Controlled expression of transgenes in human T or NK cells for cellular immunotherapy

Abstract

The present invention relates to a human T cell or NK cell comprising a nucleic acid construct comprising a transgene under control of a regulatory polynucleotide inducible by at least one essential amino acid deficiency; and a cellular immunotherapy using the human T cells or NK cells.

Description

[0001] The present invention relates to a human T cell or NK cell comprising a nucleic acid construct comprising a transgene placed under the control of a regulatory polynucleotide inducible by at least one essential amino acid deficiency; and a cellular immunotherapy using the human T cell or NK cell.

[0002] Cellular immunotherapy is currently being continuously expanded. In oncology, for example, this therapeutic approach uses immune cells to rebalance impaired antitumor immune surveillance. This principle is widely used in the treatment of hematological malignancies with older cell therapies such as allogeneic hematopoietic stem cell transplantation (allo-HSCT). Allo-HSCT fundamentally involves replacing the patient's diseased bone marrow with healthy bone marrow harvested from an HLA-matched donor. It represents a powerful cell therapy strategy that not only reconstitutes the bone marrow and immune system but also exerts the antitumor effects of the donor's lymphocytes (graft-versus-leukemia or GvL effect). Unfortunately, the procedure also exhibits high toxicity, such as graft-versus-host disease (GvHD) and infections, which limits the indications for the therapeutic strategy. These complications represent a major cause of morbidity and mortality, poor quality of life, and additional economic costs, especially due to the cost of the therapies to treat them and the increased length of hospital stay for the patients.

[0003] Recently, CAR-T (chimeric antigen receptor-T) cell therapy represents a new approach to cancer treatment. CAR-T cells are autologous T cells collected from patients and engineered ex vivo to express a chimeric antigen receptor (CAR) specific for a target tumor antigen. CARs consist of (i) an antigen-binding domain (single-chain variable fragment scFv derived from an antibody), (ii) a signaling domain of the T-cell receptor (TCR), and (iii) an additional co-stimulatory domain. These lymphocytes are then reinjected into the patient and, once they recognize their target antigen, they are able to self-activate and directly kill tumor cells bearing this antigen. For example, anti-CD19 CAR-T cells have been licensed for many years for the treatment of certain hematological malignancies that have relapsed or are refractory to conventional chemotherapy, and the development of CAR-T cells is booming worldwide, including for the treatment of solid tumors. However, this innovative treatment strategy still needs to be improved, especially in terms of efficacy, as a significant proportion of patients will find that the capacity of their CAR-T cells decreases over time, such as the phenomenon of CAR exhaustion. Furthermore, when CAR-T cell toxicity occurs (e.g., with the onset of symptoms of cytokine release syndrome or central nervous system toxicity), it may be desirable to inhibit CAR-T cell activity in a reversible manner, such as by “exhausting” the CAR-T cells so that they are reversibly immobilized rather than irreversibly destroyed.

[0004] Another major recent cell therapy strategy is TCR transgenic T cells. Similar to CAR-T cells, TCR transgenic T cells are engineered to express receptors specific for tumor antigens, but this time, the TCR looks like endogenous TCR and recognizes tumor antigens presented by the major histocompatibility complex (MHC), allowing the TCR to recognize more antigens than CAR-T cells, because CAR-T can only recognize surface antigens. This strategy has been expanded in the treatment of solid tumors in particular.

[0005] Another recent anti-tumor approach is the use of primary NK cells or engineered CAR-NK cell infusions, which have shown promising results in the field of hematological malignancies or solid tumors. This last option needs to be carefully regulated to avoid the problems highlighted in CAR-T cells.

[0006] Cellular immunotherapy is also a promising approach in all pathologies involving T cells, and numerous trials are currently underway using CAR-T cells or TCR genetically modified T cells for autoimmune or allergic diseases or transplant rejection.

[0007] However, once these cells are reinjected into the patient, controlling them to increase their therapeutic effect or limit their toxicity remains difficult to date. Therefore, it seems important to be able to provide a flexible and easily reversible gene expression regulation system to control the effects of these cells as simply as possible.

[0008] The present invention highlights a new use of the gene expression regulation technology disclosed in the international patent application publication WO2013 / 068096 (herein referred to as NUTRIREG). In mammals, essential amino acids (EAA) cannot be synthesized by the body and therefore must be provided through food. When lacking, the organism must absolutely adapt, which is why it activates a specific signaling pathway, namely the GCN2-ATF4 pathway, leading to overexpression of a transcription factor (ATF4) present in all cells of the organism. This transcription factor will then bind to the DNA at the level of the promoter of specific target genes (amino acid response-AARE sequences) and activate their transcription. These genes play a key role in the process of adapting to nutritional stress. Therefore, the GCN2-ATF4 signaling pathway is used to develop a system for regulating transgene expression. The NUTRIREG system is based on the following association: (i) an artificial promoter (based on the AARE sequence), which is strongly inducible by the lack of one EAA that controls the expression of the transgene (AARE-Gene); and (ii) a food lacking EAA, which causes a sharp drop in the blood concentration of the limiting EAA and allows the induction of the GCN2-ATF4 signaling pathway. Therefore, after delivery of the AARE-gene plasmid to the target tissue using a viral vector, expression of the transgene can be induced after consumption of a diet lacking one of the EAAs ( Figure 1 ). Interestingly, this pathway can be rapidly shut down by depleting the missing EAA, which provides a regulatory system that is easily reversible and, since it involves physiological nutrient pathways, has no side effects. Evidence for the functionality of this system has been provided in different organs of mice (liver, pancreas, brain, eye) as well as in glioblastoma tumor cells, thanks to the use of pro-apoptotic genes (Chaveroux et al., Nat Biotechnol, 2016, 34, 746–75).

[0009] The present invention uses NUTRIREG technology to control the cDNA expression of proteins with therapeutic applications in cellular immunotherapy. This structure inserted into a viral vector can be directly integrated into donor T cells or NK cells, or into modified T cells or NK cells, and then re-injected into the patient. By using a mixture lacking essential amino acids, it will allow peptide drugs to be expressed on demand when needed. Depending on the drug gene expressed and the time of its expression, the use of NUTRIREG in cellular immunotherapy opens up many preventive or therapeutic treatment possibilities, whether it is to inhibit toxicity or, conversely, stimulate the efficacy of the cell therapy being studied. Summary of the invention

[0010] The present invention relates to a human T cell or NK cell comprising a nucleic acid construct, wherein the nucleic acid construct comprises:

[0011] i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, which is activated in said T cell or NK cell when said T cell or NK cell is activated and when consuming a diet lacking at least one essential amino acid; and

[0012] ii) a transgene placed under the control of said regulatory polynucleotide.

