Enhanced NK cells for cancer immunotherapy

Abstract

Disclosed are isolated NK cells and / or an isolated population of NK cells having restored and / or enhanced autophagic activity in which transcriptional factor C / EBPp is stably or transiently inhibited in said cells and an in vitro or ex vivo method for obtaining isolated NK cells and / or an isolated population of NK cells having restored and / or enhanced and / or improved and / or increased and / or strengthened autophagic activity by pharmacological or genetic inhibition of transcription factor C / EBPp in NK cells with unaltered and / or dysfunctional autophagy, isolated NK cells and / or an isolated population of NK cells obtained by said method as well as their therapeutic uses, isolated NK cells and / or an isolated population of NK cells having restored and / or enhanced autophagic activity.

Description

[0001] ENHANCED NK CELLS FOR CANCER IMMUNOTHERAPY FIELD OF THE INVENTION

[0002] The present invention relates to the field of cancer immunotherapy, and in particular to NK cells (Natural killer cells) and an isolated population of said cells having restored and / or enhanced autophagic activity in which the transcriptional factor C / EBPp is stably or transiently inhibited in said cells, and an in vitro or ex vivo method for manipulating said NK cells (Natural killer cells) in order to restore and / or improve their killing capacity by acting on the autophagy process.

[0003] BACKGROUND OF THE INVENTION

[0004] Cancer immunotherapies, in particular adoptive T-lymphocyte transfer therapies, have shown promise in the treatment of various tumours. Since the action of such cells depends on the recognition of tumour antigens, the reduced expression of HLA molecules, necessary for antigen presentation, limits their effectiveness, just as the concomitant expression of immune checkpoints inhibits their activation. NK cells (Natural killer cells) are being studied as a therapeutic alternative, due to their ability to recognise and kill stressed or transformed cells without HLA restriction. Despite their potential, the efficacy of NK cells is often reduced in the immunosuppressive tumour microenvironment (TME). Autophagy plays a crucial role in immune cell function and requires regulation; however, its impact on NK cells is still poorly studied (references 1 to 30).

[0005] It is described in the literature that helenalin acetate (Helenalin acetate - HA) is an inhibitor of the transcription factor C / EBPp that can exert an anti-inflammatory and anti-tumour action, in particular in acute myeloid leukaemia (reference 31).

[0006] SUMMARY OF THE INVENTION

[0007] The inventors have now found that autophagy is fundamental for the activity that NK cells exert against the tumour. The same inventors have verified that tumour cells, by acting on the transcriptional factor C / EBPp (CCAAT enhancer binding protein, beta type), inhibit and / or reduce the autophagic function of NK cells. This occurs in nature in the so-called tumour microenvironment, where by tumour microenvironment (TME) is meant the cellular and extracellular environment around which the tumour exists and develops and which includes the surrounding blood vessels, the cells of the immune system that infiltrate the tumour, the connective cells and the extracellular matrix of the tumour (ECM). The inventors have verified that in vitro and ex vivo the reactivation of the autophagy process can take place through the inhibition of the transcriptional factor C / EBPp, so as to restore and / or improve the killing abilities of NK cells. Thus concluding that restoring the autophagy of NK cells with dysfunctional autophagy can be effectively employed in NK cellbased therapies.

[0008] At the same time, it is also possible to enhance autophagic activity by inhibition of the transcriptional factor C / EBPp in cells where said functionality is not altered, so as to obtain NK cells having improved and / or increased and / or enhanced and / or strengthened autophagy, which can be effectively employed in NK cell-based therapies.

[0009] The main object of the invention is therefore isolated NK cells and / or an isolated population of NK cells having restored and / or enhanced autophagic activity in which the transcriptional factor C / EBPp is stably or transiently inhibited in said cells. It is a further object of the present invention an in vitro or ex vivo method for restoring autophagy in isolated NK cells and / or an isolated population of NK cells characterised as having dysfunctional autophagy by inhibiting the transcriptional factor C / EBPp to obtain isolated NK cells and / or an isolated population of NK cells characterised as having restored and / or enhanced autophagy.

[0010] A further object of the invention is an in vitro or ex vivo method for improving and / or increasing and / or enhancing and / or strengthening autophagy in isolated NK cells and / or an isolated population of NK cells characterised as having non-dysfunctional autophagy by inhibiting the transcriptional factor C / EBPp to obtain isolated NK cells and / or an isolated population of NK cells characterised as having improved and / or increased and / or enhanced and / or strengthened autophagy.

[0011] Inhibition of the transcriptional factor C / EBPp may be pharmacological or genetic. A further object of the present invention is also a pharmaceutical composition comprising isolated NK cells and / or an isolated population of NK cells having restored autophagic activity obtained by an in vitro or ex vivo method that provides for pharmacological or genetic inhibition of the transcriptional factor C / EBPp in isolated NK cells and / or an isolated population of NK cells with dysfunctional autophagy.

[0012] A further object of the present invention is also a pharmaceutical composition comprising isolated NK cells and / or an isolated population of NK cells having improved and / or increased and / or enhanced and / or strengthened autophagic activity obtained by an in vitro or ex vivo method that provides for the pharmacological or genetic inhibition of the transcriptional factor C / EBPp in NK cells characterised as having non-dysfunctional autophagy. It is also an object of the present invention the isolated NK cells and / or the isolated population of NK cells having restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity obtained by the method of the invention or the pharmaceutical composition comprising said isolated NK cells and / or said isolated population of NK cells having restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity for use for cancer immunotherapy and for use in adoptive cell therapy (ACT).

[0013] It is a further object of the present invention the isolated NK cells and / or the isolated population of NK cells having restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity obtained by the method of the invention or the pharmaceutical composition comprising said isolated NK cells and / or said isolated population of NK cells having restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity for use as a medicament, in particular for use for the prevention and / or treatment of cancer. DETAILED DESCRIPTION OF THE INVENTION

[0014] Brief description of the drawings

[0015] Fig.1:Altered phenotype and attenuated cytotoxicity of NK cells in the prostate cancer microenvironment. Bubble diagram showing the average frequency of immune cell subpopulations in the prostate of Ptenpc+ / +and Ptenpc / _mice (n=4 mice / group).The frequency referred to the gate of the CD45+ living cells.

[0016] Fig.2:Altered phenotype and attenuated cytotoxicity of NK cells in the prostate cancer microenvironment. Cytofluorimetric analysis of the maturation status of NK cells in the prostate of Ptenpc+ / +and Ptenpc / _mice, evaluated using the CD11b marker in combination with the CD27 marker (n=10 mice / group).

