Modified human lymphocytes containing and expressing positive regulators of autophagy

Abstract

The present invention relates to a modified human lymphocyte containing and expressing a recombinant nucleic acid or a set of recombinant nucleic acids encoding at least one positive regulator of autophagy. The modified human lymphocyte according to the invention may further contain and express a recombinant nucleic acid or a set of recombinant nucleic acids encoding a T-cell receptor or a chimeric antigen receptor. The invention also relates to methods of producing such modified human lymphocytes, pharmaceutical compositions comprising the same, as well as their uses in medicine and for methods for the treatment of cancer including the immunotherapeutic treatment of cancer.

Description

[0001] Modified human lymphocytes containing and expressing positive regulators of autophagy.

[0002] FIELD OF THE I NVENTION

[0003] The present invention relates to a modified human lymphocyte containing and expressing a recombinant nucleic acid or a set of recombinant nucleic acids encoding at least one positive regulator of autophagy. The modified human lymphocyte according to the invention may further contain and express a recombinant nucleic acid or a set of recombinant nucleic acids encoding a T-cell receptor or a chimeric antigen receptor.

[0004] The invention also relates to methods of producing such modified human lymphocytes, pharmaceutical compositions comprising the same, as well as their uses in medicine and for methods for the treatment of cancer including the immunotherapeutic treatment of cancer.

[0005] BACKGROUND

[0006] Over the past decades great improvements have been made in the field of haematology and oncology with the development of intense multimodal treatments, supportive care and a better characterization of tumour biology. Nevertheless, patients suffering from recurrent or resistant tumours still maintain an unfavourable outcome1’2. For these patients there is the urgent clinical need to identify new and more efficient intervention strategies. It has been clearly shown that tumours are susceptible to targeted and immune-based therapies including adoptive cell therapy which involves harvesting immune cells (T, NK and NK-T cells), expanding them ex vivo and re-directing them to target cancer cells. Although these kinds of therapies have been studied for decades, only recently they were able to elicit clinical benefit in a wider range of patients. Observations that Epstein Barr Virus (EBV)-specific cytotoxic T cells (CTLs) from seropositive donors were able to control EBV- transformed B cells in vitro led to the first antigen specific T cell therapies that were used to treat post-transplant lymphoproliferative disorder (PTLD)3 4. Several efforts have also focused on the genetical modification of T cells to recognize tumour cells either via an exogenous T cell receptor (TCR) or with the addition of a Chimeric Antigen Receptor (CAR). The first is able to recognize peptide antigens (mainly derived from intracellular proteins) but is limited by the major histocompatibility complex (MHC) restriction and the lack of the appropriate continuation necessary for the optimal T cell activation. CARs instead contain an antibody-derived single chain variable fragment (scFv), conferring target specificity, fused to a transduction signal domain like the CD3 chain capable to activate the T cells. This structure allows the T cell to recognize antigens including non-peptide targets like glycolipids and carbohydrates, and become activated without MHC presentation5. In addition to the a / p-T cells, also the y5-T cells and NK cells are an emerging research area. While y5-T cells are able to recognize unprocessed antigens in an MHC independent manner6 7, NK cells kill through "missing-self mechanisms” by attacking cells that have a decrease or lack in the expression of MHC-I8. Both these cell platforms do not cause graft versus host disease (GvHD) making them a prime candidate for "off the shelf' therapy6 9. Recently, several of these advanced forms of approaches have been classified as Advanced Therapy Medicinal Products (ATMP) and are revolutionizing biomedicine with the establishment of new treatments for diseases for which there is currently no cure10 11. In particular, CAR immunotherapies have been an especially active area of research, but despite the health impact which some of the proposed treatments have shown on patients refractory to more traditional treatments, only 5 adoptive cell therapies targeting CD19 and the B-cell maturation antigens have been so far approved by the American and European regulatory agencies (FDA and EMA)12 13. Challenges to achieve similar responses in patients harbouring other malignancies including solid tumours and CD19nes leukaemia / lymphoma are still considerable14’16. It has been proven that this limited efficacy is mainly due to the nature of these tumours, the presence of an immunosuppressive tumour microenvironment (TME) as well as the low persistence and functionality of CAR T cells17’20.

[0007] A recent clinical study in refractory high-risk neuroblastoma patients (NCT03373097) treated with 3° generation GD2.CAR T cells, demonstrated that CAR optimization is crucial to improve safety and efficacy of this technology observing a 3-year overall survival and event free survival of 60% and 36%, respectively, with the recommended dose21, but also how the TME and its elements are responsible for the loss of function and persistence of GD2.CAR T cells affecting their metabolism and activation status20. To address this limitation, several groups including ours, investigated new strategies to metabolically reprogram CAR T cells22’27. These studies underlined that this reprogramming could be performed using different strategies including the supplementation of small lipids during the ex-vivo CAR T cell generation process which results in a better performance and memory composition of ATMPs25. Although significantly better, the long-term efficacy of these CAR T cells remains suboptimal in tumours with a hostile TME and / or which are difficult to invade25 28.