[0013] The present invention also relates to the human T cells or NK cells, which are used for cell immunotherapy.

[0014] Also provided is a method for preparing human T cells or NK cells of the present invention, wherein the human T cells or NK cells are transfected or transduced with a vector comprising a nucleic acid construct, wherein the nucleic acid construct comprises:

[0015] i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, which is activated in said T cell or NK cell when said T cell or NK cell is activated and when exposed to a deficiency of at least one essential amino acid; and

[0016] ii) a transgene placed under the control of said regulatory polynucleotide. DETAILED DESCRIPTION OF THE INVENTION

[0018] The GCN2-ATF4 signaling pathway is ubiquitous in cells, but depending on the tissue or organ, the pathway may not be activated by the nutritional regulation system. For example, EAA deficiency cannot induce activation of the GCN2-ATF4 pathway in mouse muscle. The inventors of the present application demonstrated that the GCN2-ATF4 pathway can be induced by EAA starvation in human T cells, but the initial in vitro experiments failed because the inventors of the present application also found that the activation of human T lymphocytes is essential for the activation of GCN2 kinase. Therefore, expression of the transgene is only possible when T cells are activated and EAA deprivation induces activation of the GCN2-ATF4 pathway. These results are expected to be applied to human NK cells because NK cells are basically cytotoxic T cells without TCR.

[0019] NUTRIREG T cells or NK cells

[0020] The present invention relates to a human T cell or NK cell comprising a nucleic acid construct, wherein the nucleic acid construct comprises:

[0021] i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, which is activated when the subject consumes a diet lacking at least one essential amino acid; and

[0022] ii) a transgene placed under the control of said regulatory polynucleotide.

[0023] The present invention also relates to a human T cell or NK cell comprising a nucleic acid construct, wherein the nucleic acid construct comprises:

[0024] i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, which is activated in said T cell or NK cell when said T cell or NK cell is activated and exposed to a deficiency of at least one essential amino acid; and

[0025] ii) a transgene placed under the control of said regulatory polynucleotide.

[0026] Human T cells or NK cells and transgenes

[0027] According to some embodiments, the human T cells of the present invention are chimeric antigen receptor T (CAR-T) cells (including CAR-Treg), or T cell receptor (TCR) transgenic T cells.

[0028] In these embodiments, the transgene is selected to stimulate the efficacy of cellular immunotherapy performed with the CAR-T cells or transgenic TCR T cells, or to inhibit toxicity induced by the CAR-T cells or transgenic TCR T cells. Specifically, the transgene is a transgene that prevents CAR-T cell exhaustion.

[0029] According to recent promising studies, transgenes suitable for stimulating the efficacy of cellular immunotherapy with CAR-T cells or transgenic TCR T cells include c-JUN, T-bet, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23 (to reverse or delay T cell exhaustion or stimulate those modified T cells) (Poorebrahim et al., Oncogene 40.2(2021):421-435; Pietrobon et al., International Journal of Molecular Sciences 22.19(2021):10828).

[0030] Transgenes suitable for inhibiting CAR-T cell or transgenic TCR T cell-induced toxicity include inhibitory receptors (e.g., PD-1, CTLA-4, TIM-3, LAG-3), or immunosuppressive factors (e.g., IL-35, IL-10, TGF-β, FoxP3, IDO, TOX, Eomes) (Poorebrahim et al., Oncogene 40.2(2021):421-435; Pietrobon et al., International Journal of Molecular Sciences 22.19(2021):10828).

[0031] These human T cells according to the invention are particularly intended for the treatment of cancer, such as hematological malignancies or solid cancers.

[0032] According to some embodiments, the human T cells of the invention are intended for use in allogeneic transplantation, or for the treatment of hematological cancers.

[0033] In a first aspect of these embodiments, the transgene is selected to promote differentiation of human T cells into regulatory T cells (Treg) or type 1 regulatory T cells (Tr1).

[0034] Suitable transgenes for promoting differentiation of human T cells into (i) Tr1 include transgenes encoding cytokines such as IL-10, TGF-β, IFN-α or IL-6 (Roncarolo et al., Immune-Mediated Diseases, Immunity, Volume 49, Issue 6, 2018), or (ii) Treg transgenes mainly include FoxP3. Preferably, the transgene that promotes differentiation of human T cells into Tr1 is a transgene encoding IL-10.

[0035] According to this aspect, when signs of GvHD are detected, expression of the transgene is induced to inhibit or control GvHD.

[0036] According to a second aspect of these embodiments, it is contemplated that human T cells used for the treatment of hematological cancers by allogeneic transplantation comprise a transgene that stimulates the efficacy of cellular immunotherapy in an individual (i.e., a human or non-human mammal). For example, the transgene encodes a stimulatory cytokine, such as IL-2, IFN-γ, TNF-α, IL-7, IL-15 (Ringdén et al., British journal of haematology 147.5 (2009): 614-633).

[0037] According to some embodiments, the human T cells of the present invention are intended for use in controlling allogeneic rejection of solid organ transplantation, or for treating autoimmune diseases. In these embodiments, the human T cells are human CAR-Tregs, more specifically antigen-specific CAR-Tregs, wherein the antigen is a transplant-specific alloantigen, or an autoantigen associated with an autoimmune disease.

[0038] This can be done by isolating polyclonal Tregs (from the individual's peripheral blood) and transducing them with antigen-specific CAR constructs, or by co-transducing CD4+ Tregs with antigen-specific CAR constructs and FoxP3 cDNA. + or CD3 + T cells, producing antigen-specific human CAR-Treg (Arjomandnejad et al., Biomedicines 2022, 10(2), 287).

[0039] In these embodiments, the transgenes are selected to stimulate CAR-Treg efficacy and / or prevent CAR-Treg exhaustion and include c-JUN, T-bet, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, as already mentioned above.

[0040] According to some embodiments, the human cell comprising a nucleic acid construct (including a regulatory polynucleotide and a transgene) is a human NK cell. The term NK cell used herein includes NK cells and modified NK cells, such as CAR-NK cells.

[0041] In these embodiments, a transgene is selected to stimulate the efficacy of cellular immunotherapy using the human NK cells or CAR-NK cells of the present invention. Specifically, the transgene is a transgene that enhances the activation or proliferation of NK cells or CAR-NK cells.

[0042] Suitable transgenes include NKG2D, IL-12, IL-15 or IL-18 (Shimasaki N et al., Nat Rev Drug Discov. 2020 Mar;19(3):200-218; Daher M. et al., Blood. 2021 Feb 4;137(5):624-636; Wang X. et al., Blood Adv. 2020 May 12;4(9):1950-1964).

