation of CD3+ T lymphocytes, the cells were washed twice in PBS and recultured in amino acid- or glucose-restricted media (RPMI depleted in arginine or leucine or lysine or methionine or glutamine or glucose, supplemented with 5% FBS (Gibco), IX L-Glutamine (Gibco), IX penicillin-streptomycin (Gibco), 100 U / ml recombinant human amino acid, 100 U / ml recombinant amino acid (Gibco); except in cases of glutamine restriction), IX penicillin-streptomycin (Gibco), 100 U / ml recombinant human IL-2 (Miltenyi Biotec) and an RPMI concentration of 1 / 100 of the amino acid / glucose depleted medium, or a control medium (RPMI supplemented with 10% S VF (Gibco), IX L-glutamine (Gibco), IX penicillin-streptomycin (Gibco) and 100 U / ml of recombinant human IL-2 (Miltenyi Biotec) at a concentration of 5x10 5cells per ml for 24 or 48 hours. In all experimental conditions, the cells were seeded at a concentration of 5 x 10 5 cells per ml and incubated for 24 or 48h. The cells were then harvested for other experiments such as flow cytometry (see "flow cytometry") and cytokine quantification (see "cytokine production").

[0145] Cytokine production

[0146] Cytokine secretion by CAR-T cells was assessed using the MACSPlex Mix Cytotoxic base kit (Miltenyi Biotec) according to the manufacturer's instructions. Briefly, supernatants were collected after 24 hours of co-culture of CAR-T cells and EST-109 or EST-109 melanoma cells. CD19+ or MD A, or MDA CD19+Supernatants were supersized under various tumor microenvironment conditions in 96-well flat-bottom plates with a 1:1 effector-to-target ratio. They were stored at -20°C until analysis. Fifty µL of undiluted supernatants were used per analysis. Samples were acquired using a BD Canto II (BD Biosciences) equipped with FACS Diva software and analyzed using FlowJo (10.8.1). Downstream analysis was then performed using FlowJo v.10.9.

[0147] 2D cytotoxicity assay

[0148] Five thousand EST 109 or EST-109 melanoma cells CD19+ or MD A or MD A cells CD19+Target cells were co-cultured with 20,000 CAR-T cells in 200 µL of control medium or various single-amino-acid restricted media in 96-well flat-bottom plates for 30 h. For some drug-induced experimental parameters, control media supplemented with artesunate (µM) or L-asparaginase (5 U / ml) were used. Target cells were then detached using 0.05% trypsin-EDTA (Gibco) before being acquired using a BD Canto II flow cytometer (BD biosciences) equipped with FACS Diva software and analyzed using FlowJo software (10.8.1). Specific lysis (%) was defined as (1 - (control ratio e / experimental ratio) x 100), with the control ratio equal to the number of target cells with untransduced (mock) T cells. In parallel, the supernatants were collected for cytokine quantification.

[0149] Spheroid formation

[0150] Five thousand EST-109 or EST-109 melanoma cells CD19+ or breast cancer MD A or MDA CD19+ , on t were seeded in 150 pl of spheroid medium (RPMI supplemented with 5% FBS (Gibco), IX GlutaMAX (Gibco), IX penicillin-streptomycin (Gibco) and 2.5% Matrigel basement membrane matrix (Corning)) in 96-well round-bottom ultra-low fixation (ULA) plates (PHCBI) and were then inoculated at 200xG for 3 minutes.