[0008] Therefore, the inventors further investigated how to improve CAR T cell functionality. Based on their previous studies where the inventors observed that the ex-vivo T cell manipulation, necessary for the gene-modification, is able to induce important changes including the regulation of enzymes involved in the degradation of the extracellular membrane28, the inventors investigate if other mechanisms / pathways could be affected by this procedure. They observed that the ex-vivo manipulation induces metabolic changes among which a strong and prolonged block of the autophagy pathway independently of culture conditions (cytokines used, activation: polyclonal vs. antigen specific).

[0009] This key finding is novel and has not been reported before.

[0010] Autophagy is a self-degradative process, essential for the maintenance of metabolic and genetic homeostasis in all eukaryotic organisms. By autophagy, cellular components are delivered to lysosomes in order to ensure the basal turnover of cytosolic organelles and provide energy and macromolecular precursors29. Autophagy impairment or excessive activity has been associated with several human disorders, ranging from cancer to autoimmunity and neurodegenerative disease. In cancer, autophagy is a Janus-faced process, acting both as tumour suppressor (cleaning cells from damaged organelles), and as tumour adaptive response (favouring malignant progression and chemoresistance)30. Of note, recent findings have revealed that autophagy contributes to immunosuppression-related chemoresistance and promotes the tumour's ability to avoid immune detection31 32. Moreover, enhanced autophagy in tumour cells has been observed in advanced stages of metastatic diseases, with low levels of tumour-infiltrating T lymphocytes33. Furthermore, several new studies have reported that autophagy is a critical pathway also for tumour antigen cross-presentation by major histocompatibility complexes, which is determinant for the initiation of an efficient adaptive immune response34. Moreover, the regulation of autophagy has been shown to have important effects also in protein secretion, regulating the production of both anti- and pro-inflammatory cytokines and chemokines35. However, the mechanisms whereby autophagy manipulates anticancer immunity remain to be clarified and controversial results in the context-dependent roles of autophagy need further investigations.

[0011] DESCRIPTION OF THE I NVENTION

[0012] The inventors decided to restore the autophagy machinery in immune effector cells such as lymphocytes by forcing the expression of, for instance, two exemplary autophagy key proteins in new fourth generation GD2.CAR T cells demonstrating no toxic effects and a marked improvement of the anti-tumour activity.

[0013] The inventors inter alia found that this reactivation of autophagy key proteins in in immune effector cells such as lymphocytes is not toxic and is able to maintain a more immature CAR-T cell phenotype, improves the mitochondria fitness, improves the anti-tumour response in both in vitro 2D and 3D models even in low effector: target ratios as well as in an in vivo model reducing the exhaustion profile of autophagy reprogrammed T cells, does not induce allo-reactivity, increases the extracellular vesicles production and secretion.

[0014] Other than autophagy, the inventors found that the autophagy key proteins can re-activate several other pathways involved in the response to stress, mitosis, post-protein translation, antigen presentation, activation of innate immune-system.

[0015] They further found that that the block of the autophagic pathway is not a unique feature observed during the production of CAR T cells, but also detectable in the production of antigen-specific T cells, y / 5-T cells and natural killer cells independent of a gene manipulation and the cytokines used for the ex vivo culture.

[0016] Importantly, this new strategy could revolutionize the efficacy of CAR T cells in solid tumours but also in other malignancies providing a new and prolonged intervention therapy significantly ameliorating the patients' quality of life.

[0017] Lastly, the inventors observed that this autophagic block is present also in other cellular platforms used for CAR therapy and adoptive cell therapy. Therefore, having demonstrated that the autophagic block is not due to gene manipulation but rather to the ex-vivo activation and manipulation process, crucial for the expansion of all these cellular products, the proposed strategy could be extended to other cellular products improving their long-term persistence and efficacy.

[0018] Accordingly, the invention relates to the following preferred embodiments:

[0019] 1. A modified human lymphocyte containing a recombinant nucleic acid or set of recombinant nucleic acids encoding at least one positive regulator of autophagy.

[0020] 2. The modified human lymphocyte according to item 1, wherein the at least one positive regulator of autophagy is one or more human protein(s).

[0021] 3. The modified human lymphocyte according to item 1 or 2, wherein the one or more human protein(s) are selected from the group consisting of: human TFEB, human Beclin-1, human ATG5, human AMBRA1, human ULK1, human ATG14, human WIPI1, human WIPI2, human p62, human VP34, human ATG4A, human Annexin A2, human Polycystin 2, human NFT2, and human ATG7.

[0022] 4. The modified human lymphocyte according to any one of the preceding items, wherein the at least one positive regulator of autophagy is human TFEB, human Beclin-1, and / or human ATG5.

[0023] 5. The modified human lymphocyte according to any one of items 1-4, wherein the at least one positive regulator of autophagy is human TFEB.

[0024] 6. The modified human lymphocyte according to any one of items 1-4, wherein the at least one positive regulator of autophagy is human Beclin-1 .

[0025] 7. The modified human lymphocyte according to any one of items 1-4, wherein the at least one positive regulator of autophagy is human ATG5.