[0043] These human NK cells or CAR-NK cells according to the present invention are expected to be particularly useful in the treatment of hematological malignancies or solid tumors.

[0044] Nucleic acid constructs and regulatory polynucleotides

[0045] The nucleic acid construct comprises:

[0046] i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, which is activatable or activated after the subject consumes a diet lacking at least one essential amino acid; and

[0047] ii) a transgene placed under the control of said regulatory polynucleotide.

[0048] Alternatively, the nucleic acid construct comprises:

[0049] i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, which is activated in said T cell or NK cell when said T cell or NK cell is activated and exposed to a deficiency of at least one essential amino acid; and

[0050] ii) a transgene placed under the control of said regulatory polynucleotide.

[0051] Such nucleic acid constructs have been described in International Patent Application Publications WO2013 / 068096A1 and WO2017 / 207744A1, which are incorporated herein in their entirety.

[0052] The regulatory polynucleotide comprises a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence.

[0053] As used herein, "minimal promoter" means a promoter that contains all elements required to correctly initiate transcription of a target gene located downstream. The two expressions "minimal promoter" and "core promoter" are considered equivalent expressions. It is understood by those skilled in the art that a "minimal promoter" contains at least a transcription start site, an RNA polymerase binding site, and a general transcription factor binding site (TATA box).

[0054] Suitable minimal promoters are known to those skilled in the art.

[0055] In some embodiments, the minimal promoter is selected from the group consisting of a thymidine kinase (TK) promoter, a β-globin promoter, a cytomegalovirus (CMV) promoter, a SV40 promoter, and the like.

[0056] As used herein, "AARE" or "amino acid response element" refers to a nucleic acid sequence that, after activation of the GCN2-eIF2α-ATF4 pathway due to essential amino acid (EAA) deficiency, binds to activating transcription factor 4 (ATF4) and thereby induces the expression of target genes driven by AARE.

[0057] In mammals, after consuming a diet lacking one EAA, blood concentrations of the limiting EAA drop rapidly and substantially, initiating a ubiquitous adaptive process known as the amino acid response pathway. The initial step of the pathway is the activation of the mammalian GCN2 protein kinase by uncharged tRNA. GCN2 then phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2α) on serine 51, leading to upregulation of ATF4 translation. Once induced, ATF4 activates transcription of specific target genes by binding to the AAREs. The GCN2-eIF2α-ATF4 pathway can be rapidly shut down by administration of the missing EAA.

[0058] This pathway can also be observed at the cellular level. In fact, the amino acid response pathway is also triggered in activated T cells or NK cells that are exposed to the deficiency of at least one EAA, for example when activated T cells or NK cells are cultured in a medium lacking at least one EAA.

[0059] According to some embodiments, the amino acid response element (AARE) nucleic acid sequence is selected from the group consisting of the nucleic acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

[0060] The regulatory polynucleotide comprising at least one AARE may include at least 2, at least 3, at least 4 or at least 5 AARE nucleic acid sequences. Thus, the expression "at least one AARE nucleic acid sequence" includes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 AARE nucleic acid sequences.

[0061] In certain embodiments, the regulatory polynucleotide comprises at least two AARE nucleic acid sequences. In some other embodiments, the regulatory polynucleotide comprises 1 to 20 AARE nucleic acid sequences, preferably 1 to 10 AARE nucleic acid sequences. In certain embodiments, the regulatory polynucleotide comprises 2 to 6 AARE nucleic acid sequences.

[0062] In some embodiments, the regulatory polynucleotide comprises two AARE nucleic acid sequences selected from the group consisting of nucleic acid sequences SEQ ID NO: 2 and SEQ ID NO: 4. In some embodiments, the regulatory polynucleotide comprises six AARE nucleic acid sequences of SEQ ID NO: 1.

[0063] In certain embodiments, the at least two AARE nucleic acid sequences may be the same or different.

[0064] According to one embodiment, the regulatory polypeptide includes a thymidine kinase (Tk) minimal promoter and six copies of an AARE nucleic acid sequence from the TRIB3 gene, and comprises or consists of a sequence as shown in SEQ ID NO:6.

[0065] Thus, the regulatory polynucleotide construct comprises at least one AARE nucleic acid sequence located directly upstream of a minimal promoter that controls the expression of a transgene located downstream.

[0066] The regulatory polynucleotide is activated in the T cells or NK cells of the subject after consuming a diet lacking at least one essential amino acid.

[0067] The subject is a human or non-human mammal, preferably a human. In some embodiments, the non-human mammal is selected from the group consisting of mice, rats, etc.; primates such as chimpanzees, monkeys, etc.

[0068] In mammals, nine EAAs must be supplied in the diet, and the deficiency of any one of them can induce the AARE-driven expression system.

[0069] As used herein, “essential amino acids” include histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), threonine (Thr, T), tryptophan (Trp, W), and valine (Val, V).

[0070] As used herein, a "diet lacking at least one essential amino acid" means a diet lacking 1, 2, 3, 4, 5, 6, 7, 8 or 9 essential amino acids. According to some embodiments, the diet lacks leucine.

[0071] The regulatory polynucleotide is activated in activated T cells or NK cells when exposed to a deficiency of at least one essential amino acid, such as when cultured in a medium lacking at least one essential amino acid.

[0072] The cell is a human cell or a non-human mammal cell, preferably a human cell. In some embodiments, the non-human mammal is selected from the group consisting of mice, rats, etc.; primates such as chimpanzees, monkeys, etc.

[0073] For cells to function, nine EAAs must be supplied in the diet, and the absence of any one of them can induce the AARE-driven expression system.

[0074] As used herein, "a medium lacking at least one essential amino acid" means a diet lacking 1, 2, 3, 4, 5, 6, 7, 8 or 9 essential amino acids. According to some embodiments, the diet lacks leucine.

[0075] Activation of regulatory polynucleotides

[0076] In order to activate the regulatory peptide, a diet lacking at least one essential amino acid is administered to the subject (ie a human or non-human mammal as defined above).

[0077] According to a preferred embodiment, the subject has been placed in a starvation state prior to administration of the diet lacking at least one essential amino acid.

[0078] To activate a regulatory peptide in cells in vitro, the cells are cultured in a medium lacking at least one essential amino acid, preferably for at least 2 h, at least 6 h, at least 10 h, and preferably at least 16 h.

[0079] Cellular immunotherapy using NUTRIREG T cells or NK cells

[0080] The human T cells or NK cells according to the present invention are used for cellular immunotherapy.

[0081] The present invention also provides a method for performing cellular immunotherapy on a subject in need thereof, comprising administering the T cell or NK cell according to the present invention to the subject.