[0151] Promoter induction trial in 3D spheroid models

[0152] After the formation of spheroids of melanoma EST-109 or EST-109 CD19+ or breast cancer MDA or MDA CD19+The spheroids were washed once in PB S and placed in 96-well round-bottom plates containing 200 µl of RPMI (Gibco) supplemented with 5% FBS (Gibco), IX GlutaMAX (Gibco), and IX penicillin-streptomycin (Gibco). Twenty thousand T lymphocytes were either untransduced (mock) or transduced with the pLV-2XAARE reporter vector. YB TATAGFP was then added to the well with each spheroid. Fluorescence was monitored every 2 hours with an 1Ox objective using the Incucyte system (Essen Bioscience), placed in a cell culture incubator at 37°C and 5% CO2, set to image the entire well at each time point. The total integrated intensity of GFP was quantified using Incucyte software (Essen Bioscience). All data were normalized to the first time point and represented as a change in GFP fluorescence intensity over time. After 72 h, the spheroids were collected, washed, and dissociated to quantify GFP expression by infiltrating T cells compared to 2XAARE T cells YB TATA _GFP cultured alone for 72h, by flow cytometry.

[0153] Cytotoxicity assay in 3D spheroid models

[0154] In this experimental setting, the cancer cells constitutively expressed GFP. After the formation of EST-109 melanoma or MDA-MB 231 breast cancer spheroids (see "Spheroid Formation"), the spheroids were rinsed once in PBS and transferred to 96-well round-bottom plates containing 200 µl of control medium or various single-amino-acid restricted media (see "In Vitro Studies Under Tumor Microenvironment Conditions"). For some drug-based experimental parameters, control media supplemented with artesunate (1 µM) or L-asparaginase (5 U / ml) were used. Subsequently, 20,000 untransduced (mock), 2xAARE_CAR, 2xAARE_CAR-Jun, or EFla CAR T cells were added to each spheroid.Fluorescence was assessed every 2 hours using an 1Ox objective on the Incucyte system (Essen Bioscience), located in a cell culture incubator set at 37°C with 5% CO2, capturing an image of the entire well at each time point. The size of the largest GFP object was determined using the Incucyte software (Essen Bioscience). All data were normalized to the initial time point and represented as the fold change of the largest GFP object over time. After 96 hours, the spheroids were collected, washed, and dissociated for flow cytometry analysis of the infiltrating T cells' phenotype (see "Flow Cytometry").

[0155] In vivo experiments

[0156] NXG mice (NOD-Prkdc) scld -IL2rg Tml ) 6- to 8-week-old males weighing 20 to 22 g (January laboratories) were used. EST-109, EST-109 tumor cell lines CD19+, MDA and MDA CD19+ (2x10 6Cells in PBS were injected subcutaneously (sc) into these animals. Once the tumors were palpable, their length (L) and width (W) were measured every 3 days using digital calipers, and tumor volume was calculated using the formula: (L x W²) * 0.52. The specified dose of sham T cells or CARs was injected into 150 µl of PBS via the tail vein (iv) using a 30G needle, and into 50 µl of PBS for cells injected intratumorally (it). At the end of each experiment, the mice were sacrificed, and samples were collected for further experiments. In short, blood samples were taken from the submandibular vein and transferred into 1.5 ml tubes containing 30 ml of EDTA, and healthy tissues as well as tumors were preserved in RPMI supplemented with 10% S VF (Gibco).To evaluate the efficacy of oral anti-turn CAR-T cells, tumors were weighted. Blood samples were then incubated for 5 minutes with 1 ml of ACK lysis buffer (Gibco) to remove red blood cells. Tumors and healthy tissues were chopped using sharp scissors, and individual tissue cells were obtained by incubating the chopped tissues for 1 hour at 37°C with 250 U / ml of collagenase IV (Gibco) and 0.1 mg / ml of DNase I (Gibco). The released cells were then passed through a 70 µm cell filter before antibody labeling and flow cytometry analysis of individual cells from blood, healthy tissues, and tumors.

[0157] Statistics

[0158] Statistical analyses were performed using GraphPad® Prism 10. To assess statistical variances between two groups, an unpaired Student's t-test or a Mann-Whitney U test was used. Statistical differences between three or more groups were analyzed using analysis of variance (one-way or two-way ANOVA) with appropriate multiple comparison tests. In the graphs, significance is indicated by p-values ​​less than 0.05.

