Control of Transgene Expression in Human T or NK Cells for Use in Cellular Immunotherapy

Abstract

The present invention relates to a human T cell or NK cell comprising a nucleic acid construct containing a transgene placed under the control of a regulatable polynucleotide that can be induced by the lack of at least one essential amino acid, and to a cellular immunotherapy using the above human T cell or NK cell.

Description

Technical Field

[0001] The present invention relates to human T cells or NK cells comprising a nucleic acid construct comprising a transgene placed under the control of a regulatable polynucleotide that can be induced by the lack of at least one essential amino acid, and to adoptive cell therapy using said human T cells or NK cells.

Background Art

[0002] Adoptive cell therapy is currently expanding. For example, in oncology, this treatment approach uses immune cells to restore the balance of anti-tumor immune surveillance deficiency. This principle is widely used in the treatment of hematological malignancies by old cell therapies such as allogeneic hematopoietic stem cell transplantation (allo-HSCT). Allo-HSCT basically involves replacing the bone marrow of a patient's diseased state with healthy bone marrow collected from a donor with a HLA match. This is an effective adoptive cell therapy strategy that not only enables the reconstitution of the bone marrow and immune system, but also enables the anti-tumor effect of donor lymphocytes (graft-versus-leukemia or GvL effect). Unfortunately, this approach also exhibits high toxicities such as graft-versus-host disease (GvHD) and infections, which limit the application of the curative strategy. These complications are the main causes of morbidity and mortality, poor quality of life, and additional economic costs, especially due to the expensive treatments for treating them and the increased hospital stay of the patients.

[0003] In more recent years, chimeric antigen receptor (CAR)-T cell therapy has emerged as a novel approach for the treatment of cancer. CAR-T cells are autologous T cells that are collected from a patient and modified ex vivo to express a chimeric antigen receptor (CAR) specific for the target tumor antigen. The CAR is composed of (i) an antigen-binding domain (a 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 can autoactivate after recognition of their target antigen and directly kill tumor cells bearing this antigen. For example, anti-CD19 CAR-T cells have received manufacturing approval for several years in the treatment of certain hematological malignancies that are refractory or relapsed to conventional chemotherapy treatment, and there is extensive worldwide development of CAR-T cells, including for the treatment of solid tumors. However, this innovative treatment strategy has points that need to be improved, particularly from the perspective of efficacy. This is because a significant proportion of patients experience their CAR-T cells becoming less effective over time, for example due to CAR upregulation. Furthermore, when CAR-T cell toxicity occurs (for example due to the manifestation of cytokine release syndrome symptoms or certain neurological toxicities), it may be necessary to suppress the activity of CAR-T cells in a reversible manner, for example by "depleting" the CAR-T cells so that they are reversibly inhibited 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 modified to express a receptor specific for a major antigen, but in this case the TCR appears like an endogenous TCR and can recognize tumor antigens presented by major histocompatibility antigens (MHC), enabling TCR recognition of more antigens than CAR-T cells, which only recognize surface antigens. This strategy is expanding particularly in the treatment of solid tumors.

[0005] Another recent anti-tumor approach is the use of infusion of primary NK cells or modified CAR-NK cells, which has promising results in the field of hematological diseases or solid tumors. This last option requires careful tuning to avoid the problems highlighted with CAR-T cells.

[0006] Cell immunotherapy is also a promising approach in all pathologies involving T cells, and many trials are ongoing in autoimmune or allergic diseases or transplant rejection using either CAR-T cells or TCR transgenic T cells.

[0007] However, to date, there are still many difficulties in controlling these cells after re-infusion into patients, either to increase their therapeutic action or to limit their toxicity. Thus, it seems important to be able to provide a flexible and easily reversible gene expression control system to control the action of these cells as simply as possible.

[0008] The present invention highlights a novel use of gene expression control technology, disclosed in an international patent application published as International Patent Publication No. 2013 / 068096 and referred to herein as NUTRIREG. In mammals, essential amino acids (EAAs) are not synthesized by the body and thus must be provided by food. In the case of deficiency, the organism has to adapt anyway, and for this purpose the organism activates a specific signaling pathway, the GCN2-ATF4 pathway, which results in the overexpression of a transcription factor (ATF4) present in all cells of the organism. This transcription factor then binds to DNA at the level of the promoters of specific target genes (amino acid responsive (Amino Acid Responsive)-AARE sequences) and activates their transcription. These genes play an important role in the adaptation process to nutritional stress. This GCN2-ATF4 signaling pathway was thus used to develop a system for controlling the expression of transgenes. The NUTRIREG system is based on the association of (i) an artificial promoter (based on the AARE sequence) that can be strongly induced by the deficiency of one EAA, which controls the expression of the transgene (AARE-Gene), and (ii) a food lacking in EAA, which causes a marked decrease in the blood concentration of the limiting EAA and enables the induction of the GCN2-ATF4 signaling pathway. After delivery of the AARE-Gene plasmid to the target tissue using a viral vector, the expression of the transgene can thus be induced after consumption of a food lacking one EAA (Figure 1). Interestingly, this pathway can be rapidly stopped via the consumption of the missing EAA, which provides an easily reversible control system and lacks harmful effects because it involves a physiological nutritional pathway. Proof of the functionality of this system has already been provided not only in different organs of the mouse (liver, pancreas, brain, eye) but also in glioblastoma tumor cells using a pro-apoptotic gene (Chaveroux et al, Nat Biotechnol, 2016, 34, 746‐751).