[0082] Cellular immunotherapy is intended for humans or non-human mammals, preferably humans. In some embodiments, the non-human mammal is selected from the group consisting of mice, rats, etc.; primates such as chimpanzees, monkeys, etc.

[0083] In some embodiments, the T cells or NK cells are autologous to the human subject. In some embodiments, the T cells or NK cells are allogeneic to the human subject.

[0084] Cellular immunotherapy includes, but is not limited to, treating cancer, controlling allograft rejection of solid transplants, or treating autoimmune diseases.

[0085] The cancer may be a solid cancer or a blood cancer.

[0086] At certain time points, the subject administered human T cells or NK cells is further subjected to a diet lacking at least one essential amino acid to induce transgene expression.

[0087] One advantage of the human cells according to the invention, in particular the human T cells according to the invention, is that transgene expression is induced only in activated human cells, in particular T cells, by EAA deprivation (or activation of AARE nucleic acids).

[0088] By exposing the human T cells or NK cells of the invention to EEA deficiency, this allows the transgene to be expressed only when needed. In fact, one of the major advantages of the NUTRIREG-T cell or NK cell system, in addition to induction by food-inducible endogenous signaling pathways (GCN2-ATF4), is the possibility to perform on / off regulation of transgene expression and control this expression over time.

[0089] In one embodiment of the invention, the administered T cells or NK cells of the invention are not activated. Transgene expression is induced in the cells only when the cells are activated in a subject, in particular in the case of inflammation, and only when the subject is on a diet lacking at least one essential amino acid.

[0090] The first application of the human T cells of the present invention is in the context of allogeneic stem cell transplantation (allo-SCT), which is the treatment of choice for many hematological malignancies. Allogeneic donor T cells recognize minor histocompatibility antigens (minor H antigens), which lead to the elimination of leukemic cells, a phenomenon known as the graft-versus-leukemia (GvL) effect. Since many minor H antigens are ubiquitously expressed, the GvL effect is often accompanied by the destruction of the patient's normal tissues, i.e., graft-versus-host disease (GvHD), which is the main side effect of allo-SCT.

[0091] Preventive or therapeutic control of GvHD can be achieved using human T cells of the present invention, which contain transgenes that promote the expansion of human T lymphocytes with anti-inflammatory properties to mitigate the cytotoxic response that causes GvHD. In this regard, donor T cells (present in allogeneic stem cells to be transplanted into a subject in need, or in the form of donor lymphocyte injection) are genetically modified ex vivo using a NUTRIREG system that includes a Treg or Tr1 inducible transgene and then re-injected into the subject. In this way, by simply consuming a diet lacking EAA within a few hours, (i) this Treg or Tr1 inducible transgene (usually an anti-inflammatory cytokine) can be expressed in activated T cells (which cause GvHD), and (ii) the polarization of the T response toward a T regulatory phenotype (Treg or Tr1) can be promoted.

[0092] Conversely, in order to regain the graft-versus-leukemia (GvL) effect upon relapse of a subject's hematological disease, one could also consider repriming donor T lymphocytes using stimulatory cytokines transiently expressed in the context of EEA deprivation.

[0093] Therefore, according to some embodiments, human NUTRIREG T cells are used to treat cancer, in particular hematological cancer, by allogeneic transplantation. Hematological cancer is particularly leukemia, lymphoma or multiple myeloma.

[0094] According to some embodiments, human T cells are used to treat hematological cancers by allogeneic transplantation, wherein:

[0095] - Expression of the transgene promotes differentiation of human T cells into Treg or Tr1 and prevents or treats graft-versus-host disease (GvHD), or

[0096] - Expression of the transgene stimulates the efficacy of cellular immunotherapy by inducing or stimulating the graft-versus-leukemia (GvL) effect by said human T cells.

[0097] In some aspects, the transgene promotes the differentiation of human T cells into regulatory T cells (Treg or Tr1); for example, the transgene encodes cytokines such as IL-10, TGF-β, IFN-α and IL-6 or transcription factors such as FoxP3. The expression of transgenes that promote the differentiation of human T cells into Treg or Tr1 prevents or treats graft-versus-host disease (GvHD). In this regard, it is expected that the transgenes that promote the differentiation of human T cells into Treg or Tr1 will be repeatedly induced by intermittently eating a diet lacking one EAA.

[0098] The on / off regulation that the NUTRIREG-T cell system enables is a key advantage in controlling the cytotoxic response of GvHD, as the dietary absence of EAA will, at the appropriate time, stop the expression of transgenes that promote the differentiation of human T cells into Tregs or Tr1s (e.g., anti-inflammatory cytokines). The NUTRIREG system is therefore highly advantageous as it allows for transient expression of transgenes, thereby preserving the anti-tumor effects of the graft, which is not achievable with currently used immunosuppressive drugs. Another advantage is that the transgene will only be expressed in activated T cells, as the GCN2-ATF4 pathway cannot be induced in resting T cells, which represents an essential safety control.

[0099] In other aspects, the transgene stimulates the efficacy of cellular immunotherapy with the human T cells to regain the GvL effect; for example, the transgene encodes a stimulatory cytokine, such as IL-2, IFN-γ, TNF-α, IL-7, IL-15. The expression of the transgene that stimulates the efficacy of cellular immunotherapy with the human T cells induces or stimulates the GvL effect. In this regard, when a subject's blood disease is suspected of recurring, it is expected that the transgene that stimulates the efficacy of cellular immunotherapy with the human T cells is repeatedly induced by intermittently eating a diet lacking one EAA.

[0100] The second application relates to the therapeutic strategy of human CAR-T cells or TCR transgenic T cells of the present invention, with the purpose of using transgenics encoding inhibitory receptors (e.g., PD-1, CTLA-4, TIM-3, LAG-3) or immunosuppressive factors (e.g., IL-35, IL-10, TGF-β, FoxP3, IDO, TOX, Eomes) to limit their toxicity, or conversely stimulate their activity. Human CAR-T cells or TCR transgenic T cells according to the present invention can allow transient expression of target genes to prevent T cell exhaustion. Encouraging results about c-JUN have been described in the literature, and when c-JUN is overexpressed, it will enable CAR-T cells to resist exhaustion (Lynn et al., Nature 576, 293–300 (2019)). Therefore, in CAR-T cells or TCR transgenic T cells, transient repeated expression of c-JUN under the dependence of NUTRIREG can make it resist exhaustion and promote the maintenance of its efficacy over time. Some promising results have also been obtained using T-bet, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23 to reverse or delay T cell exhaustion or stimulate modified T cells (Poorebrahim et al., Oncogene 40.2(2021):421-435; Pietrobon et al., International Journal of Molecular Sciences 22.19(2021):10828).