[0017] Fig.3:Altered phenotype and attenuated cytotoxicity of NK cells in the prostate cancer microenvironment. Cytofluorimetric analysis of Granzyme B, Perforin and IFNy expression in the prostate of Ptenpc+ / +and Ptenpc / _mice (n=10 Ptenpc+ / +mice / group, n=6 Ptenpc / _mice / group).

[0018] Fig.4:Altered phenotype and attenuated cytotoxicity of NK cells in the prostate cancer microenvironment. Cytotoxicity of splenic NK cells selected from Ptenpc+ / +mice and tumour-infiltrating NK cells from Ptenpc / _mice against YAC-1 target cells at an effectortarget ratio (E:T) of 4:1 (NK cells from 3 mice / different conditions). Fig.5:Altered phenotype and attenuated cytotoxicity of NK cells in the prostate cancer microenvironment. Prostatic tumour volume of Ptenpc / _mice at the end of the NK cell depletion experiment, by treatment with aNK1.1 antibody. Tumour sizes were estimated using the formula:(WidthA2 x Length) 12. The sum of the sizes of the two anterior lobes was considered. n=5 mice / group.

[0019] Fig.6:Altered phenotype and attenuated cytotoxicity of NK cells in the prostate cancer microenvironment. Bubble diagram showing the average frequency of immune cell subpopulations in the prostate of Ptenpc / _mice treated with aNK1.1 antibody or isotype (n=5 mice / group). The frequency referred to the gate of the CD45+ living cells.

[0020] Fig.7:Autophagy is defective in tumour-infiltrating NK cells. Graph showing the frequency of NK cells in tumour samples versus non-tumour samples (n=10 samples / group) as determined by flow cytometry. The frequency referred to the gate of the CD45+ living cells.

[0021] Fig.8:Autophagy is defective in tumour-infiltrating NK cells. Graph showing the mean fluorescence intensity (MFI) of CYTO-ID in human NK cells in tumour samples versus non-tumour samples (n=10 samples / group).

[0022] Fig.10 Cytotoxicity of control or tumour medium-conditioned NK cells, in the presence or absence of metformin, against YAC-1 target cells (n=4 biological replicates).

[0023] Fig.11 Cytotoxicity of control or tumour medium-conditioned NK-92 cells, in the presence or absence of metformin, against PC3 target cells (n=8 biological replicates).

[0024] Fig.12 Volcano plot showing the variation of autophagy gene expression folds between tumour-conditioned and control NK cells (n=2 biological replicates).

[0025] Fig.13 Flow cytometry analysis of CXCR4+ cells in tumour-conditioned NK cells vs. control NK cells (n=7 biological replicates).

[0026] Fig.14 Flow cytometry analysis of CXCR4+ cells in prostate-infiltrating NK cells Ptenpc+ / +vs. Ptenpc / _(n=6 Ptenpc+ / +mice and n=5 Ptenpc / _mice).

[0027] Fig.15 Flow cytometry analysis of CXCR4+ cells in tumour tissue-infiltrating NK cells vs. non-tumour tissue-infiltrating NK cells (n=5 samples / group).

[0028] Fig 16 Cytotoxicity of NK-92 cells conditioned by tumour (CM) or control (CTRL), treated or untreated with Helenalin acetate (HA), against the MDA-MB-231 cell line at an effectortarget (E:T) ratio of 10:1. B) Cytotoxicity of tumour-conditioned PD-L1 CAR NK-92 cells or control, treated or untreated with Helenalin acetate, against the MDA-MB-231 cell line at an E:T ratio of 1:1. One-way ANOVA analysis with Tukey post-hoc test was used for A and B.C, D) Xenograft model of human breast carcinoma obtained by subcutaneous injection of 3x10® MDA-MB-231 cells into female NSG mice.3x106NK-92 cells, treated or untreated with HA, were injected intravenously twice weekly, starting from day 3 after tumour engraftment. Tumour size was measured simultaneously. Tumour growth is shown as mean tumour volume (C) and mean percentage growth (D). One-way ANOVA analysis with Tukey post-hoc test was used for C and D, comparing the area under the curve.

[0029] NK cells and the isolated population of NK cells have restored and / or enhanced autophagic activity as the transcriptional factor C / EBPp is inhibited in said cells, either stably or transiently.

[0030] A preferred embodiment is an in vitro or ex vivo method for obtaining isolated NK cells and / or an isolated population of NK cells having restored and / or enhanced autophagic activity by inhibiting the transcriptional factor C / EBPp in isolated NK cells and / or an isolated population of NK cells having unaltered and / or dysfunctional autophagy.

[0031] Preferably, said NK cells and / or isolated population of NK cells characterised by unaltered and / or dysfunctional autophagy are selected from the group consisting of: primary NK cells from peripheral blood samples, NK cells from autologous or allogeneic apheresis, NK cells from umbilical cord blood samples, NK cells generated from CD34+ haematopoietic progenitors, NK cells generated from induced pluripotent stem cells, NK cells generated from stem cells, NK cells generated from embryonic cells, NK-92 cell line.

[0032] The NK-92 cell line is approved for clinical application by the Food and Drug Administration (FDA).

[0033] Inhibition of the transcriptional factor may be pharmacological or genetic.

[0034] In the context of the present invention, pharmacological inhibition means exposing NK cells with unaltered and / or dysfunctional autophagy to an active ingredient that inhibits the transcriptional factor C / EBPp to restore and / or improve and / or increase and / or enhance and / or strengthen autophagy.

[0035] In the context of the present invention, NK cells with unaltered autophagy are understood to mean NK cells characterised by non-dysfunctional autophagy, due to inhibition of the transcriptional factor C / EBPp, either stably or transiently, or alternatively subjected to the method of the present invention, having improved and / or increased and / or enhanced and / or strengthened autophagy.

[0036] Within the scope of the present invention, genetic inhibition means any genetic manipulation that inactivates the transcriptional factor C / EBPp. In a preferred embodiment, the in vitro or ex vivo method allows obtaining isolated NK cells or an isolated population of NK cells having restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity by inhibiting the transcriptional factor by subjecting isolated NK cells and / or an isolated population of NK cells characterised by unaltered and / or dysfunctional autophagy to pharmacological or genetic inhibition of the transcriptional factor C / EBPp in order to reactivate and / or improve and / or increase and / or enhance and / or strengthen the autophagic activity in said cells and / or cell population.