[0009] The NUTRIREG technology is used in the present invention to control the cDNA expression of proteins having therapeutic indications in cellular immunotherapy. This construct is inserted into a viral vector and can be integrated directly into donor T or NK cells, or into modified T or NK cells, and then reinjected into the patient. The use of an essential amino acid-deficient mixture allows for the expression of peptide drugs upon demand if required. The use of NUTRIREG in cellular immunotherapy expands many prophylactic or curative treatment possibilities, depending on the drug gene expressed and the time at which it is expressed, whether to suppress toxicity or, conversely, to stimulate the efficacy of the cell therapy being tested.

Summary of the Invention

[0010] The present invention relates to 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 or NK cells when said T or NK cells are activated and upon consumption of a diet deficient in at least one essential amino acid, and ii) a transgene placed under the control of said regulatory polynucleotide and relates to human T cells or NK cells comprising a nucleic acid construct.

[0011] The present invention further relates to said human T cells or NK cells for use in cellular immunotherapy.

[0012] A method for preparing human T cells or NK cells according to the present invention is also provided, wherein the human T cells or NK cells are 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 or NK cells when said T or NK cells are activated and upon exposure to a deficiency of at least one essential amino acid, and ii) a transgene placed under the control of said regulatory polynucleotide Transfected or transduced with a vector containing a nucleic acid construct comprising MODE FOR CARRYING OUT THE INVENTION

[0013] The GCN2-ATF4 signaling pathway is ubiquitous in cells, but depending on the tissue or organ, this pathway may not be activated by the nutrient regulation system. For example, deficiency of EAA cannot induce activation of the GCN2-ATF4 pathway in mouse muscle. The present inventors have shown that the GCN2-ATF4 pathway can be induced by EAA starvation in human T cells, but the first in vitro experiments failed, and the present inventors have also found that activation of human T lymphocytes is important for activation of GCN2 kinase. Thus, transgene expression is only possible when the T cells are activated and EAA deficiency induces activation of the GCN2-ATF4 pathway. These results are predicted to be transferable to human NK cells because NK cells are essentially cytotoxic T cells without TCR.

[0014] NUTRIREG-T cells or NK cells The present invention relates to a regulatory polynucleotide comprising i) a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, which is activated in a subject upon consumption of a diet lacking at least one essential amino acid, ii) a transgene placed under the control of the regulatory polynucleotide and a nucleic acid construct comprising the same, and relates to human T cells or NK cells.

[0015] The present invention also relates to human T cells or NK cells, which are i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, which is activated in the T or NK cells when the T or NK cells are activated and upon exposure to a deficiency of at least one essential amino acid, ii) a transgene placed under the control of the regulatory polynucleotide relating to human T cells or NK cells comprising a nucleic acid construct.

[0016] human T cells or NK cells, and a transgene In some embodiments, the human T cells of the present invention are chimeric antigen receptor T (CAR-T) cells (including CAR-Tregs), or T cell receptor (TCR) transgenic T cells.

[0017] In these embodiments, the transgene is selected to stimulate the efficacy of adoptive cell therapy by the CAR-T cells or transgenic TCR T cells, or to suppress the toxicity induced by the CAR-T cells or transgenic TCR T cells. In particular, the transgene is a transgene that prevents the depletion of CAR-T cells.

[0018] Suitable transgenes that stimulate the efficacy of adoptive cell therapy by CAR-T cells or transgenic TCR T cells, according to recent promising studies, include c-JUN, T-bet, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23 (to reverse or delay the depletion of T cells or to stimulate their modified T cells) (Poorebrahim, et al., Oncogene 40.2 (2021): 421-435; Pietrobon et al., International Journal of Molecular Sciences 22.19 (2021): 10828).

[0019] Suitable transgenes that suppress the toxicity induced by CAR-T cells or transgenic TCR T cells 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).

[0020] These human T cells according to the invention are particularly targeted at the treatment of cancers, such as hematological malignancies or solid tumors.

[0021] In some embodiments, the human T cells of the invention are targeted at allogeneic transplantation or at the treatment of blood cancers.

[0022] In a first aspect of these embodiments, the transgene is selected to promote the differentiation of human T cells into regulatory T cells (Tregs) or into type 1 regulatory T (Tr1) cells.

[0023] (i) Suitable transgenes that promote the differentiation of human T cells into 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) suitable transgenes that promote the differentiation of human T cells into Tregs mainly include FoxP3. Preferably, the transgene that promotes the differentiation of human T cells into Tr1 is a transgene encoding IL-10.

[0024] In this aspect, the expression of the transgene is induced when signs of GvHD are detected in order to inhibit or control GvHD.

[0025] In a second aspect of these embodiments, the human T cells intended for use in the treatment of blood cancer by allogeneic transplantation contain transgenes that stimulate the efficacy of cellular immunotherapy by 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 (Ringden, et al., British journal of haematology 147.5 (2009): 614-633).

[0026] In some embodiments, the human T cells of the present invention are intended for use in controlling allograft rejection in solid organ transplantation or in the treatment of autoimmune diseases. In these embodiments, the human T cells are human CAR-Tregs, more specifically antigen-specific CAR-Tregs, and the antigen is a transplantation-specific alloantigen or an autoantigen associated with an autoimmune disease.

[0027] Antigen-specific human CAR-Tregs can be produced by isolating polyclonal Tregs (derived from an individual's peripheral blood) and transducing them with an antigen-specific CAR construct, or by co-transducing CD4 + or CD3 + T cells with an antigen-specific CAR construct and FoxP3 cDNA (Arjomandnejad et al. Biomedicines 2022, 10(2), 287).