[0101] Similarly, therapeutic strategies may use human NK cells or CAR-NK cells according to the present invention with the aim of stimulating their activity by enhancing activation or proliferation using transgenes (e.g., NKG2D, IL-12, IL-15 or IL-18).

[0102] In the case of CAR-T cells, TCR transgenic T cells, NK cells or CAR-NK cells, the on / off regulation that the NUTRIREG system enables is also very important, as it allows both (i) the beneficial effects of genes with transient expression and (ii) limiting the toxic effects that may arise from their expression for too long (e.g. cJUN, which may have oncogenic effects).

[0103] Therefore, in some embodiments, cellular immunotherapy using human CAR-T cells or TCR transgenic T cells, or human NK cells or CAR-NK cells of the present invention is used to treat cancer, especially solid cancer.

[0104] For cancer therapy, one skilled in the art will specifically select human CAR-T cells, TCR transgenic T cells, or CAR-NK cells that are specific for tumor antigens of a subject with cancer.

[0105] In these aspects, the transgene stimulates the efficacy of cellular immunotherapy using the human CAR-T cells, transgenic TCR T cells, NK cells or CAR-NK cells, or inhibits the toxicity induced by the CAR-T cells or transgenic TCR T cells. Specifically, the transgene prevents CAR-T cell exhaustion or transgenic TCR T cell exhaustion.

[0106] In some other embodiments, cellular immunotherapy using human CAR-T cells or TCR transgenic T cells of the present invention is used to treat autoimmune diseases. Specifically, for autoimmune diseases caused by autoantibody production, human CAR-T cells or TCR transgenic T cells will target B cells that produce autoantibodies. For autoimmune diseases caused by cytotoxic T cells (such as colitis, multiple sclerosis or type 1 diabetes), human CAR-T cells or TCR transgenic T cells of the present invention will target pathogenic autoimmune T cells.

[0107] According to these aspects, human T cells are chimeric antigen receptor T (CAR-T) cells or T cell receptor (TCR) transgenic T cells. Depending on the therapeutic use, CAR-T cells or TCR transgenic T cells are specific for autoantibodies or pathogenic autoimmune T cells. In these aspects, transgenics stimulate the efficacy of cellular immunotherapy performed with the CAR-T cells or transgenic TCR T cells, or inhibit the toxicity induced by the CAR-T cells or transgenic TCR T cells. Specifically, transgenics prevent CAR-T cell exhaustion or transgenic TCR T cell exhaustion.

[0108] In some embodiments, cellular immunotherapy is used to control allograft rejection of solid transplants.

[0109] In addition, cell therapy using autologous Tregs expanded in vitro is one of the most promising methods for regulating alloimmunity and reducing immunosuppression. Preclinical studies have shown that the effectiveness of Treg therapy can be significantly enhanced by using Tregs (i.e., donor-specific (ds) Tregs) that are specific to donor alloantigens. CAR-Treg technology is used to generate dsCAR-Tregs, and it is found that these cells can suppress humoral immunity and delay allogeneic transplant rejection in unsensitized immunocompetent recipients (Sicard et al., Am J Transplant.2020; 20: 1562–1573). Therefore, in some embodiments, cell immunotherapy is used to control allogeneic transplant rejection using human CAR-T cells or TCR transgenic T cells that are specific to donor alloantigens according to the present invention. In some aspects, transgenics prolongs the life span of the donor-specific CAR-T cells or donor-specific transgenic TCR T cells, particularly preventing dsCAR-T cell exhaustion or transgenic dsTCR T cell exhaustion, as described above.

[0110] Method for preparing NUTRIREG T cells or NK cells

[0111] A method for preparing a human T cell or NK cell according to the present invention is provided, wherein the human T cell or NK cell is transfected or transduced by a vector comprising a nucleic acid construct, wherein the nucleic acid construct comprises:

[0112] i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, which is activated after the subject consumes a diet lacking at least one essential amino acid; and

[0113] ii) a transgene placed under the control of said regulatory polynucleotide.

[0114] Also provided is a method for preparing a human T cell or NK cell according to the present invention, wherein the human T cell or NK cell is transfected or transduced by a vector comprising a nucleic acid construct, wherein the nucleic acid construct comprises:

[0115] i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, which is activated in said T cells or NK cells when said T cells or NK cells are activated, and when exposed to a deficiency of at least one essential amino acid, for example when cultured in a medium lacking at least one essential amino acid; and

[0116] ii) a transgene placed under the control of said regulatory polynucleotide.

[0117] The vector may be a synthetic vector (cationic lipid, polymer liposome, etc.), a plasmid or a viral vector.

[0118] If necessary, it can be combined with one or more substances that improve the transfection efficiency and / or stability of the vector. These substances are widely recorded in the literature available to those skilled in the art (for example, see Felgner et al., 1987, Proc. West. Pharmacol. Soc. 32, 115-121; Hodgson and Solaiman, 1996, Nature Biotechnology 14, 339-342; Remy et al., 1994, Bioconjugate Chemistry 5, 647-654). By way of non-limiting example, they can be polymers, cationic lipids, liposomes, nucleoproteins or neutral lipids. These substances can be used alone or in combination. A possible combination is a recombinant plasmid vector combined with a cationic lipid (DOGS, DC-CHOL, spermine-cholesterol, spermidine-cholesterol, etc.) and a neutral lipid (DOPE).

[0119] In the case of the present invention, a wide selection of plasmids can be used. They can be cloning and / or expression vectors. In general, they are known in the art, and many of them are commercially available, but they can also be constructed or modified using genetic manipulation techniques. For example, we can mention plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript (Stratagene), pREP4, pCEP4 (Invitrogene) or pPoly (Lathe et al., 1987, Gene 57, 193-201). Preferably, the plasmid implemented by the present invention contains a replication origin that ensures that replication begins in a production cell and / or a host cell (for example, if a plasmid is to be produced in Escherichia coli, the CoIEI origin will be used, and if self-replication is to be performed in a mammalian host cell, the oriP / EBNAI system will be used, Lupton and Levine, 1985, Mol. Cell. Biol. 5, 2533-2542; Yates et al., Nature 313, 812-815). It may also include selection genes for selection or identification of transfected cells (complementation of auxotrophic mutations, genes encoding antibiotic resistance, etc.) It may also include additional elements that improve its maintenance and / or stability in a given cell.