[0037] In a further embodiment of the present invention, the in vitro or ex vivo method for obtaining isolated NK cells and / or an isolated population of NK cells having restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity comprises the following steps:

[0038] a) Collect an isolated sample of NK cells having unaltered and / or dysfunctional autophagy;

[0039] b) Cultivating the isolated sample referred to in step a) in a cell culture medium supplemented with an inhibitor of the transcriptional factor C / EBPp for a time sufficient to restore and / or improve and / or increase and / or enhance and / or strengthen the autophagic activity of NK cells;

[0040] a) Collecting the isolated NK cells and / or an isolated population of NK cells with restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity obtained at the end of step b).

[0041] Preferably, in step a) the NK cells with unaltered and / or dysfunctional autophagy are selected from the group consisting of: primary NK cells from peripheral blood samples, NK cells from autologous or allogeneic apheresis, NK cells from umbilical cord blood samples, NK cells generated from CD34+ haematopoietic progenitors, NK cells generated from induced pluripotent stem cells, NK cells generated from stem cells, NK cells generated from embryonic cells, NK-92 cell line.

[0042] At the end of the process, isolated NK cells with restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity are thus obtained, preferably selected from the group consisting of: isolated NK cells obtained from primary NK cells with unaltered and / or dysfunctional autophagy from peripheral blood samples, isolated NK cells obtained from NK cells with unaltered and / or dysfunctional autophagy from autologous or allogeneic apheresis, isolated NK cells obtained from NK cells with unaltered and / or dysfunctional autophagy from umbilical cord blood samples, isolated NK cells obtained from NK cells with unaltered and / or dysfunctional autophagy generated from CD34+ haematopoietic progenitors, isolated NK cells obtained from NK cells with unaltered and / or dysfunctional autophagy generated from induced pluripotent stem cells, isolated NK cells obtained from NK cells with unaltered and / or dysfunctional autophagy generated from stem cells, isolated NK cells obtained from NK cells with unaltered and / or dysfunctional autophagy generated from embryonic cells, isolated NK-92 cells obtained from NK-92 cells with unaltered and / or dysfunctional autophagy. Alternatively, isolated cell lines of NK cells with restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity are obtained, selected from the group consisting of: isolated cell line of NK cells obtained from primary NK cells with unaltered and / or dysfunctional autophagy from peripheral blood samples, isolated cell line of NK cells obtained from NK cells with unaltered and / or dysfunctional autophagy from autologous or allogeneic apheresis, isolated cell line of NK cells obtained from NK cells with unaltered and / or dysfunctional autophagy from umbilical cord blood samples, isolated NK cell line obtained from NK cells with unaltered and / or dysfunctional autophagy generated from CD34+ haematopoietic progenitors, isolated NK cell line obtained from NK cells with unaltered and / or dysfunctional autophagy generated from induced pluripotent stem cells, isolated NK cell line obtained from NK cells with unaltered and / or dysfunctional autophagy generated from stem cells, isolated NK cell line obtained from NK cells with unaltered and / or dysfunctional autophagy generated from embryonic cells, isolated NK-92 cell line obtained from NK-92 cells with unaltered and / or dysfunctional autophagy.

[0043] In the context of the present invention, ex vivo means that the method is performed on cells isolated from a subject.

[0044] In the case of NK-92 cells, these cells represent a cell line.

[0045] In one embodiment of the present invention, isolated NK cells and / or an isolated population of NK cells having restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity are obtained by inhibiting the transcriptional factor C / EBPp by exposing NK cells with unaltered and / or dysfunctional autophagy to an inhibitor of the transcriptional factor C / EBPp.

[0046] In one embodiment of the present invention, the isolated population of NK cells having restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity can be obtained by a method comprising the following steps: a) Collect an isolated sample of NK cells having unaltered and / or dysfunctional autophagy;

[0047] b) Cultivating the isolated sample according to step a) in a cell culture medium supplemented with an inhibitor of the transcriptional factor C / EBPp for a time sufficient to restore and / or improve the autophagic activity of NK cells; c) Collecting the isolated NK cells or an isolated population of NK cells with restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity obtained at the end of step b).

[0048] Preferably, in step a) the NK cells with unaltered and / or dysfunctional autophagy are selected from the group consisting of: primary NK cells from peripheral blood samples, NK cells from autologous or allogeneic apheresis, NK cells from umbilical cord blood samples, NK cells generated from CD34+ haematopoietic progenitors, NK cells generated from induced pluripotent stem cells, NK cells generated from stem cells, NK cells generated from embryonic cells, NK-92 cell line.

[0049] In the context of the present invention, Natural Killer (NK) cells are understood to be innate lymphoid cells that mediate resistance against pathogens and contribute to the activation and orientation of adaptive immune responses, mediate resistance against haematopoietic neoplasms and are involved in the carcinogenesis of solid tumours.

[0050] In the context of the present invention, NK cells with both unaltered autophagy and dysfunctional autophagy are understood to mean NK cells that can be obtained from various sources, including primary NK cells from peripheral blood or apheresis products (autologous or allogeneic) and umbilical cord blood. NK cells can also be generated from CD34+ haematopoietic progenitors, induced pluripotent stem cells, embryonic stem cells, and cell lines, including the NK-92 cell line, approved for clinical application by the Food and Drug Administration (FDA).

[0051] According to the method of the invention, NK cells with unaltered and / or dysfunctional autophagy are exposed to a pharmacological inhibitor of the transcriptional factor C / EBPp, and by exposed it is meant that the cells are brought into contact with an inhibitor of the transcriptional factor C / EBPp.

[0052] The pharmacological inhibitor of the transcriptional factor C / EBPp may be any molecule and / or compound and / or active ingredient capable of inactivating the transcriptional factor, and mixtures thereof.