[0028] In these embodiments, the transgene is selected to stimulate the efficacy of Tregs and / or prevent the depletion of CAR-Tregs and includes c-JUN, T-bet, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23 as already described above.

[0029] In some embodiments, the human cells comprising a nucleic acid construct comprising a regulatory polynucleotide and a transgene are human NK cells. As used herein, the term NK cells includes modified NK cells such as NK cells and CAR-NK cells.

[0030] In these embodiments, the transgene is selected to stimulate the efficacy of cellular immunotherapy by the human NK cells or CAR-NK cells of the present invention. In particular, the transgene is a transgene that enhances the activation or proliferation of NK cells or CAR-NK cells.

[0031] 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.).

[0032] These human NK cells or CAR-NK cells according to the present invention are particularly targeted at the treatment of hematological malignancies or solid tumors.

[0033] Nucleic Acid Construct and Regulatory Nucleotide The nucleic acid construct i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, the regulatory polynucleotide being activatable or activated in a subject upon consumption of a diet lacking at least one essential amino acid, ii) a transgene placed under the control of the regulatory polynucleotide and comprises.

[0034] Alternatively, the nucleic acid construct 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 or NK cells when the T or NK cells are activated and upon exposure to a deficiency of at least one essential amino acid, and ii) A transgene placed under the control of said regulatory polynucleotide comprising.

[0035] Such nucleic acid constructs are described in international patent applications published as international patent publication nos. WO 2013 / 068096 and WO 2017 / 207744, which are incorporated herein by reference in their entirety.

[0036] The regulatory polynucleotide comprises a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence.

[0037] As used herein, "minimal promoter" is intended to mean a promoter that contains all the necessary elements for appropriately initiating transcription of the gene of interest located downstream. The expressions "minimal promoter" and "core promoter" are considered equivalent expressions. One of ordinary skill in the art understands that a "minimal promoter" includes at least one transcription start site, a binding site for RNA polymerase, and a binding site for general transcription factors (TATA box).

[0038] Suitable minimal promoters are known to those of ordinary skill in the art.

[0039] In some embodiments, the minimal promoter is selected from the group consisting of the promoter of thymidine kinase (TK), the promoter of β-globin, the promoter of cytomegalovirus (CMV), the SV40 promoter, and the like.

[0040] As used herein, "AARE" or "amino acid response element" refers to a nucleic acid sequence that is bound by activating transcription factor 4 (ATF4) after activation of the GCN2-eIF2α-ATF4 pathway due to deficiency of essential amino acids (EAAs), thereby inducing the expression of target genes driven by the AARE.

[0041] In mammals, after consumption of a diet lacking one EAA, the blood concentration of the limiting EAA rapidly and significantly decreases, inducing a universal adaptive process called the amino acid response pathway. The first step of this pathway is the activation of mammalian GCN2 protein kinase by uncharged tRNA. GCN2 then phosphorylates the α subunit (eIF2α) of eukaryotic initiation factor 2 at serine 51, leading to upregulation of ATF4 translation. Once induced, ATF4 activates the transcription of specific target genes via binding to AARE. The GCN2-eIF2α-ATF4 pathway can be rapidly halted by administration of the deficient EAA.

[0042] This pathway can also be observed at the cellular level. Indeed, the amino acid response pathway is also induced in activated T or NK cells exposed to deficiency of at least one EAA, such as when activated T or NK cells are cultured in a medium lacking at least one EAA.

[0043] In some embodiments, the amino acid response element (AARE) nucleic acid sequence is selected from the group consisting of nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

[0044] A regulatory polynucleotide comprising at least one AARE can comprise at least two, at least three, at least four, or at least five 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.

[0045] In certain embodiments, the regulatory polynucleotide comprises at least two AARE nucleic acid sequences. In some other embodiments, the regulatory polynucleotide comprises from 1 to 20 AARE nucleic acid sequences, preferably from 1 to 10 AARE nucleic acid sequences. In certain embodiments, the regulatory polynucleotide comprises from 2 to 6 AARE nucleic acid sequences.

[0046] In some embodiments, the regulatory polynucleotide comprises two AARE nucleic acid sequences selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4 of the nucleic acid sequences. In some embodiments, the regulatory polynucleotide comprises six AARE nucleic acid sequences of the sequence of SEQ ID NO: 1.

[0047] In certain embodiments, the at least two AARE nucleic acid sequences may be the same or different.

[0048] In one embodiment, the regulatory polypeptide comprises a thymidine kinase (Tk) minimal promoter and six copies of an AARE nucleic acid sequence derived from the TRIB3 gene, and comprises or consists of the sequence shown in SEQ ID NO: 6.

[0049] The regulatory polynucleotide construct thus comprises at least one AARE nucleic acid sequence located immediately upstream of a minimal promoter that controls the expression of a downstream transgene. The regulatory polynucleotide is activated in the T or NK cells of the subject upon consumption of a diet lacking at least one essential amino acid.

[0050] The subject is human or non-human, preferably human. In some embodiments, the non-human mammal is selected from the group consisting of mice, rats, etc.; primates such as chimpanzees, monkeys, etc.

[0051]

[0052] ​In mammals, nine EAAs must be supplied in the diet, and a deficiency of any one of these can induce an AARE-driven expression system.

[0053] As used herein, "essential amino acid" includes 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).

[0054] As used herein, "diet lacking at least one essential amino acid" is intended to mean a diet lacking 1, 2, 3, 4, 5, 6, 7, 8, or 9 essential amino acids. In some embodiments, the diet lacks leucine. .