[0026] 8. The modified human lymphocyte according to item 1, wherein the at least one positive regulator of autophagy is one or two peptides selected from the following group: TNVFNATFHIWHSGQFGT (SEQ ID NO: 1) and TNVFNATFEIWHDGEFGT (SEQ ID NO: 2).

[0027] 9. The modified human lymphocyte according to any one of the preceding items, wherein the lymphocyte expresses said at least one positive regulator of autophagy from said recombinant nucleic acid or set of recombinant nucleic acids.

[0028] 10. The modified human lymphocyte according to item 9, wherein the expression of said at least one positive regulator of autophagy from said recombinant nucleic acid or set of recombinant nucleic acids is constitutive.

[0029] 11 . The modified human lymphocyte according to item 9, wherein the expression of said at least one positive regulator of autophagy from said recombinant nucleic acid or set of recombinant nucleic acids is inducible.

[0030] 12. The modified human lymphocyte according to any one of the preceding items, wherein the lymphocyte is a T-cell, an NK cell, or an NKT cell. The modified human lymphocyte according to any one of the preceding items, wherein the lymphocyte is a T-cell. The modified human lymphocyte according to any one of the preceding items, wherein the lymphocyte is an op T-cell. The modified human lymphocyte according to any one of items 1-13, wherein the lymphocyte is an y5 T- cell. The modified human lymphocyte according to any one of the preceding items, wherein the lymphocyte is a CD8+T-cell. The modified human lymphocyte according to any one of items 1-15, wherein the lymphocyte is a CD4+T- cell. The modified human lymphocyte according to any one of items 1-12, wherein the lymphocyte is an NK cell. The modified human lymphocyte according to any one of items 1-12, wherein the lymphocyte is a NKT cell. The modified human lymphocyte according to any one of the preceding items, wherein the lymphocyte further contains and expresses a recombinant nucleic acid or a set of recombinant nucleic acids encoding a T-cell receptor or a chimeric antigen receptor. The modified human lymphocyte according to item 20, wherein the lymphocyte contains and expresses a recombinant nucleic acid or a set of recombinant nucleic acids encoding a T-cell receptor. The modified human lymphocyte according to item 20, wherein the lymphocyte contains and expresses a recombinant nucleic acid or a set of recombinant nucleic acids encoding a chimeric antigen receptor. The modified human lymphocyte according to any one of items 20-22, wherein the T-cell receptor or chimeric antigen receptor binds to a cancer cell antigen. The modified human lymphocyte according to item 23, wherein the cancer cell antigen is an antigen of a solid cancer. The modified human lymphocyte according to item 24, wherein the solid cancer is a brain cancer. The modified human lymphocyte according to item 25, wherein the brain cancer is a neuroblastoma. The modified human lymphocyte according to item 25, wherein the brain cancer is a medulloblastoma. The modified human lymphocyte according to any one of items 23-27, wherein the cancer cell antigen is an antigen of a pediatric cancer. The modified human lymphocyte according to any one of the preceding items, wherein the recombinant nucleic acid or set of recombinant nucleic acids encoding said at least one positive regulator of autophagy and / or the recombinant nucleic acid or set of recombinant nucleic acids encoding said T-cell receptor or chimeric antigen receptor is integrated into the genome, preferably the chromosomal genome, of the lymphocyte. The modified human lymphocyte according to any one of the preceding items, wherein the recombinant nucleic acid or set of recombinant nucleic acids encoding said at least one positive regulator of autophagy and / or the recombinant nucleic acid or set of recombinant nucleic acids encoding said T-cell receptor or chimeric antigen receptor is an expression cassette. The modified human lymphocyte according to item 30, wherein the expression cassette is from a retroviral vector. The modified human lymphocyte according to item 30 or 31, wherein the expression cassette contains a selection marker. The modified human lymphocyte according to item 32, wherein the selection marker is ACD19. A method for obtaining a modified human lymphocyte according to any one of the preceding items, the method comprising a step of introducing the recombinant nucleic acid or set of recombinant nucleic acids encoding said at least one positive regulator of autophagy into a human lymphocyte, in order to obtain the modified human lymphocyte. The method according to item 34, further comprising a step of introducing the recombinant nucleic acid or set of recombinant nucleic acids encoding the T-cell receptor or chimeric antigen receptor into the human lymphocyte, wherein the modified human lymphocyte is a modified human lymphocyte according to any one of items 20-33. The method according to item 34 or 35, wherein the method comprises a step of expanding said modified human lymphocyte ex vivo. The method according to any one of items 34-36, wherein the method is carried out in vitro. A pharmaceutical composition comprising a modified human lymphocyte according to any one of items 1- 33 or a modified human lymphocyte obtainable by the method of any one of items 34-37. A modified human lymphocyte according to any one of items 1-33, for use in medicine. A modified human lymphocyte according any one of items 1-33 or a pharmaceutical composition according to item 38, for use in a method for the treatment of cancer in a human patient, wherein the modified human lymphocyte is a modified human lymphocyte according to item 23. The modified human lymphocyte or pharmaceutical composition for use according to item 40, wherein the treatment of cancer is an immunotherapeutic treatment of cancer. The modified human lymphocyte or pharmaceutical composition for use according to item 41 , wherein the cancer is a solid cancer and the modified human lymphocyte is a modified human lymphocyte according to item 24. The modified human lymphocyte or pharmaceutical composition for use according to item 42, wherein the solid cancer is a brain cancer and the modified human lymphocyte is a modified human lymphocyte according to item 25. The modified human lymphocyte or pharmaceutical composition for use according to item 43, wherein the brain cancer is a neuroblastoma and the modified human lymphocyte is a modified human lymphocyte according to item 26. The modified human lymphocyte or pharmaceutical composition for use according to item 44, wherein the brain cancer is a medulloblastoma and the modified human lymphocyte is a modified human lymphocyte according to item 27. 46. The modified human lymphocyte or pharmaceutical composition for use according to any one of items 41- 45, wherein the cancer cell antigen is an antigen of a pediatric cancer and the modified human lymphocyte is a modified human lymphocyte according to item 28.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : Blockage of the autophagy machinery in ex-vivo manipulated primary human T cells.