[0053] The pharmacological inhibitor may also be any antibody that inactivates the transcriptional factor C / EBPp and mixtures thereof. Preferably, the pharmacological inhibitor is selected from the group consisting of: i) molecules that inhibit the interaction between the transcription factor C / EBPp and its co-activator p300, such as, for example, Helenalin acetate, which is a naturally derived molecule that binds the N-terminal part of C / EBPp, thus interrupting the cooperation of C / EBPp with the coactivator p300, or, for example, Withaferin A, which is a natural compound that inhibits the transcription factor C / EBPp by covalent binding with specific cysteine residues and consequent inhibition of the interaction with the co-activator p300, or, for example, celastrol, which is a natural compound that inhibits the activity of C / EBPp by interrupting its interaction with the Taz2 domain of the p300 co-activator, or, for example, any synthetic mimetic molecule of Helenalin that acts as a covalent inhibitor of C / EBPp by binding to cysteine residues in the transactivation domain by inhibiting the activation of C / EBPp by the p300 co-activator, as well as its derivatives, i.e. molecules synthesised from said prototype molecule characterised by having an inhibitory effect on C / EBPp analogous to and / or greater than that of the prototype molecule;

[0054] ii) molecules that inhibit the transcriptional activity of C / EBPp by a mechanism other than that mentioned above, such as quercetin, which is a flavonoid that binds the protein C / EBPp, inhibiting its transcriptional activity;

[0055] iii) molecules that cause the degradation of the transcription factor C / EBPp, such as ST101, which is an antagonist peptide of C / EBPp, which binds its leucine zipper domain, preventing its dimerization and increasing its proteasome-dependent degradation;

[0056] iv) TRAnscription Factor TArgeting Chimeras (TRAFTACs), which are hetero-bifunctional molecules consisting of a chimeric oligonucleotide that binds simultaneously to the transcription factor of interest and to the dCas9 protein fused with HaloTag to induce the degradation of the transcription factor through the proteasomal pathway;

[0057] v) Oligonucleotide-based PROTACs (Oligo-PROTACs), a class of non- canonical PROTACs (proteolysis targeting chimeras) that use a decoy consisting of a double-stranded oligonucleotide capable of binding to the binding domain between DNA and the transcription factor; vi) TF-PROTACs, a class of PROTACs that use a DNA oligonucleotide linked to an E3 ligase ligand via a click chemistry reaction, to selectively degrade the transcription factor of interest.

[0058] In one preferred embodiment, the transcriptional factor C / EBPp inhibitor is Helenalin acetate (HA).

[0059] Within the scope of the present invention, genetic inhibition means any genetic manipulation that has the effect of activating autophagy, and preferably that inactivates the transcriptional factor C / EBPp.

[0060] Inactivation by genetic manipulation which results in knockout of the transcriptional factor C / EBPp can take place by means of any gene silencing method such as CRISPR / Cas9, TALENs, siRNA, shRNA, or genetic manipulation which inactivates the transcriptional factor C / EBPp is carried out by CRISPR / Cas9-mediated genome editing to obtain knockout of the transcriptional factor C / EBPp.

[0061] Preferably, the pharmacological or genetic inhibitor of the transcriptional factor C / EBPp is added to the cell culture medium.

[0062] Cell culture medium, within the scope of the present invention, is any cell culture medium known to the person skilled in the art for the growth of NK cells and in particular NK cells. The cell culture medium may further optionally comprise suitable components and elements which are commonly used for the growth of NK cells and in particular of NK cells according to the general knowledge in the field, such as fetal bovine serum (FBS), antibiotics, such as penicillin-streptomycin, and / or amino acids, such as L-glutamine.

[0063] NK cells with unaltered and / or dysfunctional autophagy can be exposed to the transcriptional factor C / EBPp inhibitor for a suitable time that can be chosen by the person skilled in the art or, in stage b), the cells are cultured until confluence, where confluence means growth until reaching a cell density such as to completely cover the culture substrate.

[0064] NK cells with dysfunctional autophagy subjected to the method of the present invention may previously be isolated from tumour tissue, for example, isolated from a subject, human or animal, according to common methods known in the art, preferably human.

[0065] NK cells with unaltered and / or dysfunctional autophagy may be isolated from peripheral blood and / or tumour tissue of the same subject treated or from a different subject. Isolated NK cells and / or an isolated population of NK cells having restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity obtained by the method of the invention can be included in a composition, such as a pharmaceutical composition.

[0066] The present invention therefore also relates to a pharmaceutical composition comprising isolated NK cells and / or an isolated population of NK cells having restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity obtained by the method of the invention and at least one pharmaceutically acceptable carrier and / or vehicle and / or pharmacologically acceptable excipients.

[0067] The carrier and / or vehicle may be any of those conventionally used for the administration of cells. Pharmaceutically acceptable carriers and vehicles and pharmacologically acceptable excipients are well known to those skilled in the art and are commercially available.

[0068] The cells and / or the population of cells and / or the pharmaceutical composition of the invention may be administered in any suitable manner. In one embodiment they are administered as an injectable formulation. The requirements for effective pharmaceutically acceptable carriers for injectable compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, ed., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4thed., pages 622-630 (1986). Methods for preparing administrable compositions (e.g., parenterally administrable) are known or will be apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science (17thed., Mack Publishing Company, Easton, PA, 1985).

[0069] The dose of the cells to be administered, e.g. the number of cells, is an amount sufficient to achieve the desired effect, in particular the prevention or treatment or control of cancer. The physician may determine a suitable amount depending on the severity of the disease, the patient's condition, and other suitable parameters according to general knowledge in the art.

[0070] The invention comprises the isolated cells and / or the isolated population of cells and / or obtained by the method of the present invention and / or the pharmaceutical composition comprising them for use as a medicament.

[0071] The invention comprises the isolated cells and / or the isolated population of cells and / or obtained by the method of the present invention and / or the pharmaceutical composition comprising them for use for anti-tumour immunotherapy and for use in adoptive cell therapy (ACT).

[0072] The invention comprises the isolated cells and / or the isolated population of cells and / or obtained by the method of the present invention and / or the pharmaceutical composition comprising them for use for the prevention and / or treatment of cancer. According to the present invention, by "prevention" is meant that administration of the agent reduces the likelihood of developing a disease or condition, i.e. decreases the likelihood of developing cancer. In some embodiments, by "prevention" is meant that administration of the agent arrests or slows the progression of a disease that has already begun. For example, in some embodiments the agent of the invention is administered to a subject who already has cancer and the cancer does not develop to a more advanced stage.

[0073] According to the present invention, "treatment" means that administration of the agent ameliorates or cures or reverses a condition or disease, i.e., ameliorates or cures or reverses cancer. In some embodiments, by “treatment” is meant that the disease, such as cancer, is not completely cured but returns to a less advanced stage. Any amount of any level of treatment or prevention of cancer may be provided by administration of the cells of the invention. In addition, the treatment or prevention provided by the invention can include treatment or prevention of one or more conditions or one or more symptoms of the disease, e.g., cancer, to be treated or prevented.