[0055] A regulatory polynucleotide is activated in activated T 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.

[0056] The cells are human or non-human cells, preferably human cells. In some embodiments, the non-human mammal is selected from the group consisting of mice, rats, etc.; primates such as chimpanzees, monkeys, etc.

[0057] In cells, nine EAAs must be supplied in the diet, and a deficiency of any one of these can induce an AARE-driven expression system.

[0058] As used herein, "medium lacking at least one essential amino acid" is intended to mean a diet lacking 1, 2, 3, 4, 5, 6, 7, 8, or 9 essential amino acids. In some embodiments, the diet lacks leucine.

[0059] Activation of Regulatory Polynucleotides For the activation of the regulatory peptide, a diet lacking at least one essential amino acid is administered to a subject, i.e., a human or a non-human mammal as defined above.

[0060] In a preferred embodiment, the subject was in a starved state prior to being administered a diet lacking at least one essential amino acid.

[0061] For the activation of the 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 hours, at least 6 hours, at least 10 hours, and preferably for at least 16 hours.

[0062] Cell immunotherapy with NUTRIREG T cells or NK cells The human T cells or NK cells according to the invention are for use in cell immunotherapy.

[0063] Also provided is a cell immunotherapy for a subject in need thereof, comprising the step of administering the T cells or NK cells according to the invention to the subject.

[0064] Cell immunotherapy is directed to a subject that 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.

[0065] 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.

[0066] Cell immunotherapy includes, but is not limited to, treating cancer, controlling allograft rejection of solid organ transplants, or treating autoimmune diseases.

[0067] The cancer can be a solid cancer or a hematological cancer.

[0068] At some point, a subject administered with human T cells or NK cells further consumes a diet lacking at least one essential amino acid to induce transgene expression.

[0069] The advantage of the human cells according to the present invention, particularly the human T cells according to the present invention, is that the expression of the transgene is induced only by EAA deficiency (or activation of AARE nucleic acid) in activated human cells, particularly T cells.

[0070] This allows the expression of the transgene only when necessary by exposing the human T cells or NK cells of the present invention to EAA deficiency. In fact, in addition to being induced by the diet-inducible endogenous signaling pathway (GCN2-ATF4), one of the major advantages of the NUTRIREG-T cell or NK cell line is the possibility of on / off regulation of the transgene that controls this expression over time.

[0071] In one embodiment of the present invention, the T cells or NK cells of the present invention to be administered are not activated. The expression of the transgene is induced for the first time in the cells only when they are activated in the subject, particularly in the case of inflammation, and when the subject consumes a diet lacking at least one essential amino acid.

[0072] 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 first choice therapy for many hematological malignancies. Allogeneic donor T cells recognize minor histocompatibility antigens (minor H antigens), which results in the elimination of leukemia cells, a phenomenon called the graft-versus-leukemia effect (GvL). Since many minor H antigens are ubiquitously expressed, the GvL effect often involves the destruction of the patient's normal tissues, graft-versus-host disease (GvHD), which is a major side effect of allo-SCT.

[0073] The prophylactic or curative control of GvHD can be achieved by the human T cells of the present invention comprising a transgene that promotes the proliferation of human T lymphocytes having an anti-inflammatory profile in order to delay the cytotoxic response that causes GvHD. In this embodiment, donor T cells (present in the form of allogeneic hepatocytes transplanted in a subject in need thereof or in the form of donor lymphocyte infusion) are genetically modified ex vivo by the NUTRIREG system comprising a Treg or Tr1-inducing transgene prior to reinfusion into the subject. This enables (i) the expression of this Treg or Tr1-inducing transgene (usually an anti-inflammatory cytokine) in activated T cells, which are the cause of GvHD, and (ii) the polarization of the T response towards a T regulatory phenotype (Treg or Tr1) by the mere consumption of a few hours of a diet deficient in a certain EAA.

[0074] Conversely, in the event of recurrence of the subject's hematological disease, it may also be considered to boost the donor T lymphocytes again using a stimulatory cytokine that is transiently expressed under the dependence of EEA deficiency in order to regain the graft-versus-leukemia (GvL) effect.

[0075] Thus, in some embodiments, the human NUTRIREG T cells are for use in treating cancer, particularly hematological cancer, by allogeneic transplantation. Hematological cancers are particularly leukemia, lymphoma, or multiple myeloma.

[0076] In some embodiments, the human T cells are for use in treating hematological cancer by allogeneic transplantation, the expression of the transgene promotes the differentiation of human T cells into Treg or Tr1 and prevents or treats graft-versus-host disease (GvHD), or the expression of the transgene stimulates the efficacy of cellular immunotherapy by the human T cells that induce or stimulate the graft-versus-leukemia (GvL) effect.

[0077] In some embodiments, the transgene promotes the differentiation of human T cells into regulatory T cells (Tregs 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. Expression of the transgene that promotes the differentiation of human T cells into Tregs or Tr1 prevents or treats graft-versus-host disease (GvHD). In this embodiment, repeated induction of the transgene that promotes the differentiation of human T cells into Tregs or Tr1 is intended by the intermittent consumption of a diet lacking one essential amino acid (EAA).

[0078] Under the circumstances of controlling the cytotoxic response of GvHD, the on / off regulation enabled by the NUTRIREG-T cell line is an important advantage because the consumption of the deficient EAA will eventually stop the expression of the transgene that promotes the differentiation of human T cells into Tregs or Tr1, such as anti-inflammatory cytokines. The NUTRIREG system is therefore very beneficial because it allows for transient expression of the transgene and thereby preserves the anti-tumor effect of the graft, which is not possible with currently used immunosuppressive agents. Another advantage is that the transgene is expressed only in activated T cells because the GCN2-ATF4 pathway cannot be induced in resting T cells, which represents an important safety control.