[0033] A) Quantitative real time PCR (qPCR) analysis of the fold change in RNA levels of four autophagy key genes (LC3, AMBRA1 , p62, ULK1) in polyclonally activated CD3+primary human T cells at different timepoints after isolation from human peripheral blood compared to TO (CD3+T cells just after isolation); n=4. B) Volcano plot representation of 85 autophagy related mRNA modulation. C) qPCR analysis of the fold change in RNA levels of four autophagy key genes (LC3, AMBRA1 , p62, ULK1) in polyclonally activated CD4+primary human T cells at different timepoints after isolation from human peripheral blood compared to TO (fresh isolated T cells); n=3. D) qPCR analysis of the fold change in RNA levels of four autophagy key genes (LC3, AMBRA1 , p62, ULK1) in polyclonally activated CD8+primary human T cells at different timepoints after isolation from human peripheral blood compared to TO (fresh isolated T cells); n=3. E) qPCR analysis of the fold change in RNA levels of four autophagy key genes (LC3, AMBRA1 , p62, ULK1) in polyclonal activated CD3+primary human T cells (NT) and CAR-T cells compared to TO (fresh isolated T cells); n=3. F) Representative Western Blot analysis of the protein levels of ACTIN, p62, LC3, mTOR, ULK1 and AMPK in polyclonally activated CD3+primary human T cells (NT) and CAR-T cells cultured using IL2 compared to TO (fresh isolated T cells); n=3. G) Representative Western Blot analysis of the protein levels of ACTIN, p62, LC3, mTOR and ULK1 in the CD4+and CD8+CAR+ T cells at day 14 of culture with IL-2 compared to TO; n=2. H) Representative Western Blot analysis of the protein levels of ACTIN, p62, LC3 and ULK1 in polyclonally activated CD4+and CD8+CAR+T cells at day 14 of culture with IL7 / 15 compared to TO; n=2. 1) Representative Western Blot analysis of the protein levels of ACTIN, p62, LC3II, p-mTOR and p-ULK1 in CMV-specific cytotoxic T lymphocytes at day 14 of culture (two rounds of stimulation with pp65 pulsed autologous dendritic cells) compared to TO; n=2. J) Western blot analysis of T cells cultured with IL2 (+IL) or without (UT), with or without the addition of mTOR-inhibitor (Torin 1 ) for 6h or 24h. K) Summary table for interpretation of autophagic flux shown in western blot panels. Data are shown as mean ± SEM. L) qPCR analysis of the fold change in RNA levels of five autophagy key genes (LC3, AMBRA, p62, ULK, Beclinl , ATG5) in NK and y5-T cells compared to TO (fresh isolated in NK and y5-T cells); n=3. M) Representative Western Blot analysis of the protein levels of ACTIN, ULK1, ATG14, pULK1 , pATG14 and LC3 in NK cells after 14 days of culture compared to TO; n=3.

[0034] Figure 2: Transduction of HEK 293T cells using retroviral vectors encoding for 1CD19, TFEB+X1CD19 and Beclinl +X1CD19.

[0035] A) Transduction efficiencies in HEK 293T cells at different timepoints after transduction monitored by FACS; n=1 B) GFP MFIs in HEK 293T cells at different timepoints after transduction monitored by FACS; n=1 C) qPCR analysis of the fold change in RNA levels of Beclinl and TFEB in wild-type and transduced HEK 293T cells; technical n=3 D) Representative Western Blot analysis of protein levels of TFEB, ACTIN and Beclinl in wildtype and transduced HEK 293T cells (21CD19, #116, TFEB+Z1CD19: #276, Beclin 1 +zlCD 19: #277). Data are shown as mean ± SEM. P values shown are from two-way ANOVA.

[0036] Figure 3: Transduction of primary human T cells using retroviral vectors encoding for 1CD19, TFEB+Z1CD19 and Beclinl +Z1CD19.