[0120] When it comes to viral vectors, it can be a vector derived from adenovirus, lentivirus, retrovirus, adenovirus-associated virus (AAV), herpes virus, alphavirus, parvovirus, poxvirus (fowlpox, canarypox, vaccinia virus, in particular MVA (Modified Ankara virus) or Copenhagen strain, etc.) or foamy virus. Preferably, a non-replicative and optionally non-integrative vector is used. Retroviruses have the property of infecting and mainly integrating into dividing cells, and are therefore particularly suitable for anticancer therapy applications. Retroviral vectors suitable for implementing the present invention include LTR (long terminal repeat) terminal sequences and encapsidation regions. It can be derived from retroviruses of any source (murine, primate, feline, human, etc.), and in particular, can be derived from a retrovirus selected from the group including MoMuLV (Moloney murine leukemia virus), MVS (murine sarcoma virus) or Friend murine retrovirus (Fb29). It is propagated in an encapsidated line that is capable of trans-providing gag, pol and / or env viral polypeptides required to constitute viral particles. These types of lines are described in the literature (PA317, Psi CRIP GP+Am-12, etc.). The retroviral vectors of the invention may comprise modifications, in particular at the LTR (replacement of the promoter region with a eukaryotic promoter) or at the encapsidation region (replacement with a heterologous encapsidation region (e.g. of the VL30 type).

[0121] In a preferred embodiment of the present invention, the vector is a lentiviral vector, an adenoviral vector, or a vector derived from an adeno-associated virus (AAV).

[0122] According to an advantageous embodiment, the viral vector used according to the invention may be in the form of a DNA vector or an infectious viral particle.

[0123] The present invention will be further illustrated by the following figures and examples.

[0124] BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 : General principle of NUTRIREG.

[0126] Figure 2 : Conditions for the inducibility of target genes (TRB3) of the GCN2-ATF4 pathway in human T cells after EAA starvation.

[0127] Figure 3 : Inducible expression of the luciferase transgene under the control of NUTRIREG. NT cells: non-transduced cells; LTAARE-TK-LUC: T cells transduced with the 2xAARE-TK-LUC construct.

[0128] Figure 4: Inducible expression of the GFP transgene under the control of NUTRIREG. NT cells: non-transduced cells; LT AARE-TK-eGFP: T cells transduced with the 2xAARE-TK-eGFP construct.

[0129] Figure 5 : Co-expression analysis of activation markers of T cells and eGFP protein by flow cytometry.

[0130] Figure 6 : Principle of using NUTRIREG to induce IL-10 expression in T cells.

[0131] Figure 7 : Induction of mRNA (A) and protein (B) expression of the IL-10 transgene under the control of NUTRIREG. NT cells: non-transduced cells; LT AARE-BG-IL10: T cells transduced with the 2xAARE-BG-IL10 construct.

[0132] Figure 8 : Reversibility of NUTRIREG-mediated IL-10 transgenic mRNA (A) and protein (B) expression.

[0133] Fig. 9 :Effect of NUTRIREG-dependent IL-10 expression on mRNA expression of characteristic cytokines in type 1 regulatory T cells (Tr1).

[0134] Fig.10 : Schematic diagram of the experimental design. NXG immunodeficient mice received 10.10 6 Human T cells transduced with a viral vector containing the AARE-TK-IL-10 construct and 5.10 6 Seven days later, and then weekly until the third week, mice were fed a control diet or a diet lacking leucine for 16 h, after which blood samples were collected for flow cytometric analysis and IL-10 measurement.

[0135] Fig.11 : Monitoring human CD45 in mouse blood + Cell expansion. PBMCs were analyzed weekly by flow cytometry from week 1 to week 3, 16 h after mice had consumed a control diet or a diet lacking leucine. Results are expressed as human CD45 + The percentage of cells (left) or human CD45 + CD4 + or CD8 + Percentage of cells (Student's t-test - Leu(16h) vs Ctl: ns not significant; n=4-5 for each condition; error bars, sem).

[0136] Fig.12 : Monitoring of human IL-10 in mouse plasma. As per the Methods section, human IL-10 concentrations in plasma were measured by ELISA every week from week 1 to week 3, immediately after mice consumed a control diet or a diet lacking leucine for 16 hours (n=3-4 per condition; error bars, sem; right: linear regression analysis).

[0137] Fig.13 : Application of NUTRIREG-T cells with FoxP3 therapeutic gene. (A) FoxP3 mRNA levels were analyzed by RT-qPCR in non-transduced T cells (NT cells) and T cells transduced with the AARE-TK-FoxP3 construct (LT) (MOI 10), which were cultured in control medium (Ctl) or in the absence of leucine during 16 hours (-Leu (16h)). Expression was normalized according to actin expression. ANOVA test (-leu vs ctl et NT cells vs LT AARE-TK-FoxP3), ns not significant, ***p<0.001, n=3 / condition. (B) Western blot analysis of FoxP3 protein levels, eIF2α protein levels, and phosphorylated eIF2α protein levels in non-transduced T cells (NT cells) and T cells transduced with the AARE-TK-FoxP3 construct (LT) (MOI 10) and cultured in control medium (Ctl) or in the absence of leucine during 16 hours (-Leu (16h)).

[0138] Fig.14 : TGF-β mRNA levels were analyzed by RT-qPCR in non-transduced T cells (NT cells) and T cells transduced with the AARE-TK-FoxP3 construct (LT) (MOI 10), which were cultured in control medium (Ctl) or in the absence of leucine during 16 hours (-Leu (16h)). Expression was normalized according to actin expression. ANOVA test (-leu vs ctlet NT cells vs LT AARE-TK-FoxP3), ns not significant, *p<0.05, n=3 / condition. Example

[0139] Example 1: Verification of the inducibility of the GCN2-ATF4 pathway in human T cells after EAA starvation ( Figure 2 )

[0140] In order to be able to use NUTRIREG in human T cells later, we first verified that the GCN2-ATF4 pathway can be induced by EAA starvation in this cell type. Previous literature data emphasized that mouse T cells activated this signaling pathway in response to IDO (indoleamine 2.3-dioxygenase), halofuginone or asparaginase (Munn et al., Immunity 2005, Vol. 22, 633–642; Van de Velde et al., Cell reports 17.9 (2016): 2247-2258; Sundrud et al., Science 324.5932 (2009): 1334-1338.; Bunpo et al., Journal of Nutrition 2010, 140, 2020-2027), but there is little data on the activation of the GCN2-ATF4 pathway in response to AA deficiency in human T cells.

[0141] We therefore performed in vitro experiments using human T cells purified from "buffy coats" from the French Blood Establishment intended for research use. We cultured these cells in control medium or in medium lacking leucine.