[0159] RESULTS

[0160] Induction of a CAR by the promoter 2XAARE YB - TATA due to various deficiencies

[0161] To achieve ATF4-regulated CAR expression, a second-generation, CD19-specific anti-CD19-41BBZ CAR (SEQ ID NO: 14) was placed under the control of the 2XAARE promoter. YB TATA(SEQ ID NO: 1). This mini-promoter includes six copies of the "Amino Acid Responsive Element" (AARE) sequence from the TRB3 gene cell promoter followed by a Yb TATA box, allowing greater transgene expression than with the TATA box from the Thymidine Kinase (TK) gene of the Herpes virus (Figure 1).

[0162] This 2XAARE expression cassette YB - TATA -CARCD19 (SEQ ID NO: 20) was cloned into a lentivral vector, used to transduce donor lymphocytes or NK-92 cell line.

[0163] Under these conditions, it was observed that in complete medium, genetically modified T lymphocytes did not express the CAR on their surface. Conversely, it was observed that these same T lymphocytes, cultured in conditions depleted of an amino acid, induced CAR expression at the membrane, but at varying levels depending on the missing amino acid. In particular, it was observed that methionine deficiency was less effective at inducing CAR expression than arginine, leucine, lysine, tryptophan, or glutamine deficiencies, and that the deficiencies inducing greater CAR expression were those of lysine and arginine (Figure 2A). Furthermore, the reversibility of the expression system was evaluated, and it was observed that after CAR induction in media deficient in different amino acids, T lymphocytes could lose CAR expression if cultured in complete medium.Furthermore, these genetically modified T lymphocytes could subsequently re-express CAR if they were again cultured in amino acid-deficient media (Figure 2B). This demonstrated that after being induced in an amino acid-deficient environment, a lymphocyte returning to a "normal" medium loses CAR expression, thus avoiding a "target-but-non-tumor" CAR effect and limiting the occurrence of destruction of healthy tissues expressing the tumor antigen.

[0164] In addition, a second cell model was used, Natural Killer (NK) lymphocytes and more specifically the NK-92 cell line, considered safer than T lymphocytes in an allogeneic context and approved in clinical trials for CAR-NK therapies.

[0165] NK-92 cells cultured in complete medium did not induce CAR expression on their surfaces, but unlike T lymphocytes, they also did not induce it under amino acid deficiency conditions. In contrast, glucose deficiency, present in the tumor microenvironment, allowed CAR induction in these cells (Figures 3A, 3B). Similar to T lymphocytes, CAR expression was induced by the 2XAARE promoter. YB - TATA(SEQ ID NO: 1) was reversible (Figure 3C). Here, two distinct cell models were developed, inducing the ATF4 pathway and thus transgene expression under different conditions: amino acid restriction for T lymphocytes and glucose restriction for NK-92 cells. The use of these two cell types under different tumor microenvironment conditions can therefore be implemented. Since some tumors are characterized as being more restricted in amino acids and others in glucose, the invention makes it possible to offer a form of personalized medicine tailored to the specific needs of each patient.

[0166] Induction of a CAR by the promoter 2XAARE YB - TATAIn a 3D in vitro tumor model: After validating CAR induction by tumor microenvironment conditions in our two cell types, the ability of these same cell types to induce CAR in an in vitro model closely resembling a solid tumor was investigated. Various in vitro models allow us to approximate the actual physiology of a solid tumor. Here, 3D spheroid models formed from the CD19 melanoma cell line were used. + (EAST-

[0167]

[0168] 231 CD19+ ).

[0169] It was observed that T lymphocytes infiltrating both spheroid models induced the expression of a fluorescent transgene (GFP: Green Fluorescent Protein SEQ ID NO: 15) under the control of the 2XAARE promoter YB TATA(SEQ ID NO: 1) (Figure 4A). It was also observed that NK-92 cells infiltrating spheroids also induced GFP expression, but with a lower proportion of induced cells compared to T lymphocytes (Figure 4B).