[0037] A) T cell ex-vivo expansion from day 0 (transduction) until day 11 . B) Transduction efficiencies in primary human T cells at different timepoints after transduction monitored by FACS; n=4. C) zlCD 19 MFIs in primary human T cells at different timepoints after transduction monitored by FACS; n=4. D) qPCR analysis of the fold change in RNA levels of Beclinl and TFEB in primary human T cells; n=3. E) Representative Western Blot analysis of protein levels of TFEB, ACTIN and Beclinl in primary human T cells (21CD19: #116, TFEB+Z1CD19: #276, Beclinl +Z1CD 19: #277). F) Distribution between CD4+and CD8+cells in primary human T cells monitored by FACS; n=4. G) FACS-based analysis of the fold change in MFI of mitochondrial potential in primary human T cells; n=4. H) Representative Western Blot analysis of a starvation experiment using control Z1CD19 and Beclin 1 +Z1CD 19 primary human T cells cultured in growth media (GM) or nutrient-free EBS with or without the addition of Chloroquine (CQ) using p62, LC3II, ACTIN and TFEB antibodies. I) Quantitative Western Blot intensity-based analysis of autophagic flux (determined by the intensity-quotient of LC3II and ACTIN) in nontransduced and TFEB-modified primary human T cells; n=4. J) % of tumour killing of the Z1CD19, TFEB+Z1CD19 and Beclin 1 +zlCD 19 versus CHLA255 neuroblastoma cell line line at ratio 1 :1 in a 24h killing assay. Data are shown as mean ± SEM (A-C, I) or mean ± SD (D, F, G).

[0038] Figure 4: Generation of CAR-T cells from primary human T cells using retroviral vectors encoding GD2.CAR, GD2.CAR+TFEB and GD2.CAR+Beclin1.

[0039] A) Cell-count-based fold expansion of primary human T cells at different timepoints during culture; n=4. B) Transduction efficiencies in primary human T cells at different timepoints after transduction monitored by FACS; n=5. C) CAR MFIs in primary human T cells at different timepoints after transduction monitored by FACS; n=5. D) qPCR analysis of the fold change in RNA levels of Beclinl and TFEB in primary human T cells; n=3. E) Distribution between CD4+and CD8+cells in primary human T cells monitored by FACS; n=3. F) Representative Western Blot analysis of protein levels of TFEB, ACTIN and Beclinl in primary human T cells. G) Oxygen consumption rate in primary human T cells monitored by Seahorse XF-96e extracellular flux analyzer; n=4. H) Extracellular acidification rate in primary human T cells monitored by Seahorse XF-96e extracellular flux analyzer; n=4. I) Representative Western Blot analysis of protein levels of LC3II and ACTIN in #203 Beclinl +GD2.CAR-T cells and #50 GD2.CAR T cells for the monitoring of autophagic flux. J) Representative Western Blot analysis of protein levels of p-ATG14, ULK1, ATG14 and ACTIN in #203 Beclin 1 +GD2. CAR-T cells and #50 GD2.CAR T cells. K) Representative Western Blot analysis of protein levels of LC3II and ACTIN in #203 Beclinl +GD2.CAR-T cells and #50 GD2.CAR T cells with or without the addition of BafA1 for the monitoring of autophagic activity. L) Representative Western Blot analysis of protein levels of LC3II and ACTIN in #202 TFEB+GD2.CAR-T cells and #50 GD2.CAR T cells with or without the addition of BafA1 for the monitoring of autophagic activity. M) Immune fluorescence puncta assay and quantification of LC3 accumulation in #203 Beclin1 +GD2.CAR-T cells and #50 GD2.CAR T cells in EBS media or EBS media + BafA1. Data are shown as mean ± SEM (G, H, M) or mean ± SD (A-EE, F).

[0040] Figure 5: Immunophenotype characterization of autophagy-modified CAR-T cells.

[0041] A) FACS-based analysis of the memory phenotype of CD4+primary human T cells, (CM central memory, EM effector memory, ET effector terminal); n=3 B) FACS-based analysis of the memory phenotype of CD8+primary human T cells, (CM central memory, EM effector memory, ET effector terminal); n=3 C) FACS-based analysis of the SCMT (stem-cell-like memory T cells) population in CD4+and CD8+primary human T cell; n=3 D) FACS- based analysis of exhaustion marker expression (Lag3, PD1 , CD95, Tim3) and exhausted cells (all four markers expressed) in CD4+primary human T cells; n=3 E) FACS-based analysis of exhaustion marker MFI (Lag3, PD1 , CD95, Tim3) in CD4+primary human T cells; n=3 F) FACS-based analysis of exhaustion marker expression (Lag3, PD1, CD95, Tim3) and exhausted cells (all four markers expressed) in CD8+primary human T cells; n=3 G) FACS-based analysis of exhaustion marker MFI (Lag3, PD1 , CD95, Tim3) in CD8+primary human T cells; n=3 H) FACS-based analysis of activation-associated marker expression (CD28, CD25, HLA-DR) in CD4+primary human T cells; n=3. 1) FACS-based analysis of activation-associated marker MFI (CD28, CD25, HLA- DR) in CD4+primary human T cells; n=3. J) FACS-based analysis of activation-associated marker expression (CD28, CD25, HLA-DR) in CD8+primary human T cells; n=3. K) FACS-based analysis of activation-associated marker MFI (CD28, CD25, HLA-DR) in CD8+primary human T cells; n=3. Data are shown as mean ± SD.