[0079] In other embodiments, the transgene stimulates the efficacy of cellular immunotherapy by the human T cells to regain the graft-versus-leukemia (GvL) effect; for example, the transgene encodes stimulatory cytokines such as IL-2, IFN-γ, TNF-α, IL-7, and IL-15. Expression of the transgene that stimulates the efficacy of cellular immunotherapy by the human T cells induces or stimulates the GvL effect. In this embodiment, repeated induction of the transgene that stimulates the efficacy of cellular immunotherapy by the human T cells is intended by the intermittent consumption of a diet lacking one EAA when recurrence of a hematological disease is suspected in the subject.

[0080] A second application relates to the therapeutic strategy of the human CAR-T cells or TCR transgenic T cells of the invention for limiting their toxicity or, conversely, stimulating their activity using transgenes 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). The human CAR-T cells or TCR transgenic T cells according to the invention allow for transient expression of the gene of interest when preventing cell depletion of T cells. Promising results have been described in the literature for c-JUN, which, when overexpressed, renders CAR-T cells resistant to depletion (Lynn et al., Nature 576, 293‐300 (2019)). Transient and repeated expression of c-JUN under the dependence of NUTRIREG in CAR-T cells TCR transgenic T cells can therefore render it resistant to depletion and promote the maintenance of its efficacy over time. Some results are also promising for T-bet, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, for reversing or delaying T cell depletion or for stimulating modified T cells (Poorebrahim, et al., Oncogene 40.2 (2021): 421-435; Pietrobon et al., International Journal of Molecular Sciences 22.19 (2021): 10828).

[0081] Similarly, the therapeutic strategy can use the human NK cells or CAR-NK cells according to the invention for stimulating their activity by enhancing their activation or proliferation by transgenes such as NKG2D, IL-12, IL-15, or IL-18.

[0082] In the context of CAR-T cells, TCR transgenic T cells, NK cells, or CAR-NK cells, the on / off regulation enabled by the NUTRIREG system is also very important to enable both (i) having a useful effect on genes that are transiently expressed and (ii) limiting its potentially toxic effects (e.g., cJUN which may have carcinogenic effects) if it is expressed for too long.

[0083] Thus, in some embodiments, the adoptive cell therapy with the human CAR-T cells or TCR transgenic T cells, or human NK cells or CAR-NK cells of the present invention is for treating cancer, particularly solid tumors.

[0084] For cancer therapy, those skilled in the art specifically select human CAR-T cells, TCR transgenic T or CAR-NK cells that are specific for the tumor antigens of a subject having cancer.

[0085] In these embodiments, the transgene stimulates the efficacy of the adoptive cell therapy with the above human CAR-T cells, transgenic TCR T cells, NK cells, or CAR-NK cells, or suppresses the toxicity induced by the above CAR-T cells or transgenic TCR T cells. In particular, the transgene prevents the depletion of CAR-T cells or transgenic TCR T cells.

[0086] In some other embodiments, the adoptive cell therapy with the human CAR-T cells or transgenic T cells of the present invention is for treating autoimmune diseases. In particular, in autoimmune diseases caused by the production of autoantibodies, the human CAR-T cells or TCR transgenic T cells target autoantibody-producing B cells. In autoimmune diseases caused by cytotoxic T cells (e.g., colitis, multiple sclerosis, or type 1 diabetes), the human CAR-T cells or TCR transgenic T cells of the present invention target pathogenic autoimmune T cells.

[0087] In these embodiments, the human T cells are chimeric antigen receptor T (CAR-T) cells or T cell receptor (TCR) transgenic T cells. Depending on the therapeutic use, the CAR-T cells or TCR transgenic T cells are specific for autoantibodies or pathogenic autoimmune T cells. In these aspects, the transgene stimulates the efficacy of cellular immunotherapy by the above CAR-T cells or transgenic TCR T cells, or suppresses the toxicity induced by the above CAR-T cells or transgenic TCR T cells. In particular, the transgene prevents the depletion of CAR-T cells or the depletion of transgenic TCR T cells.

[0088] In some embodiments, the cellular immunotherapy is for controlling allograft rejection of solid transplants.

[0089] Furthermore, cell therapy with ex vivo-expanded autologous Tregs is one of the most promising approaches for modulating allogeneic immunity and reducing immunosuppression. Preclinical studies have shown that the efficacy of Treg therapy can be significantly enhanced by the use of Tregs specific for donor alloantigens (i.e., donor-specific (ds) Tregs). CAR-Treg technology has been used to generate dsCAR-Tregs, and these cells have been found to suppress humoral immunity and delay allograft rejection in naive immunocompetent recipients (Sicard et al., Am J Transplant. 2020;20:1562‐1573). Thus, in some embodiments, the cellular immunotherapy is for controlling allograft rejection by the human CAR-T cells or TCR transgenic T cells according to the present invention that are specific for donor alloantigens. In some aspects, the transgene extends the lifespan of the above donor-specific CAR-T cells or donor-specific transgenic TCR T cells and, as already described above, particularly prevents the depletion of dsCAR-T cells or the depletion of transgenic dsTCR T cells.