[0170] The promoter 2XAARE YB TATA (SEQ ID NO: 1) was therefore indeed induced by T lymphocytes and to a lesser extent by NK-92, in an in vitro spheroid model.

[0171] Regulated expression of a transcription factor by the 2XAARE promoter YB - TATA in T lymphocytes

[0172] Having demonstrated that it was possible to induce the expression of a GFP or CAR transgene under the control of our 2XAARE promoter YB TATA (SEQ ID NO: 1) Under tumor microenvironment conditions, the induction of the c-Jun transcription factor by the 2XAARE promoter was tested. YB TATA (SEQ ID NO: 1) in T lymphocytes.

[0173] Expression of c-Jun in lymphocytes genetically modified by pLV-2XAARE vectors YB TATA -CARCD19 (SEQ ID NO: 24) or 2xAARE YB TATA -CARCD19_p2A_c-Jun (SEQ ID NO: 26) was quantified after culturing them in different amino acid-deficient media. Under these conditions, overexpression of c-Jun was observed in 2xAARE cells YB - TATA -CARCD19_p2A_c-Jun but not in the 2XAARE cells YB TATA -CARCD19 (Figure 5A). Similar to CAR expression under the control of the 2XAARE promoter. YB TATA (SEQ ID NO: 1), c-Jun overexpression was reversible and reinducible (Figure 5B). The reversibility of this c-Jun overexpression thus prevents T-cell overactivation in healthy tissues. Optimization of T-cell expansion prior to in vivo injection using the 2XAARE promoter YB - TATA

[0174] In clinical practice, the lymphocyte expansion phase prior to injection into the patient is a key step in therapeutic efficacy. Indeed, the phenotype of the injected cells has a major impact on the success of CAR-T cell therapy.

[0175] Therefore, the phenotype of 2XAARE T lymphocytes was compared YB TATA -CARCD19 and EFla-CARCD19 (constitutively expressing CAR) after 10 days of expansion in culture. It was observed that, unlike EFla-CARCD19 lymphocytes, 2XAARE T lymphocytes YB TATA CARCD19 did not express CAR during the expansion protocol, thus preventing the tonic CAR signal during this procedure and cell depletion. Indeed, it was observed that with regulated CAR expression (2XAARE) YB - TATA-CARCD19), these lymphocytes contained a higher proportion of cells conducive to a more favorable treatment response, namely central stem-memory (SCM) and central memory (CM) cells, as well as a decrease in terminal effector memory cells, which are detrimental to a favorable treatment response compared to EFla-CARCD19 cells (Figure 6A). Furthermore, the expression of the cellular exhaustion markers PD-1, LAG-3, and TIM-3 was observed to be significantly reduced in 2XAARE cells. YB TATA -CARCD19 compared to EFla-CARCD19 lymphocytes (Figure 6B).

[0176] Thus, the regulated expression of CAR, using the 2XAARE promoter YB TATA (SEQ ID NO: 1) enabled the optimization of the preclinical expansion phase of CAR-T cells.

[0177] Efficacy and cytotoxicity of CAR under the control of the 2XAARE promoter YB - TATA

[0178] Having validated the capacity of the promoter 2XAARE YB TATA (SEQ ID NO: 1) To regulate CAR expression in T lymphocytes and NK-92 cells under tumor microenvironment conditions, 2D and 3D co-culture models with CD19 tumor cells were established. + of melanoma (EST109 CD19+ ) and breast cancer (MDA-MB 231 CD19+ ), in different deficient environments, to evaluate the cytotoxic response of the cells of the invention against their targets in this context.

[0179] It has been observed that the 2XAARE T lymphocytes YB TATA CARCD19 showed no cytotoxic effect on its targets in a complete culture medium, which is consistent with the absence of CAR expression under the control of the 2XAARE promoter. YB TATA (SEQ ID NO: 1) in complete medium (Figure 7A). However, under amino acid deficiency conditions, in this 2D model, 2XAARE T lymphocytes YB TATA-CARCD19 exhibited significant cytotoxic activity against their targets, which was even more pronounced with 2xAARE T lymphocytes. YB - TATA -CARCD19_p2A_c-Jun, (Figure 7A).