[0042] Figure 6: Functional 2D killing assay of autophagy-modified CAR-T cells.

[0043] Percentage of improvement in tumour killing of GD2.CAR+TFEB and GD2.CAR+Beclin1 versus GD2.CAR control in a short-term killing assay (24h) (A-F) after 12 days (A-C) and 24 days (D-F) of ex-vivo culture using SHSy5Y (A, D), CHLA255 (B, E) and D341 (C, F) tumour cell lines at different E:T ratio.

[0044] Percentage of improvement in tumour killing of GD2.CAR+TFEB and GD2.CAR+Beclin1 versus GD2.CAR control in a long-term killing assay (4 days) (G-L) after 12 days (G-l) and 24 days (J-L) of ex-vivo culture using SHSy5Y (G, J), CHLA255 (H, K) and D341 (I, L) tumour cell lines at different E:T ratio, n=3.

[0045] Figure 7: Functional 3D spheroid killing assay using autophagy-modified CAR-T cells.

[0046] A) Representative fluorescence-based 3D spheroid killing assay using #50 and #203 GD2-specific CAR-T cells in an E:T ratio of 1 :3 against GFP+SH-SY5Y neuroblastoma cells monitored at different timepoints B) Average GFP intensity of the fluorescence-based 3D spheroid killing assay using #50 and #203 GD2-specific CAR-T cells in an E:T ratio of 1 :3 against GFP+SH-SY5Y neuroblastoma cells monitored at different timepoints; n=2. C) Representative fluorescence-based 3D spheroid killing assay using #50 and #203 GD2-specific CAR-T cells in an E:T ratio of 1 :3 against GFP+D341 medulloblastoma cells monitored at different timepoints. D) Average GFP intensity of the fluorescence-based 3D spheroid killing assay using #50 and #203 GD2-specific CAR-T cells in an E:T ratio of 1 :3 against GFP+D341 medulloblastoma cells monitored at different timepoints; n=2.

[0047] Figure 8: Validation of autophagy restoration using ATG5 and Beclinl peptide or apply with a different CAR targeting the B7-H3 antigen.

[0048] A) Cell-count-based fold expansion of primary human T cells at different timepoints during culture; cells were transducted with GD2.CAR or GD2.CAR+ATG5 or GD2.CAR+Beclin1_peptide; n=3. B) Transduction efficiencies of primary human T cells at day 10 after transduction monitored by FACS; n=3. C-D) Percentage of improvement in tumour killing of GD2.CAR+ATG5 and GD2.CAR+Beclin1_peptide versus GD2.CAR control in a long-term killing assay (4 days) using D341 (C) or SHSy5Y (D) at different E:T ratio, n=3. E) Cell-count-based fold expansion of primary human T cells at different timepoints during culture; cells were transducted with B7- H3.CAR or B7-H3.CAR+TFEB or B7-H3.CAR+Beclin1 or B7-H3.CAR+ATG5 or GD2.CAR+Beclin1.peptide; n=3. F-G) Percentage of improvement in tumour killing of B7-H3.CAR+TFEB or B7-H3.CAR+Beclin1 or B7- H3.CAR+ATG5 or GD2.CAR+Beclin1.peptide versus B7-H3.CAR control in a long-term killing assay (4 days) using A673 (F, Ewing Sarcoma) and CT-10 (G, Rhabdomyosarcoma) cell lines at different E:T ratio, n=1.

[0049] Figure 9. Proteomic, transcriptome and metabolome profile of autophagy reprogrammed CAR T cells.

[0050] A) The heatmap shows the different protein profiles of cells transduced with GD2.CAR, GD2.CAR+TFEB (#202), and GD2.CAR+Beclin1 (#203) after 48h from antigen stimulation via CAR activation (1A7, anti-idiotype). B) Venn diagram representation showing the distribution of proteins modulated by TFEB and Beclinl after 48h from antigen stimulation via CAR activation (1 A7, anti-idiotype). C) Pathway enrichment analysis on modulated proteins detected only in GD2.CAR+TFEB after comparison with GD2.CAR T-cells. D) Pathway enrichment analysis on modulated proteins detected only in GD2.CAR+Beclin1 after comparison with GD2.CAR T-cells. E) Pathway enrichment analysis on modulated proteins detected both in GD2.CAR+TFEB and GD2.CAR+Beclin1 after comparison with GD2.CAR T-cells. F) Pathway enrichment analysis on modulated mRNA detected in GD2.CAR+TFEB or GD2.CAR+Beclin1 after comparison with GD2.CAR T-cells after 96h from antigen stimulation via CAR activation (1A7, anti-idiotype). G) Metabolites found significance up- or down-regulated in GD2.CAR, GD2.CAR+TFEB, GD2.CAR+Beclin1, GD2.CAR+ATG5 and GD2.CAR+Beclin1 peptide after 96h from antigen stimulation via CAR activation (1 A7, anti-idiotype).