[0090] Method for preparing NUTRIREG T cells or NK cells A method for preparing human T cells or NK cells according to the present invention is provided, wherein the human T cells or NK cells are i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, which is activated in a subject upon consumption of a diet lacking at least one essential amino acid, the regulatory polynucleotide, ii) a transgene placed under the control of the regulatory polynucleotide and is transfected or transduced with a vector comprising a nucleic acid construct containing the same.

[0091] A method for preparing human T cells or NK cells according to the present invention is also provided, wherein the human T cells or NK cells are i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, which is activated in the T or NK cells when the T or NK cells are activated and upon exposure to a lack of at least one essential amino acid, for example when cultured in a medium lacking at least one essential amino acid, the regulatory polynucleotide, ii) a transgene placed under the control of the regulatory polynucleotide and is transfected or transduced with a vector comprising a nucleic acid construct containing the same.

[0092] The vector is a synthetic vector (cationic lipid, polymeric liposome, etc.), plasmid, or viral vector.

[0093] If desired, it may be combined with one or more substances that improve the efficacy and / or stability of vector transfection. These substances are widely discussed in the literature available to those skilled in the art (see, for example, 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, these can be polymers, cationic lipids, liposomes, nucleoproteins, or neutral lipids. These substances can be used alone or in combination. One possible combination is a recombinant plasmid vector combined with a cationic lipid (such as DOGS, DC-cholesterol, spermine-cholesterol, spermidine-cholesterol, etc.) and a neutral lipid (DOPE).

[0094] A wide selection of plasmids can be used in the context of the present invention. They can be cloning vectors and / or expression vectors. Generally, they are known in the art and many of them are commercially available, but it is also possible to construct or modify them using genetic engineering techniques. By way of example, the Applicants may 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 plasmids used in the present invention contain an origin of replication that ensures the initiation of replication in the producing cells and / or host cells (for example, the origin of CoIEI is used for plasmids produced in Escherichia coli, and the oriP / EBNAI system is used when it self-replicates in mammalian host cells, Lupton and Levine, 1985, Mol. Cell. Biol. 5, 2533-2542; Yates et al., Nature 313, 812-815). It may also contain a selectable gene for selecting or identifying the transfected cells (such as complementation of auxotrophic mutations, genes encoding resistance to antibacterial agents, etc.). It may also contain additional elements that improve its maintenance and / or its stability in a given cell.

[0095] When a viral vector is involved, it can be a vector derived from adenovirus, lentivirus, retrovirus, adeno-associated virus (AAV), herpes virus, alphavirus, parvovirus, poxvirus (fowlpox, canarypox virus, vaccinia virus, especially MVA (Modified Virus Ankara) or vaccinia virus of the Copenhagen strain, or a foamy virus). Preferably, a non-replicating and optionally non-integrating vector is used. Retroviruses have the property of preferentially integrating into infected and dividing cells, and thus are particularly suitable for applications in anti-cancer therapy. Suitable retroviral vectors for the practice of the present invention include LTR (long terminal repeat) terminal sequences and a capsid-forming region. It can be derived from retroviruses of any origin (mouse, primate, cat, human, etc.), and in particular, can be derived from a retrovirus selected from the group including MoMuLV (Moloney murine leukemia virus), MVS (mouse sarcoma virus), or Friend murine retrovirus (Fb29). It can be propagated in a capsid-forming line that can trans-provide the viral polypeptides of gag, pol, and / or env required for the constitution of viral particles. These types of lines are described in the literature (PA317, Psi CRIP GP+Am-12, etc.). The retroviral vectors of the present invention can include, in particular, modifications at the LTR (replacement of the promoter region by a eukaryotic promoter) or modifications at the capsid-forming region (for example, replacement by a heterologous capsid-forming region of VL30 species).

[0096] In a preferred embodiment of the present invention, the vector is a vector derived from a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV).

[0097] In a preferred embodiment, the viral vector used according to the present invention can be in the form of a DNA vector or an infectious viral particle.

[0098] The present invention is further illustrated by the following drawings and examples.

Brief Description of Drawings

[0099]

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Example

[0100] Example 1: Verification of the inducibility of the GCN2-ATF4 pathway in human T cells after EAA starvation (Figure 2) To enable the next use of NUTRIREG in human T cells, the applicants first verified that the GCN2-ATF4 pathway can be induced by EAA starvation in this cell type. Data from the literature have previously highlighted the activation of this signaling pathway in mouse T cells 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 only a few data were available regarding the GCN2-ATF4 pathway in response to AA deficiency in human T cells.

[0101] The applicants thus conducted in vitro experiments using human T cells purified from "buffy coats" manufactured by the French Blood Establishment. The applicants cultured these cells in control medium or in leucine-deficient medium.

[0102] The applicants showed the following (Figure 2): (1) The expression of a known target gene (TRB3) of the GCN2-ATF4 pathway can be significantly induced by 6 hours of EAA starvation (leucine or any other EAA) in T cells. (2) The induction of the GCN2-ATF4 pathway by short-term leucine starvation is rapidly reversible in T cells starting from 16 hours after the addition of leucine in the starvation medium. (3)Importantly, activation of T cells (by anti-CD3 and anti-CD28 antibodies in the presence of IL-2) is important for the induction of the GCN2-ATF4 pathway in response to EAA starvation. The fact that activation of T lymphocytes is important 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 information regarding human T lymphocytes was not available. (4)The induction of the expression of TRB3, a target gene of the GCN2-ATF4 pathway, in response to short-term leucine starvation is completely dependent on GCN2 kinase; this was demonstrated by the use of a pharmacological inhibitor of GCN2.