[0180] Similar results were observed with the 3D co-culture model, with lysis of spheroids by 2XAARE T lymphocytes YB TATA -CARCD19, which was also more important with 2xAARE T lymphocytes YB - TATA -CARCD19_p2A_c-Jun, equivalent to the efficacy of EFla-CARCD19 T lymphocytes (Figures 7B and 7C). It is important to note that in this 3D model, even with a complete medium, a reduction in the size of the spheroids was observed, which is explained by the potential presence within these spheroids of a tumor-like microenvironment as previously demonstrated (see Figure 4A).

[0181] Using the same 2D and 3D tumor cell destruction models with genetically modified NK-92 cells, but this time in a glucose-deficient medium, results similar to those obtained with the previously described T lymphocytes were observed. Indeed, with the 2D co-culture model, no cytotoxic activity was observed in complete medium, whereas with a reduction in glucose concentration, the cytotoxic activity of NK-92 2xAARE cells increased. YB TATA CARCD19 against their target was induced (Figure 8A). Similarly, the cytotoxicity of NK-92 2xAARE cells YB TATA -CARCD19 towards spheroids EST109 CD19+ and MDA-MB 231 CD19+was significant under glucose deficiency conditions and approached that of NK-92 EFla-CARCD19 cells, particularly with the melanoma cell line (Figures 8B and 8C). It is important to note that in the presence of complete medium, as with T lymphocytes, no cytotoxic activity was observed with NK-92 2XAARE cells. YB - TATA -CAR, which is consistent with the results previously observed (Figure 4B).

[0182] Induction of a CAR by the promoter 2XAARE YB - TATA in different tissues in vivo Having demonstrated the regulation of a transgene by the 2XAARE promoter YB - TATA(SEQ ID NO: 1) Under tumor microenvironment conditions, these in vitro models allowed the study of different promoter-inducing conditions depending on the cell type (T lymphocyte and NK92). These in vitro conditions also allowed for the partial study of in vivo tissue phenomena, without, however, perfectly reproducing the tumor microenvironment or the physiological conditions of healthy tissues. Therefore, the inducibility of the 2XAARE promoter was investigated. YB TATA (SEQ ID NO: 1) in T lymphocytes injected into the bloodstream of immunodeficient mice that had received a tumor cell transplant (EST109 CD19+ ) or (MDA CD19+ ), in order to analyze them in different tumor and healthy tissues.

[0183] Various tissues, as well as the tumor, were isolated from these mice that had received an injection of 2XAARE T lymphocytes. YB TATA-CAR. It was observed in all tissues that the proportion of T lymphocytes expressing CAR was low, or even non-existent, particularly in the lungs. Conversely, T lymphocytes infiltrating the tumor strongly expressed CAR on their surfaces (Figure 9).

[0184] This experiment has thus demonstrated the induction of the 2XAARE promoter YB TATA (SEQ ID NO: 1) specifically in the tumor microenvironment, with relatively low induction in the healthy tissues analyzed.

[0185] The 2xAARE system YB - TATA -CAR-Jun enhances the anti-tumor efficacy of CAR-T cells. To confirm the unprecedented potential of the 2xAARE system YB TATA -CARCD19_p2A_c-Jun (SEQ ID NO: 22) as a therapeutic strategy against solid tumors, NXG mice bearing EST-109 tumors CD19+ were injected with control T cells, 2XAARE YB - TATA-CARCD19_p2A_c-Jun or EFla-CAR. Tumor size was monitored every 3-4 days until 23 days post-injection. It was observed that, compared to control T cells, 2xAARE T cells YB TATA -CARCD19_p2A_c-Jun effectively reduced tumor growth, while EFla-CARCD19 T cells were less effective (Figure 10). The 2xAARE construct YB TATA CARCD19_p2A_c-Jun (SEQ ID NO: 22) therefore promoted the infiltration of activated CAR T cells into solid tumors and enhanced their anti-tumor activity, thus demonstrating the full efficacy of the invention. Induction of GFP expression by ULBP1, HSPAlb, and CHAC1 in a 3D in vitro tumor model