[0051] Figure 10. Extracellular detection in GD2.CAR T cells.

[0052] A) Electron microscopy analysis of GD2.CAR, GD2.CAR+TFEB, GD2.CAR+Beclin1. B) Quantification of extracellular vesicle / l in culture supernatant of GD2.CAR, GD2.CAR+TFEB, GD2.CAR+Beclin1 , GD2.CAR+ATG5 without antigen stimulation. C) Quantification of extracellular vesicle / l in culture supernatant of GD2.CAR, GD2.CAR+TFEB, GD2.CAR+Beclin1 , GD2.CAR+ATG5 with antigen stimulation by 1A7.

[0053] Figure 11 . In vivo validation of autophagy restored GD2.CAR T cells. A) In vivo bioluminescence imaging of NSG mice bearing SHSY5Y-FF-Luc.GFP cells treated with NT, GD2.CAR, GD2.CAR+TFEB, GD2.CAR+Beclin1 T cells. B) Distribution of human CD3 and CAR T cells in the peripheral blood of mice treated with GD2.CAR, GD2.CAR+TFEB, GD2.CAR+Beclin1 T cells at 12 days after CAR T cell treatment. C) Percentage of CD4 and CD8 in the peripheral blood of mice treated with GD2.CAR, GD2.CAR+TFEB, GD2.CAR+Beclin1 T cells at 12 days after CAR T cell treatment. D) Frequency of resting CD4 and CD8 T cells (CD27ne9 / CD28ne9) in the peripheral blood of mice treated with GD2.CAR, GD2.CAR+TFEB, GD2.CAR+Beclin1 T cells at 12 days after CAR T cell treatment. Median fluorescence intensities of CAR, CXCR3, PD1, Lag3 and HLA-DR in both CD8 (E) and CD4 (F) compartment.

[0054] DETAILED DESCRIPTION OF THE INVENTION

[0055] Unless otherwise defined below, the terms used in the present invention shall be understood in accordance with their common meaning known to the person skilled in the art. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. Publications referred to herein may be cited either by specifying the full literature reference in the text, or by specifying a reference number and adding the corresponding full literature reference to the references section at the end of the description.

[0056] Definitions

[0057] In accordance with the invention, the term "autophagy” has its known meaning. It is understood that the meaning of this term encompasses a cellular process that involves the degradation and recycling of cellular components, such as proteins, organelles, and macromolecules, through a highly regulated mechanism. It is a fundamental process for maintaining cellular homeostasis and is involved in various physiological and pathological conditions.

[0058] In accordance with the invention, a "modified human lymphocyte” refers to a human lymphocyte, that has been modified (e.g., altered or engineered), e.g., to fulfil a particular purpose. The modification includes the modification according to the claims, i.e., the presence of a set of recombinant nucleic acids encoding at least one positive regulator of autophagy according to claim 1 , and it optionally also includes the presence of a recombinant nucleic acid or a set of recombinant nucleic acids encoding a T-cell receptor or a chimeric antigen receptor. The modification can further include, but is not limited to, genetic manipulation, the introduction of specific receptors or proteins, and / or other modifications, e.g., modifications to modify the lymphocyte's function or targeting capabilities.

[0059] It is understood that the modified human lymphocyte of the invention is preferably suitable for use in a human patient. Methods for preparing modified human lymphocytes such that they are suitable for use in a human patient are known in the art. The modified human lymphocyte of the invention is preferably suitable for use in the treatment of cancer in a human patient, e.g., the immunotherapeutic treatment of cancer in a human patient. In accordance with the invention, a human lymphocyte that has been isolated from a healthy donor or a patient having cancer, can be modified by introducing a recombinant nucleic acid or a set of recombinant nucleic acids as defined by the claims, and administered to the patient to treat said cancer. The human lymphocyte is preferably selected from an NK cell, a NKT cell, and a T cell. T cells are preferably CD8+T cells, CD4+T cells, op T cells, and / or yd T cells.

[0060] An immunoreceptor according to the invention is a transmembrane receptor, which, when expressed by an immune cell, is capable of mediating an immune response. The immunoreceptor can be an endogenous immunoreceptor or a non-natural immunoreceptor, i.e. genetically engineered. Exemplary immunoreceptors are T-cell receptors (TCRs) and chimeric antigen receptor (CARs) in accordance with the invention, e.g., recombinant T-cell receptors (TCRs) and recombinant chimeric antigen receptors (CARs). The CAR and / or the TCR according to the invention in its monomeric form may either consist of a single molecule comprising all of its domains or consist of a heterodimer that comprises all of its domains. The CAR and / or the TCR can bind to its antigen either directly, or it can bind indirectly through an adapter.