[0103] Example 2: Verification of the functionality of NUTRIREG in human T cells using a luciferase reporter gene (Figure 3). The inventors next evaluated the functionality 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 via a lentiviral vector. Transduction was performed 24 hours after activation of T cells with magnetic beads loaded with anti-CD2 / CD3 / CD28 antibodies in the presence of IL-2, followed by measurement of luciferase activity.

[0104] The inventors thus showed that 16-hour leucine starvation actually induces the expression of the luciferase transgene via NUTRIREG in T cells, compared to (i) transduced T cells cultured in control medium and (ii) untransduced T cells starved of leucine for 16 hours. This induction was detectable from 3 hours of leucine deprivation and the expression of the transgene increased as the duration of EAA deprivation increased (6h, 9h) (Figure 3).

[0105] Example 3: Verification of the functionality of NUTRIREG in human T cells in vitro using the eGFP reporter gene (Figs. 4-5). The second reporter gene evaluated was the eGFP gene, which was inserted into the 2xAARE-TK-eGFP construct and transduced via a lentiviral vector. Transduction was performed 24 hours after activation of T cells with magnetic beads loaded with anti-CD2 / CD3 / CD28 antibodies in the presence of IL-2. These cells were then leucine-starved 8 days after their activation, and the fluorescence emitted by eGFP was measured by flow cytometry.

[0106] The inventors showed that 16 hours of leucine starvation actually induced the expression of the eGFP transgene via NUTRIREG in T cells, compared to (i) transduced T cells cultured in control medium and (ii) untransduced T cells starved of leucine for 16 hours. This induction was not possible with shorter leucine starvation times, probably due to problems with the stability of the eGFP protein (Fig. 4).

[0107] Furthermore, analysis of the co-expression of T cell activation markers (CD69 as an early activation marker and CD25 as a late activation marker) and the eGFP protein in flow cytometry made it possible to confirm the important role of T cell activation in inducing the expression of the transgene under the control of NUTRIREG. Indeed, non-activated cells (CD25 - CD69 - ) did not express eGFP, moderately activated cells (CD69 - CD25 + or CD69 + CD25 - ) weakly expressed it, and strongly activated cells (CD69 + CD25 + ) were the cells that expressed the most eGFP protein (Fig. 5).

[0108] Example 4: Application of NUTRIREG-T cells with interleukin-10 (IL-10) therapeutic gene (Figs. 6-9) and in vivo results (Figs. 10-12). To use the NUTRIREG technology for therapeutic purposes in a model for the treatment or prevention of graft-versus-host disease (GvHD), the inventors designed a 2xAARE-BG-IL10 construct in which interleukin-10 (IL-10) cDNA and a 2XAARE sequence were inserted downstream of the minimal beta-globin (BG) promoter (Fig. 6). Indeed, transduction of T cells with IL-10 lentivirus has been shown in the literature to enable (i) directing the immune response towards a Tr1-type anti-inflammatory response (type 1 regulatory T cells), and (ii) preventing the onset of GvHD in a mouse model (Andolfi et al., Molecular Therapy 2012, vol. 20, 1778-1790; Locafaro et al., Molecular Therapy 25.10 (2017): 2254-2269).

[0109] There are also initial human clinical trials aimed at redirecting the immune response towards a Tr1 phenotype for the purpose of preventing GvHD. For this purpose, donor T cells are made anergic to host cells by culturing these donor T cells with host CD3-depleted mononuclear cells (PBMCs) in the presence of IL-10. The anergic T cells are then reinfused into the recipient a few weeks after allogeneic hematopoietic stem cell transplantation (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 TGFβ, GZMb, IFNγ, and IL-22. On the other hand, they do not express IL-2, IL4, and IL17, nor the FoxP3 transcription factor (Gregori et al., Frontiers in immunology 6(2015): 593.).

[0110] In the experiments of the present inventors, transduction with a lentivirus carrying the 2xAARE-BG-IL-10 construct was performed 24 hours after activation of T cells by magnetic beads filled with anti-CD2 / CD3 / CD28 antibodies in the presence of IL-2. The cells were then leucine-starved 8 days after their activation, and the expression of (i) IL-10 mRNA by RT-qPCR, (ii) IL-10 protein by ELISA or flow cytometry, and (iii) cytokines with different Tr1 phenotypes by RT-qPCR was measured (Figure 6).

[0111] The present inventors have thus shown that 16-hour leucine starvation actually induces the expression of the IL-10 transgene at the mRNA and protein levels via NUTRIREG, as compared to (i) transduced T cells cultured in control medium and (ii) untransduced T cells lacking leucine (Figures 7A - B).

[0112] The present inventors have also confirmed that the addition of leucine to the culture medium after starvation actually stops the expression of the transgene in transduced T cells at 24 hours for mRNA expression and at 48 hours for IL-10 protein expression.

[0113] Finally, the present inventors have shown that the transient expression of the IL-10 transgene via NUTRIREG can direct the immune response towards a Tr1-type response, as indicated by the cytokine and transcription factor profile (mRNA): IL-10 ++ GZMb + IL22 + IFNγ + FoxP3 - as shown by IL4, which was able to direct the immune response towards a Tr1-type response (Figure 9).