[0074] By "adoptive cell therapy" is meant a type of immunotherapy in which NK cells having restored and / or enhanced autophagic activity are administered to a patient to help the body fight a disease, such as cancer. In adoptive cell therapy for cancer, NK cells are usually isolated from the patient's blood or tumour tissue, cultured in the laboratory, and then administered to the patient to help the immune system fight the cancer. Adoptive cell transfer, adoptive cell immunotherapy, and NK cell transfer therapy are synonymous.

[0075] Cells administered to a subject in need thereof may be allogeneic or autologous for the host.

[0076] The cancer can be any type of cancer, including any of the sarcomas (e.g., synovial sarcoma, osteogenic sarcoma, uterine leiomyosarcoma, and alveolar rhabdomyosarcoma), lymphomas (e.g., Hodgkin lymphoma and non-Hodgkin lymphoma), hepatocellular carcinoma, glioma, head and neck cancer, acute lymphocytic cancer, acute myeloid leukaemia, bone cancer, brain cancer, breast carcinoma, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck, gallbladder, or pleural cancer, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, vulvar cancer, chronic lymphatic leukaemia, chronic myeloid cancer, colon cancer (e.g., colon carcinoma), oesophageal cancer, cervical cancer, gastrointestinal carcinoid tumour, hypopharyngeal tumour, laryngeal tumour, liver tumour, lung tumour, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, peritoneal, omentum, and mesenteral cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and urinary bladder cancer.

[0077] Preferably they are solid tumours, more preferably bladder cancer.

[0078] Further active ingredients may be administered to the subject together with the isolated NK cells and / or an isolated population of NK cells having restored and / or improved autophagic activity obtained by the method of the invention.

[0079] Therefore, a further subject of the invention is the aforementioned pharmaceutical composition optionally comprising and further comprising at least one active ingredient.

[0080] Preferably, such additional active ingredient is a chemotherapeutic agent.

[0081] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Any chemotherapeutic agent known in the art may be used, in particular agents which are known to be effective in the cancer to be treated or which are known to potentiate NK cell activity.

[0082] When the cells of the invention are administered with one or more additional therapeutic agents, one or more additional therapeutic agents can be coadministered to the mammal. By “coadministering” is meant administering one or more additional therapeutic agents and the cells of the invention sufficiently close in time such that the cells of the invention can enhance the effect of one or more additional therapeutic agents. In this regard, the cells of the invention can be administered first and the one or more additional therapeutic agents can be administered second, or vice versa. Alternatively, the cells of the invention and the one or more additional therapeutic agents can be administered simultaneously. In light of the above detailed description and with reference also to the appended claims the invention therefore relates to:

[0083] An in vitro or ex vivo method for restoring and / or re-establishing or improving and / or increasing and / or enhancing and / or strengthening autophagic activity and obtaining isolated cells and / or an isolated population of NK cells having restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity by pharmacological or genetic inhibition of the transcriptional factor C / EBPp in NK cells with unaltered and / or dysfunctional autophagy.

[0084] In one embodiment of said method, the inhibition is pharmacological and therefore the method comprises the following steps:

[0085] a) collecting an isolated sample of NK cells with unaltered and / or dysfunctional autophagy;

[0086] b) culturing the isolated sample referred to in step a) in a cell culture medium supplemented with an inhibitor of transcriptional factor C / EBPp for a time such as to restore and / or restore or improve and / or increase and / or enhance and / or strengthen the autophagic activity of NK cells; c) Collect the isolated NK cells or an isolated population of NK cells with restored and / or improved autophagic activity obtained at the end of step b).

[0087] Preferably, the pharmacological inhibitor is at least one molecule and / or compound and / or active ingredient capable of inactivating the transcriptional factor C / EBPp and mixtures thereof and / or at least one antibody that inactivates the transcriptional factor C / EBPp.

[0088] In said method, alternatively, the inhibition is genetic and is obtained by any genetic manipulation technique or method resulting in the inactivation of the transcriptional factor C / EBPp, preferably gene silencing or genome editing.

[0089] The invention also relates to isolated NK cells or an isolated population of NK cells having re-established and / or restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity in which the transcriptional factor C / EBPp is stably or transiently inhibited in said cells.

[0090] In isolated NK cells or an isolated population of NK cells having re-established and / or restored and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity in which the transcriptional factor C / EBPp is stably or transiently inhibited in said cells, the transient or stable inhibition of the transcriptional factor C / EBPp is genetic or pharmacological.

[0091] When the transient or stable inhibition of the transcriptional factor C / EBPp is pharmacological, preferably the pharmacological inhibitor is at least one molecule and / or compound and / or active ingredient capable of inactivating the transcriptional factor C / EBPp and mixtures thereof and / or at least one antibody that inactivates the transcriptional factor C / EBPp.

[0092] When the transient or stable inhibition of the transcriptional factor C / EBPp is genetic, it is achieved by any genetic manipulation technique or method which results in the inactivation of the transcriptional factor C / EBPp, for example gene silencing or genome editing.

[0093] In one embodiment, said cells are obtained from NK cells with unaltered and / or dysfunctional autophagy by said method.

[0094] The invention also relates to a pharmaceutical composition comprising said cells and / or population of cells and at least one pharmaceutically acceptable carrier and / or vehicle and / or pharmacologically acceptable excipients, as well as their use as a medicament, for anti-tumour immunotherapy, for use in adoptive cell therapy (ACT), for use in the prevention and / or treatment of cancer.

[0095] The invention will now be described with the aid of illustrative examples.

[0096] EXAMPLES

[0097] In the prostate, NK cells show significant dysfunction, although the mechanisms behind this alteration are not fully understood. Characterization of the immune infiltrate of a murine model of prostate cancer revealed a marked decrease in the number of NK cells within the TME and a marked increase in the subpopulation of NK cells characterized by a more immature state and with lower lytic capacities. In addition, tumour NK cells have lower levels of Granzyme B and Perforin, and a decreased ability to kill tumour cells when tested in vitro. Surprisingly, the depletion of NK cells in already established tumours does not lead to a significant alteration of tumour growth or TME characteristics, suggesting that NK cells acquire a dysfunctional state in step with tumour progression (Fig.1-6).