[0186] To investigate the potential of other promoters for tissue-specific expression in the tumor microenvironment, the fluorescent transgene GFP (GFP: Green Fluorescent Protein SEQ ID NO: 15) was cloned under the promoter control of the ULBP1 (SEQ ID NO: 4), HSPAlb (SEQ ID NO: 6), and CHAC1 (SEQ ID NO: 7) genes into lentiviral vectors, pLV-ULBP1-GFP (SEQ ID NO: 29), pLV-HSPAlb-GFP (SEQ ID NO: 30), and pLV-CHACl-GFP (SEQ ID NO: 31), and these were used to transduce donor CD3+ lymphocytes. These genetically modified lymphocytes were maintained in culture alone or co-cultured with an in vitro solid tumor model (EST109 melanoma cells).

[0187] It was observed that T lymphocytes infiltrating these spheroids induced GFP expression approximately 12-fold when under the control of the HSPAlb promoter (SEQ ID NO: 6), approximately 7-fold when controlled by the ULBP1 promoters (SEQ ID NO: 4), or under the control of the 2XAARE promoter. YB TATA (SEQ ID NO: 1) and 3 times with the CHAC1 promoter (SEQ ID NO: 7). In this experiment, it was also observed that the induction of transgene expression is faster with the HSPAlb (SEQ ID NO: 6) and ULBP1 (SEQ ID NO: 4) promoters than with the 2XAARE promoters. YB TATA (SEQ ID NO: 1) or CHAC1 (SEQ ID NO: 7) (Figure 11).

[0188] This experiment has therefore demonstrated that these new cellular promoters are of great interest for inducing the specific expression of a transgene in the tumor microenvironment.

Claims

DEMANDS 1. Animal eukaryotic cell for use in the treatment of a solid tumor in a patient who requires it, wherein said animal eukaryotic cell: ■ is chosen from: the T lymphocyte and the NK cell; and ■ includes an expression cassette comprising from end 5' to end 3': - an inducible promoter whose sequence has at least 90%, in particular 95%, identity with the SEQ ID NO: 1 (2XAARE) sequence YB - TATA ), 2 (2XAARE TK ), 3 (miniCHACl YB TATA ), 4 (ULBP1), 6 (HSPA1B) or 7 (CHAC1); - at least one initial therapeutic transgene, in which the expression of said at least one first therapeutic transgene is reversible and is induced by the tumor microenvironment of said solid tumor to be treated, said patient who needs it, being free from deficiency in essential or non-essential amino acids, and in which said expression cassette is in particular devoid of a coding or non-coding sequence having regulatory properties at the post-transcriptional level.

2. Animal eukaryotic cell for use according to claim 1, wherein said at least a first therapeutic transgene is a chimeric antigen receptor (CAR), in particular a CAR chosen from: an anti-A-folate receptor CAR, an anti-Carbonic anhydrase IX CAR, an anti-CD171 CAR, an anti-CD 19 CAR, an anti-CD20 CAR, an anti-CD276 CAR, an anti-319 CAR, an anti-CEA CAR, an anti-cMet CAR, an anti-EGFR CAR, an anti-EGFRIII CAR, an anti-EGFRn CAR, an anti-FAP CAR, an anti-GD2 CAR, an anti-gpl 00 CAR, an anti-GPC3 CAR, an anti-HER2 CAR, an anti-IL13Ra2 CAR, an anti-MAGE CAR, an anti-MART-1 CAR, an anti-MSLN CAR, an anti-MuclC CAR, an anti-Muclô CAR, an anti-NKG2D CAR, an anti-PSMA CAR, an anti-TRP-1 CAR, an anti-TRP2 CAR, a CAR anti-VEGFR, a CAR anti-HLA-G, an anti-CLDN18.2 CAR, an anti-EPCAM CAR, an anti-FAP CAR, an anti-RORl CAR, an anti-ROR2 CAR, an anti-PDI CAR and an anti-PSC A CAR.