[0114] Finally, the inventors investigated the nature of a leucine-deficient diet that upregulates the expression of AARE-driven IL-10 in human T cells injected into mice. Indeed, the lack of any of the EAAs in the mammalian diet is a potential inducer of the expression of the AARE-transgene. Since the activation of T cells is required for the EAA-induction of the GCN2-ATF4 pathway, the inventors generated a mouse model and injected transduced human T cells conjugated with irradiated autologous human PBMCs. The inventors hypothesized that the transduced human T cells are transiently activated by the irradiated PBMCs and that this activation is sufficient to be induced by the nutritional deficiency of EAAs for the GCN2-ATF4 pathway. Briefly, immunodeficient NXG mice were injected intravenously with (i) human T cells transduced with a lentivirus carrying the 2xAARE-TK-IL-10 construct and (ii) irradiated human PBMCs, and then fed either a control diet or a diet lacking leucine once a week for 16 hours until week 3 (Figure 10). Weekly flow cytometry analysis of PBMCs enabled the accurate tracking of the proliferation of human CD45 + cells, and there was no significant effect of the leucine-deficient diet on cell proliferation or the CD4 + or CD8 + phenotype (Figure 11). Human IL-10 increased linearly over time in the leucine-deficient group (R 2 = 0.581; P = 0.004) and did not change significantly over time in the control group (Figure 12).

[0115] In summary, these results indicate that the NUTRIREG technology can regulate the expression of therapeutic genes such as IL-10 in activated human T cells in vitro and in vivo.

[0116] Example 5: Application of NUTRIREG-T cells by the FoxP3 transcription factor (Figures 13-14). Several publications in the literature have shown that transduction of T lymphocytes with the FoxP3 gene enables the phenotype of these T lymphocytes to be directed towards the Treg FoxP3 + phenotype. For example, injection of CD4 - / - CD25 + T cells (without constitutive expression of FoxP3 cells) transduced with a retrovirus carrying FoxP3 into immunodeficient RAG - mice can direct these cells towards the phenotypic CD4 + CD25 + and suppress the cytotoxic CD8 + response and prevent the onset of inflammatory colitis. These results in mice were confirmed in vitro in human cells by transduction with a lentivirus of FoxP3. Under this situation, the inventors overexpressed the transcription factor Foxp3 via NUTRIREG to temporarily and reversibly polarize T lymphocytes into the Treg phenotype. The inventors thus designed the 2xAARE-TK-FoxP3 (human) transgene and transduced it into activated (activated for 24 hours) human T lymphocytes. Eight days after transduction, these cells were transferred into control medium or leucine-deficient medium for 16 hours. The inventors thus observed that transduction of FoxP3 under the control of NUTRIREG enables the expression of this transgene to be induced after culture in leucine-deficient medium for 16 hours. This expression is induced at the transcriptional level (Figure 13A) and at the translational level (Figure 13B). This expression of FoxP3 is associated with an increase in the expression of the anti-inflammatory cytokine TGF-β (Figure 14), which is a characteristic presented by FoxP3 + Treg, as well as the cytotoxic molecules granzyme and perforin 1.

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 the T cell or NK cell when the T cell or NK cell is activated and upon exposure to a deficiency of at least one essential amino acid; and ii) A transgene placed under the control of the regulatory polynucleotide comprising a human T cell or NK cell.

2. The human T cell or NK cell according to claim 1, which is a human T cell, and 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 adoptive cell therapy by the CAR-T cell, transgenic TCR T cell, NK cell or CAR-NK cell, or the transgene suppresses the 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 the transgene reverses or delays the depletion of the CAR-T cell or the transgenic TCR T cell, or encodes an inhibitory receptor or an immunosuppressive factor.

6. The human T cell or NK cell according to claim 1, which is a human T cell for the purpose of allotransplantation or for the purpose of controlling allograft rejection of solid organ transplantation.

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

8. The human T cell according to claim 6, which is for the purpose of allotransplantation, and the transgene stimulates the efficacy of adoptive cell therapy by the human T cell.

9. a) The amino acid response element (AARE) nucleic acid sequence is selected from the group consisting of the sequences 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, The human T cell or NK cell according to any one of claims 1 to 8. **Claim 10** The human T cell or NK cell according to any one of claims 1 to 9 for use in cellular immunotherapy. **Claim 11** The human T cell or NK cell according to claim 10, wherein the cellular immunotherapy is for treating cancer, controlling allograft rejection of solid organ transplantation, or treating autoimmune diseases. **Claim 12** The human T cell or NK cell 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 for use in treating cancer. **Claim 13** A human T cell for use in treating hematological cancer by allotransplantation or for controlling allograft rejection of solid organ transplantation, wherein the human T cell is as defined in any one of claims 5 to 9, the human T cell or NK cell according to claim 10 or 11. **Claim 14** A human T cell for use in treating hematological cancer by allotransplantation, - the expression of the transgene promotes the differentiation of human T cells into Treg or Tr1 and prevents or treats graft-versus-host disease (GvHD), or - the expression of the transgene stimulates the efficacy of cellular immunotherapy using the human T cells that induce or stimulate the graft-versus-leukemia (GvL) effect, The human T cell or NK cell according to claim 12 or 13. **Claim 15** A human CAR-Treg for use in controlling allograft rejection of solid organ transplantation or for treating autoimmune diseases, the human T cell or NK cell according to claim 10, 11 or 13. **Claim 16** The human T cell or NK cell according to any one of claims 10 to 15, wherein the subject administered with the T cell or NK cell further undergoes a dietary therapy lacking at least one essential amino acid to induce the expression of the transgene in the activated human T cell or NK cell. **Claim 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 i) is a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, A regulatory polynucleotide that is activated in said T cells or NK cells upon activation of said T cells or NK cells and upon exposure to a deficiency of at least one essential amino acid, and ii) a transgene placed under the control of said regulatory polynucleotide is transfected or transduced with a vector comprising a nucleic acid construct comprising a method.

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