[0098] Further investigation into the mechanisms of NK cell dysfunction revealed that autophagy is significantly deregulated in these cells. In fact, we analysed samples of human prostate cancer and adjacent non-tumour tissue, and confirmed that the abundance of NK cells is lower in the tumour than in control tissue (Fig.7). In addition, scRNA-seq analysis of prostate samples showed alterations in biological processes related to stress response and autophagy in tumour NK cells. This data was validated by cytofluorimetric analysis in human tumours and in a mouse model (Fig.8-9). In vitro experiments then revealed that the activation of autophagy, in NK cells exposed to tumour, results in a reactivation of the same. In fact, activation of autophagy with Metformin in NK cells improved their ability to kill target tumour cells (Fig.10-11).

[0099] To understand the mechanisms behind the observed decreased autophagy, we assessed the expression of autophagy-associated genes in NK cells exposed to prostate tumour cell-conditioned medium. In this experiment we observed a significant inhibition of the expression of various genes that positively regulate autophagy, including Irgm, Mapk8 and Tp53 (Fig.12), as well as molecules involved in the lysosomal degradation process. In contrast, this analysis highlighted the positive modulation of a limited number of molecules, including Fadd, Igf1, Ins2 and the chemokine receptor CXCR4 (Fig.12). Upon further analysis, CXCR4 expression was increased in murine NK cells (Fig.13-14) and human NK cells exposed to tumour.

[0100] To verify the role of CXCR4 in the decrease of autophagy observed in tumour NK cells, we applied a scRNA-seq strategy to analyse the tumour infiltrate of murine prostate tumours. Analysis of intercluster interactions through CellphoneDB predicted the CXCR4-CXCL12 axis as an existing interaction between tumour cells and NK cells. Analysis through NicheNet revealed that C / EBPp is a potential transcription factor activated by the CXCL12 / CXCR4 axis.

[0101] Transmission electron microscopy (TEM) analysis of control and tumour-exposed NK cells was performed, and investigation of mitochondrial architecture revealed the loss of outer membrane integrity, widespread cristolysis, and matrix disruption in NK cells exposed to the tumour medium. Overall, these data indicate that NK cells exposed to the tumour secretome accumulated damaged mitochondria, as confirmed by FACS staining, which revealed a reduced percentage of cells containing functional mitochondria and a concomitant increase in cells accumulating dysfunctional mitochondria. As a result, exposure to the tumour led to the alteration of multiple parameters associated with mitochondrial fitness, including mitochondrial membrane potential (Ai m), as assessed by TMRM staining, mitochondrial production of reactive oxygen species, as assessed by MitoSox staining, and intracellular lactate levels. To assess whether impairment of autophagy is associated with mitochondrial damage in tumour NK cells, we performed FACS analysis of NK92 cells in which Beclin-1 (BECN1 OE NK92) protein expression was induced, in the presence and absence of tumour medium. BECN1 OE NK-92 cells showed higher TMRM levels when exposed to the tumour secretome, compared to BECN1 WT cells. Furthermore, genetic activation of autophagy by overexpression of BECN1 prevented the accumulation of mitochondrial ROS in tumour-conditioned NK cells, suggesting that autophagy may regulate oxidative stress by removing damaged mitochondria that produce ROS.

[0102] A CXCR4 knockout NK-92 cell line (CXCR4 KO) was generated by CRISPR / Cas9-mediated genome editing. The lack of the CXCR4 receptor induces in NK cells a marked resistance to the tumour-mediated inhibitory effect on both autophagic flux and cytolytic functions. The abrogation of CXCL12 inhibitory effects in CXCR4 KO cells confirmed the role of the CXCR4 receptor in the regulation of both autophagy and lytic capacity. NK-92 CXCR4 KO cells were used in vivo and showed both an increased ability to control tumour growth and resulted in a marked reduction in tumour volume.

[0103] The above data suggest that CXCR4 is increased in expression on NK cells that have been exposed to factors released by tumour cells, and that activation of this receptor leads to dysfunctional autophagy in NK cells, through the involvement of the transcriptional factor C / EBPp. To confirm this prediction, exposure of NK cells to the supernatant of tumour cells is able to activate the transcription factor C / EBPp in vitro, and this activation is abolished by administration of the drug Plerixafor, which blocks the CXCR4 receptor. It has also been demonstrated in vitro that a specific compound, Helenalin acetate (HA), capable of inhibiting C / EBPp activation, activates the autophagic programme and improves the killing capacity of NK cells. To confirm this, we developed an in vivo prostate cancer model, based on subcutaneous injection of prostate cancer cells (PC3 cells). The mice that develop the tumour are then exposed to successive infusions of NK cells, to mimic the NK-based cell therapy, under clinical investigation in patients with cancer. In this experiment, NK-92 cells, a human NK cell line, were exposed to Helenalin acetate. The compound was then washed, and the cells resuspended in cell culture medium were infused into the animals. Treatment of NK-92 cells with HA leads to a significant improvement in tumour growth control. Importantly, the number of tumour-infiltrating NK cells increases upon exposure to HA, and the autophagy of these infiltrating cells is increased compared to the control. These data demonstrate that in prostate cancer, NK cells are dysfunctional due to activation of the CXCR4 receptor, which, once engaged, activates the transcriptional factor C / EBPp. Activation of C / EBPp results in dysfunctional autophagy, and consequent inactivation of the lytic capacity of NK cells. Inhibition of C / EBPp in NK cells restores their functional capacity in the tumour and increases the efficacy of NK-based cell therapy against prostate cancer. To confirm previous results on the inhibitory effect of Helenalin acetate (HA) on C / EBPp activity in NK cells in different tumour settings, the impact on NK cell functionality was tested using a breast carcinoma model. In particular, both unmodified NK-92 cells and NK-92 cells engineered with PD-L1 CAR were used, together with MDA-MB-231 breast carcinoma cells as a tumour model in vitro and in vivo.

[0104] The cytotoxic activity of NK-92 cells cultured under control (CTRL) or tumour-conditioned (CM) conditions, with or without HA treatment, was initially assessed compared to MDA-MB-231 target cells at an effectortarget (E:T) ratio of 10:1. Tumour-conditioned NK-92 cells exhibited reduced cytotoxicity compared to control cells, while HA treatment significantly restored their ability to kill tumour cells (Fig.9A). Similarly, PD-L1 CAR NK-92 cells were tested under control conditions or tumour-conditioned with an E:T ratio of 1:1. Consistently, exposure to the tumour-conditioned medium impaired their cytotoxic activity, while HA treatment restored their anti-tumour function (Fig.9B).