[0142] We have proved that ( Figure 2 ):

[0143] (1) Six hours of EAA starvation (leucine or any other EAA) in T cells significantly induced the expression of a known target gene (TRB3) of the GCN2-ATF4 pathway.

[0144] (2) The induction of the GCN2-ATF4 pathway in T cells by short-term leucine starvation can be rapidly reversed 16 hours after the addition of leucine to the starvation medium.

[0145] (3) Importantly, activation of T cells (by anti-CD3 and CD28 antibodies in the presence of IL-2) is essential for the induction of the GCN2-ATF4 pathway in response to EAA starvation. The fact that activation of T lymphocytes is essential for the activation of GCN2 kinase has been described in mouse T cells after culture in tryptophan-deficient medium (Munn et al., Immunity 2005, Vol. 22, 633–642; Van de Velde et al., Cell reports 17.9 (2016): 2247-2258), but there is no information on human T lymphocytes.

[0146] (4) In response to short-term leucine starvation, the induction of expression of TRB3, a target gene of the GCN2-ATF4 pathway, is completely dependent on the GCN2 kinase; this was confirmed by using a pharmacological inhibitor of GCN2.

[0147] Example 2: Verification of NUTRIREG function in human T cells using luciferase reporter gene ( Figure 3 ).

[0148] We then evaluated the function of NUTRIREG in human T cells using two reporter genes. The first reporter gene used was the luciferase gene, which was inserted into the 2xAARE-TK-LUC construct and transduced by a lentiviral vector. Transduction was performed 24 hours after activation of T cells by magnetic beads loaded with anti-CD2 / CD3 / CD28 antibodies in the presence of IL-2. The cells were then deprived of leucine for 8 days after their activation, and luciferase activity was then measured.

[0149] Thus, we demonstrated that 16 h of leucine starvation indeed induced expression of the luciferase transgene in T cells via NUTRIREG, compared to (i) transduced T cells cultured in control medium and (ii) non-transduced T cells deprived of leucine for 16 h. This induction was detectable starting from 3 h of leucine deprivation, and transgene expression increased when the duration of EAA deprivation increased (6 h, 9 h). Figure 3 ).

[0150] Example 3: In vitro validation of the function of NUTRIREG in human T cells using the eGFP reporter gene ( Figure 4-5 ).

[0151] The second reporter gene evaluated was the eGFP gene, which was inserted into the 2xAARE-TK-eGFP construct and transduced by a lentiviral vector. Transduction was performed 24 hours after activation of T lymphocytes with magnetic beads loaded with anti-CD2 / CD3 / CD28 antibodies in the presence of IL-2. The cells were then deprived of leucine 8 days after their activation, and the fluorescence emitted by the eGFP protein was measured by flow cytometry.

[0152] We demonstrated that 16 h of leucine starvation indeed induced expression of the eGFP transgene in T cells via NUTRIREG, compared to (i) transduced T cells cultured in control medium and (ii) non-transduced T cells deprived of leucine for 16 h. The lack of such induction with shorter leucine starvation times may be due to stability issues of the eGFP protein ( Figure 4 ).

[0153] Furthermore, by analyzing by flow cytometry the co-expression of T cell activation markers (CD69 as an early activation marker and CD25 as a late activation marker) and eGFP protein, we were able to confirm the key role of T cell activation in inducing the expression of the transgene under the control of NUTRIREG.- CD69-) do not express eGFP, and those moderately activated cells (CD69 - CD25 + or CD69 + CD25 - ) weakly expressed eGFP, while strongly activated cells (CD69 + CD25 + ) are the cells that express the most eGFP protein ( Figure 5 ).

[0154] Example 4: Application of NUTRIREG-T cells and interleukin-10 (IL-10) therapeutic gene ( Figure 6-9 ) and body Internal results ( Figure 10-12 ).

[0155] For the purpose of using the NUTRIREG technology for treatment or prevention of GvHD (graft versus host disease) models, we designed a 2xAARE-BG-IL10 construct in which the interleukin 10 (IL-10) cDNA ( Figure 6 ). In fact, it has been demonstrated in the literature that lentiviral transduction of T cells with IL-10 can (i) direct the immune response to a Tr1-type anti-inflammatory response (T regulatory type 1) and (ii) prevent the appearance of GvHD in mouse models (Andolfi et al., Molecular Therapy 2012, vol. 20, 1778-1790; Locafaro et al., Molecular Therapy 25.10(2017):2254-2269).

[0156] Early human clinical trials also aim to direct immune response to Tr1 phenotype to prevent GvHD. For this reason, in the presence of IL-10, by culturing donor T cells with mononuclear cells (PBMC) exhausted by host CD3, these donor T cells lose reactivity to host cells. Then, a few weeks after allogeneic hematopoietic stem cell transplantation, anergic T cells are re-injected into recipients (Bacchetta et al., Frontiers in immunology 5 (2014): 16.). These type 1 regulatory T cells (Tr1) have a specific cytokine profile because they strongly express IL-10, and to a lesser extent express TGFβ, GZMb, IFNγ and IL-22. On the other hand, they do not express IL-2, IL4 and IL17, nor do they express FoxP3 transcription factors (Gregori et al., Frontiers in immunology 6 (2015): 593).

[0157] In our experiments, T cells were transduced with lentivirus carrying the 2xAARE-BG-IL-10 construct 24 h after activation with magnetic beads loaded with anti-CD2 / CD3 / CD28 antibodies in the presence of IL-2. The cells were then deprived of leucine for 8 days after their activation and (i) the expression of IL-10 mRNA was measured by RT-qPCR, (ii) the expression of IL-10 protein was measured by ELISA or flow cytometry, and (iii) the expression of different cytokines of the Tr1 phenotype was measured by RT-qPCR ( Figure 6 ).

[0158] Thus, we demonstrated that 16 h of leucine starvation indeed induced mRNA and protein expression of the IL-10 transgene via NUTRIREG, compared to (i) transduced T cells cultured in control medium and (ii) non-transduced T cells subjected to leucine starvation ( Figure 7 AB).

[0159] We also checked that the addition of leucine to the culture medium after starvation indeed shuts down transgene expression in transduced T cells, shutting down mRNA expression at 24 hours and IL-10 protein expression at 48 hours ( Figure 8 AB).

[0160] Finally, we were able to show that transient expression of the IL-10 transgene via NUTRIREG can direct the immune response towards a Tr1-type response, as indicated by cytokine and transcription factor profiles (mRNA): IL-10 ++ G JZ + IL22 + IFNγ + FoxP3 - IL4 - ( Fig. 9 ).