3. Animal eukaryotic cell for use according to claim 1 or 2, wherein said expression cassette further comprises a second transgene downstream of said first transgene, the expression of said second transgene being reversible and being induced by the tumor microenvironment of said solid tumor to be treated, said second transgene being in particular: ■ a transcription factor, in particular a transcription factor chosen from: c-JUN, F0X01, B ATF, TBET and NRF2; ■ a cytokine, in particular a cytokine chosen from: IL2, IFNγ, TNFα, IL18, and IL12; or ■ a carrier, in particular the GLUT1 carrier.

4. Animal eukaryotic cell for use according to claim 3, wherein said second transgene is c-JUN, the nucleic acid of which has a sequence having at least 90% identity with the nucleic acid of sequence SEQ ID NO: 9 or the nucleic acid of which codes an amino acid sequence having at least 90% identity with the sequence SEQ ID NO:

10.

5. Animal eukaryotic cell comprising an expression cassette including from the 5' end to the 3' end: ■ an inducible promoter whose sequence has at least 90%, in particular 95%, identity with the SEQ ID NO: 1 (2XAARE) sequence YB - TATA ), 2 (2xAARE TK ), 3 (miniCHACl YB TATA ), 4(ULBP1), 6 (HSPA1B) or 7 (CHAC1); and ■ at least one first therapeutic transgene, said at least one first therapeutic transgene being a chimeric antigen receptor (CAR), said expression cassette being in particular devoid of a coding or non-coding sequence having post-transcriptional regulatory properties and said animal eukaryotic cell being selected from: the T lymphocyte and the NK cell.

6. Animal eukaryotic cell according to claim 5, wherein said at least a first therapeutic transgene is a chimeric antigen receptor (CAR), in particular a CAR selected from: an anti-A-folate receptor CAR, an anti-Carbonic anhydrase IX CAR, an anti-CD171 CAR, an anti-CD19 CAR, an anti-CD20 CAR, an anti-CD276 CAR, an anti-319 CAR, an anti-CEA CAR, an anti-cMet CAR, an anti-EGFR CAR, an anti-EGFRIII CAR, an anti-EGFRn CAR, an anti-FAP CAR, an anti-GD2 CAR, an anti-gpl O0 CAR, an anti-GPC3 CAR, an anti-HER2 CAR, an anti-IL13Ra2 CAR, an anti-MAGE CAR, an anti-MART-1 CAR, an anti-MSLN CAR, an anti-Mucl CAR, an anti-Muclô CAR, an anti-NKG2D CAR, an anti-PSMA CAR, an anti-TRP-1 CAR, an anti-TRP2 CAR, an anti-VEGFR CAR, an anti-HLA-G CAR, an anti-CLDN18.2 CAR, an anti-EPCAM CAR, an anti-FAP CAR, an anti-RORl CAR, an anti-ROR2 CAR, an anti-PDI CAR and an anti-PSC A CAR.

7. An animal eukaryotic cell according to claim 5 or 6, wherein said expression cassette further comprises a second transgene downstream of said first transgene, said second transgene being in particular: ■ a transcription factor, in particular a transcription factor chosen from: c- JUN, FOXO 1, B ATF, TBET and NRF2; ■ a cytokine, in particular a cytokine chosen from: IL2, IFNγ, TNFα, IL18, and IL12; or ■ a carrier, in particular the GLUT1 carrier.

8. Animal eukaryotic cell according to claim 7, wherein said second transgene is c-JUN, the nucleic acid of which has a sequence having at least 90% identity with the nucleic acid of sequence SEQ ID NO: 9 or the nucleic acid of which codes an amino acid sequence having at least 90% identity with the sequence SEQ ID NO: 10.

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