[0105] Further, to evaluate the in vivo efficacy of HA-treated NK-92 cells, a xenograft model of human breast carcinoma was established by subcutaneously injecting MDA-MB-231 cells into female NSG mice. NK-92 cells, whether or not treated with HA, were administered intravenously twice weekly starting from the third day after tumour implantation. Tumour growth was monitored overtime and reported as mean tumour volume and percentage growth. Treatment with HA greatly improved the anti-tumour activity of NK-92 cells, resulting in a significant reduction in tumour growth compared to untreated NK-92 cells (Fig.9C, 9D). Statistical analysis was performed using ANOVA with Tukey's post-hoc test.

[0106] These results confirm that inhibition of C / EBPp by Helenalin acetate enhances the anti-tumour activity of both conventional and CAR-engineered NK cells, supporting its potential as an immunomodulatory strategy in different tumour settings.

[0107] Materials and methods

[0108] Experimental model, animals and ethical statement

[0109] All mice used in this study were housed in individually ventilated cages in a specific pathogen-free (SPF) animal housing facility of the Humanitas Clinical and Research Centre (Rozzano, Milan, Italy). Procedures related to animal handling and care were conducted according to protocols approved by the Humanitas Clinical and Research Centre, in accordance with national and international legislation and policies. The study was approved by the Italian Ministry of Health. Efforts were made to minimise the number of animals used and their suffering, in accordance with the 3R principle. Ptenpc / _mice were obtained by crossing ptenloxp / loxpmice with Probasin-Cre transgenic mice, to obtain a specific deletion of Pten in the prostate. Male C57BL / 6 mice were purchased from Charles River Laboratories or are siblings of Ptenpc / _mice, not having the Pten gene deletion. Seven-week-old NSG male mice (NOD.Cg-Prkdcscid||2rgtm1SzJ) were purchased from Charles River Laboratories.

[0110] Cell lines

[0111] Prostate cancer cell lines, including the murine cell line PTEN-Cap8 (Pten_ / “), and the human cell lines PC3 and LNCaP were obtained from ATCC. The prostate cancer cell line Pten_ / “ Trp53_ / “ was provided by Prof. Ronald De Pinho, MD Anderson. Cells were maintained regularly in RPMI medium supplemented with 10% heat-inactivated FBS, 1% penicillin / streptomycin solution, 1% L-glutamine and 1% sodium pyruvate solution at 37°C in 5% CO2.For the culture of Pten_ / “ prostate cancer cells, 25 pg / ml of bovine pituitary extract, 5 pg / ml of recombinant human insulin and 6 ng / ml of recombinant human epidermal growth factor were added to the complete medium. For Pten_ / _Trp53_ / _prostate cancer cells, dihydrotestosterone was added to the complete medium.

[0112] The MDA-MB-231 and NK-92 cell lines were obtained from ATCC. The PD-L1 CAR NK-92 cell line was donated by Prof. Tonn Torsten. Cells were routinely maintained in RPMI cell culture medium supplemented with 10% heat-inactivated FBS, 1% penicillin / streptomycin solution, 1% L-glutamine, and 1% sodium pyruvate solution, at 37°C in an atmosphere with 5% CO2.

[0113] NK-92 and PD-L1 CAR NK-92 cell lines were maintained in X-VIVO 20 cell culture medium supplemented with 12.5% heat-inactivated FBS, 12.5% heat-inactivated horse serum, 1% penicillin / streptomycin solution, 1% L-glutamine, 1% sodium pyruvate solution and 1% non-essential amino acid solution, 500 U / mL IL-2.

[0114] HEK293T cells were cultured in DMEM cell culture medium, supplemented with 10% heat-inactivated FBS, 1% penicillin / streptomycin solution, and 1% L-glutamine. Mouse procedures

[0115] The xenograft model of prostate carcinoma in an immunodeficient background was obtained by subcutaneous injection of 3*10A6 cells of the Pten_ / _Trp53_ / _prostate cancer cell line into male NSG mice. The xenograft model of human prostate carcinoma was obtained by injecting 3*10A6 cells of the PC3 cell line into male NSG mice. The tumour size was measured with a caliper and quantified according to the formula:Volume = (DxdA2) / 2, where D = largest tumour diameter and d = smallest tumour diameter. At the end of the experiment, tumour size was measured by applying the following formula:Volume = (WidthA2 x Length) I 2. For adoptive cell transfer of NK-92 cells, NK-92 cells were stimulated in complete X-VIVO cell culture medium supplemented with IL-2 (500 U / mL), in the presence or absence of HA After 24 hours, the cells were collected and cell viability was estimated by Trypan Blue exclusion.3*10A6 cells were injected intravenously into tumour-bearing mice. For the NK cell depletion experiment, tumour-bearing mice were injected intraperitoneally with either the aNK1.1 antibody (clone PK136) or the recommended isotype control antibody.

[0116] The xenograft model of breast carcinoma in an immunodeficient setting was generated by subcutaneous injection of MDA-MB-231 cells into female NSG mice. The tumour size was measured with a caliper and quantified according to the formula:Volume = (D x d2) 12, where D is the largest and d the smallest diameter of the tumour. At the end of the experiment, tumour size was also calculated with the formula: Volume = (Width2x Length) 12.

[0117] For adoptive cell transfer of NK-92 cells, these were stimulated in complete X-VIVO cell culture medium supplemented with IL-2 (500 U / mL), in the presence or absence of HA (1 pM). After 24 hours, the cells were collected and viability was assessed by Trypan Blue exclusion. A total of 3x106cells was injected intravenously into tumourbearing mice.

[0118] Analysis of the tumour infiltrate by FACS

[0119] For analysis of tumour-infiltrating leukocytes, tumours were harvested, cut into small pieces, and digested with Collagenase I (1 mg / mL for murine tissues and 0.5 mg / mL for human tissues) for 45 minutes at 37°C on a moving platform. After rapid digestion in 2.5% trypsin and DNase I, a single-cell suspension was obtained by mechanical dissociation through a syringe needle (18G) and subsequent filtration through a 40 pm cell strainer. The composition of the tumour infiltrate was determined by flow cytometry. The samples were analysed with the FACSymphony™ A5 Cell Analyzer.