[0161] Finally, we investigated the ability of a leucine-deficient diet to upregulate AARE-driven IL-10 expression in human T cells injected into mice. Indeed, the absence of any of the EAAs in a mammalian diet could be a potential inducer of AARE transgene expression. Since T cell activation is essential for EAA induction of the GCN2-ATF4 pathway, we generated a mouse model in which we injected transduced human T cells and autologous irradiated human PBMCs. We hypothesized that transduced human T cells would be transiently activated by irradiated PBMCs and that this activation would be sufficient for the induction of the GCN2-ATF4 pathway by EAA nutritional deprivation. Briefly, immunodeficient NXG mice were injected into the tail vein with (i) lentivirally transduced human T cells carrying the 2xAARE-TK-IL-10 construct and (ii) human irradiated PBMCs and then fed once a week with a control diet or a leucine-free diet for 16 h until week 3 ( Fig.10 Weekly flow cytometric analysis of PBMCs allowed us to track human CD45 + The correct expansion of cells, while a diet lacking leucine has an adverse effect on cell expansion and CD4 + or CD8 + There was no significant effect on phenotype ( Fig.11 ). Human IL-10 increased linearly over time in the leucine-deficient group (R 2 =0.581; P = 0.004), while human IL-10 in the control group did not change significantly over time ( Fig.12 ).

[0162] Collectively, these results demonstrate the ability of NUTRIREG technology to modulate expression of therapeutic genes, such as IL-10, in activated human T cells both in vitro and in vivo.

[0163] Example 5: Application of NUTRIREG-T cells and FoxP3 transcription factor ( Figure 13-14 ).

[0164] Several publications in the literature have shown that transduction of T lymphocytes with the FoxP3 gene can shift the phenotype of these T lymphocytes toward a Treg FoxP3+ phenotype. + CD25 - T cells (without constitutive expression of FoxP3) were injected into immunodeficient RAG - / - In mice, these cells can be switched to a CD4 + CD25 +, inhibiting cytotoxic CD8+ responses and preventing the emergence of inflammatory colitis. These results in mice were confirmed in vitro in human cells by lentiviral transduction of FoxP3. In this context, we investigated the overexpression of the transcription factor Foxp3 via NUTRIREG with the aim of transiently and reversibly polarizing T lymphocytes to a Treg phenotype. Therefore, we designed the 2xAARE-TK-FoxP3 (human) transgene, which we transduced into activated human T lymphocytes (24 h of activation). Eight days after transduction, we transferred the cells to control medium or to medium lacking leucine for 16 h. Thus, we observed that the transduction of FoxP3 under the control of NUTRIREG made it possible to induce the expression of this transgene after 16 hours of culture in medium lacking leucine. This expression was at the transcriptional level ( Fig.13 A) and translation level ( Fig.13 This FoxP3 expression is induced by the anti-inflammatory cytokine TGF-β ( Fig.14 ) and increased expression of the cytotoxic molecules granzyme B and perforin 1, all of which are FoxP3 + Characteristics exhibited by Tregs.

Claims

1. A human T cell or NK cell comprising a nucleic acid construct, wherein the nucleic acid construct comprises: i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, which is activated in said T cell or NK cell when said T cell or NK cell is activated and after exposure to a deficiency of at least one essential amino acid; and ii) a transgene placed under the control of said regulatory polynucleotide.

2. The human T cell or NK cell according to claim 1, which is a human T cell, and wherein the human T cell is a chimeric antigen receptor T (CAR-T) cell or a T cell receptor (TCR) transgenic T cell.

3. The T cell or NK cell according to claim 1, which is a human NK cell or a human CAR-NK cell.

4. The human T cell or NK cell according to claim 2 or 3, wherein the transgene stimulates the efficacy of cellular immunotherapy performed with the CAR-T cell, transgenic TCR T cell, NK cell or CAR-NK cell, or the transgene inhibits toxicity induced by the CAR-T cell or transgenic TCR T cell.

5. The human T cell or NK cell according to claim 2 or 4, which is a human CAR-T cell or a transgenic TCR T cell, and wherein the transgene reverses or delays CAR-T cell exhaustion or transgenic TCR T cell exhaustion, or encodes an inhibitory receptor or an immunosuppressive factor. The human T cell or NK cell according to claim 1 , which is a human T cell intended for use in allogeneic transplantation or for controlling allogeneic transplant rejection of a solid transplant.

7. The human T cell of claim 6, wherein the transgene promotes the differentiation of the human T cell into a regulatory T cell (Treg or Tr1).

8. The human T cell of claim 6, wherein the human T cell is intended for use in allogeneic transplantation and the transgene stimulates the efficacy of cellular immunotherapy using the human T cell.

9. The human T cell or NK cell according to any one of claims 1 to 8, wherein: a) an amino acid response element (AARE) nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and / or b) The regulatory polynucleotide comprises at least two AARE nucleic acid sequences.

10. The human T cell or NK cell according to any one of claims 1 to 9, for use in cellular immunotherapy.

11. The human T cell or NK cell for use according to claim 10, wherein the cell immunotherapy is used to treat cancer, control allogeneic transplant rejection of solid transplants or treat autoimmune diseases.

12. The human T cell or NK cell for use according to claim 10 or 11, wherein the human T cell or NK cell is as defined in any one of claims 1 to 9 and is used for treating cancer.

13. The human T cell or NK cell for use according to claim 10 or 11, which is a human T cell for treating hematological cancer by allogeneic transplantation, or for controlling allogeneic transplant rejection of a solid transplant, and the human T cell is defined in any one of claims 5 to 9.

14. The human T cell or NK cell for use according to claim 12 or 13, which is a human T cell for treating hematological cancer by allogeneic transplantation, and wherein: - Expression of the transgene promotes differentiation of human T cells into Treg or Tr1 and prevents or treats graft-versus-host disease (GvHD), or - Expression of the transgene stimulates the efficacy of cellular immunotherapy using said human T cells to induce or stimulate the graft versus leukemia (GvL) effect.

15. The human T cell or NK cell for use according to claim 10, 11 or 13, which is a human CAR-Treg for controlling allogeneic transplant rejection of solid transplants, or for treating autoimmune diseases.

16. The human T cell or NK cell for use according to any one of claims 10 to 15, wherein the subject to whom the T cell or NK cell is administered further receives a diet lacking at least one essential amino acid to induce transgene expression in the activated human T cell or NK cell.

17. A method for preparing a human T cell or NK cell as defined in any one of claims 1 to 9, wherein the human T cell or NK cell is transfected or transduced with a vector comprising a nucleic acid construct comprising: i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, which is activated in said T cell or NK cell when said T cell or NK cell is activated and exposed to a deficiency of at least one essential amino acid; and ii) a transgene placed under the control of said regulatory polynucleotide.

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