[0120] Single-cell RNA sequencing (single-cell RNA sequencing)

[0121] The raw sequencing data in bcl format were converted to fastq files and aligned to the mm10 murine reference genome, using the Cell Ranger Pipeline version 3.0.1 provided by 10X Genomics. After quality control, a total of 13,843 cells was obtained from the two biological replicates. Cell Ranger-filtered gene expression matrices were used as input for clustering analysis using the Seurat package in R (version 3.2.2; R version 4.0.3). We first processed each dataset individually, considering thresholds of 200, 50,000, and 0.25 for gene number, number of unique molecular identifiers (UMIs), and mitochondrial content, respectively. For each dataset, we selected the 2,000 most variable genes. Next, we used the FindlntegrationAnchors function to combine the data sets, choosing 2,000 anchor genes for the integration. After integration, we performed a principal component analysis (PCA) and used the first 72 principal components (PCs) to perform Louvain clustering and LIMAP embedding. Finally, we obtained a total of 24 clusters (resolution level = 0.4). The analysis of marker genes was performed using the FindAIIMarkers function (with default parameters set).

[0122] Quantitative RT-PCR

[0123] For screening alterations in autophagy-related genes, NK cells were treated with tumour medium. Total RNA was extracted from the NK cells using the RLT lysis solution. Subsequently, the RNA was further purified using the RNA RNeasy Mini Isolation Kit according to the manufacturer's protocol. cDNA was synthesized using 0.5-1 pg of total RNA by reverse transcription with the High Capacity cDNA Reverse Transcription Kit. Finally, gene expression was assessed using a predefined panel of 384 autophagic genes (SAB Target List) M384, according to the manufacturer's protocol.

[0124] Transcription factor analysis

[0125] Fortranscription factor analysis, NK-92 cells were cultured for 4 hours in serum-free RPMI medium and IL-2 to improve the signal / background ratio. Subsequently, the cells were treated with tumour medium, in the presence or absence of Plerixafor. Nuclear extracts of NK-92 cells were prepared using a commercially available nuclear extraction kit, according to the manufacturer's instructions. These nuclear extracts were then subjected to the TransAM C / EBPp Transcription Factor ELISA Kit, according to the manufacturer's instructions. Briefly, 10 pg of nuclear proteins were incubated with a specific plate coated with oligonucleotides. Activation of the C / EBPp transcription factor was detected by incubation with specific primary antibodies and an HRP-conjugated secondary antibody. The colorimetric reading was measured as optical density (OD, absorbance 450 nm) and is proportional to the activity of the transcription factor. The specific activity of the transcription factor was expressed as follows:OD450nm (treated condition) - OD450nm (untreated condition).

[0126] In vitro cytotoxicity assay of NK cells Murine NK cells used in the in vitro cytotoxicity assays were obtained, as previously described, from naive or tumour-bearing mice. Tumour-conditioned NK cells were tested after a 24-hour in vitro stimulation, while NK cells from tumour-bearing mice were used immediately after isolation. For selected experiments, NK cells were selected (CD3“F4 / 80“B220“Ly6G“NK1.1+) and immediately used for co-culture with target cells.

[0127] To distinguish effector cells from target cells, target cells were stained with CellTrace Violet (1 pM final concentration in PBS) for 20 minutes at room temperature.10,000 non-adherent cells (YAC-1) were seeded in 96-well Il-bottom plates in complete RPMI medium, while 20,000 adherent cells (Pten- / -, Pten- / - Trp53- / -, LNCaP and PC3) were seeded in 96-well flat-bottom plates in complete RPMI medium. For adherent cells, the co-culture was set up after 2 hours of incubation to promote cell adhesion.

[0128] NK cells were added to wells containing target cells, at the specified effectortarget ratio, and incubated for 4 hours at 37°C. The cells were then collected and stained with a mixture of Helix NP™ NIR and Annexin V FITC in Annexin V binding buffer for 15 minutes at room temperature. FACS analysis was performed immediately with BD FACSCanto II.

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Claims

CLAIMS1) Isolated NK cells and / or isolated population of NK cells having restored and / or enhanced autophagic activity wherein the transcriptional factor C / EBPp is stably or transiently inhibited in said cells.2) Isolated NK cells and / or isolated population of NK cells according to claim 1 wherein the inhibition of the transcriptional factor C / EBPp is genetic or pharmacological.3) In vitro or ex vivo method for restoring and / or re-establishing or improving and / or increasing and / or enhancing and / or strengthening autophagic activity and obtaining isolated cells and / or an isolated population of NK cells having restored and / or re-established and / or improved and / or increased and / or enhanced and / or strengthened autophagic activity by pharmacological or genetic inhibition of the transcriptional factor C / EBPp in NK cells with unaltered and / or dysfunctional autophagy.4) Method according to claim 3, wherein the inhibition is pharmacological and it comprises the following steps:d) collecting an isolated sample of NK cells with unaltered and / or dysfunctional autophagy;e) culturing the isolated sample referred to in step a) in a cell culture medium supplemented with an inhibitor of transcriptional factor C / EBPp for a time such as to restore and / or restore or improve and / or increase and / or enhance and / or strengthen the autophagic activity of NK cells;f) Collect the isolated NK cells or an isolated population of NK cells with restored and / or improved autophagic activity obtained at the end of step b).5) The method according to claim 3 or 4 wherein the pharmacological inhibitor is at least one molecule and / or compound and / or active ingredient capable of inactivating the transcriptional factor C / EBPp and a mixture thereof and / or at least one antibody that inactivates the transcriptional factor C / EBPp. 6) Method according to claim 3 wherein the genetic inhibition is obtained by any genetic manipulation technique or method resulting in the inactivation of the transcriptional factor C / EBPp.7) The method according to claim 6, wherein the genetic manipulation is gene silencing or genome editing.8) Isolated NK cells or an isolated population of NK cells having restored and / or enhanced and / or improved and / or increased and / or strengthened autophagicactivity obtained from NK cells with unaltered and / or dysfunctional autophagy by the method of any one of claims 3 to 7.9) A pharmaceutical composition comprising cells and / or a population of cells according to any one of claims 1 or 2 or 8 and at least one pharmaceutically acceptable carrier and / or vehicle and / or pharmacologically acceptable excipients.10) Cells and / or a population of cells according to any one of claims 1 or 2 or 8 and a pharmaceutical composition according to claim 9 for use as a medicament.11 ) Cells and / or a population of cells according to any one of claims 1 or 2 or 8 and a pharmaceutical composition according to claim 9 for use in anti-tumour immunotherapy.12) Cells and / or a population of cells according to any one of claims 1 or 2 or 8 and a pharmaceutical composition according to claim 9 for use in adoptive cell therapy (ACT).13) Cells and / or a population of cells according to any one of claims 1 or 2 or 8 and a pharmaceutical composition according to claim 9 for use in the prevention and / or treatment of cancer.

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