[0061] It will be understood that a CAR and / or a TCR in accordance with the invention binds to an antigen, preferably a cancer cell antigen. While a CAR typically binds to an extracellular domain of a cancer cell antigen, i.e., a cancer cell surface antigen, a TCR typically binds to an intracellular cancer cell antigen presented by a HLA molecule.

[0062] As used in connection with the invention, the terms "binds”, "binding” or "bind” refer to specific binding to the antigen of interest. It is to be understood that where the terms "binds”, "binding” or "bind” are mentioned, they refer to the intrinsic capability of the CAR or TCR to specifically bind to the antigen without further modification, but they do not require that the antigen must actually be present.

[0063] In accordance with the invention, the term "T-cell receptor (TCR)” has the meaning known in the art. Typically, a TCR is understood as a heterodimeric cell surface protein of the immunoglobulin superfamily that participate in the activation of T cells in response to the binding of an antigen. The TCR complex can consist of TCRo / B chains and CD3y / 5 / E / subunits, which can associate through hydrophobic interactions. Somatic VDJ recombination allows to generate distinct TCRo and TCRS chains, and TCRoB heterodimers are generally responsible for antigen recognition by binding to peptide-MHC complexes. CD3 can transmit the TCR-triggered signal through immunoreceptor tyrosine-based activation motifs (ITAMs) in its cytoplasmic tail, but it is generally not directly involved in antigen recognition. ITAMs are tandem duplications of a tyrosine-containing sequence (YXXL / I), and the CD3y / 5 / E chains each contain one ITAM, while the CD3 chain contains three. As a consequence of TCR engagement, ITAM phosphorylation can be induced by protein tyrosine kinases (PTKs), which allow other effector molecules to interact with the TCR complex. A TCR can be found on the surface of a cell or in soluble form. The TCR can be an intact or full-length TCR, including but not restricted to a TCR in the oB form or yd form, as a dimeric TCR (dTCR), a single-chain TCR (scTCR). The TCR is an antigen-binding portion that is less than a full-length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC-peptide complex. In some cases, an antigen-binding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as MHC-peptide complex, to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable a chain and variable B chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of a TCR contain complementarity determining regions involved in recognition of the peptide, MHC and / or MHC-peptide complex. A TCR can contain a constant domain, a transmembrane domain and / or a short cytoplasmic tail. Each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end. A TCR can be associated with invariant proteins of the CD3 complex involved in mediating signal transduction.

[0064] In accordance with the invention, the term "chimeric antigen receptor (CAR)” has the meaning known in the art. Typically, a CAR is understood as a receptor protein that have been engineered to give T cells the new ability to target a specific antigen. The receptor is chimeric in that it combines both antigen-binding and T cell activating functions into a single receptor. CAR T cells can be derived either from T cells in a patient's own blood (autologous) or from the T cells of another, healthy, donor (allogeneic). Once isolated from a person, these T cells can be genetically engineered to express a specific CAR, which programs them to target an antigen, e.g., an antigen that is present on the cancer cell surface.

[0065] A CAR is designed to enhance the recognition and targeting of cancer cells or other diseased cells. A CAR according to the invention typically comprises, but is not limited to, three main components: an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signalling domain. The extracellular domain, often derived from an antibody fragment, provides specificity for the target antigen. The transmembrane domain anchors the receptor in the lymphocyte (e.g., T cell) membrane, while the intracellular signalling domain initiates activation signals upon antigen binding. When expressed in lymphocytes (e.g., T cells), the CAR can allow the modified lymphocytes (e.g., T cells) to recognize and bind to the target antigen, leading to the activation and killing of the target cells expressing the target antigen.

[0066] It will also be understood that the CAR can be any known type of CAR, e.g., a first-generation CAR, a second- generation CAR, a third-generation CAR, a fourth-generation CAR or a fifth-generation CAR. A first-generation CAR generally has an intracellular signalling domain comprising an intracellular signalling domain of CD3 , FcyRI, or other ITAM-containing activating domain to provide a T cell activation signal. Second and third- generation CARs further comprise a costimulatory signalling domain (e.g., a costimulatory signalling domain from an endogenous T cell costimulatory receptor, such as CD28, 4-1 BB, or ICOS) or two costimulatory signalling domains, respectively. A fourth generation CAR, instead, may express one or two costimulatory molecules together with a constitutive or inducible expression cassette containing a transgenic protein such as a cytokine or enzyme. A fifth generation CAR is known in the art and may comprise an additional intracellular domain compared to the first to fourth generation CAR. The CAR may comprise, but is not limited to, truncated intracellular domains of cytokine receptors (e.g., IL-2R chain fragment) with a motif for binding transcription factors such as STAT-3 / 5.

[0067] In accordance with the invention, the term "Natural Killer (NK) cell” has the meaning known in the art and is typically understood as a type of cytotoxic lymphocyte which is a subset of white blood cells that play a crucial role in the innate immune system. NK cells are known for their ability to recognize and eliminate target cells, such as virus-infected cells and tumour cells, without prior sensitization or the need for specific antigen recognition. Human NK cells can be identified by the presence of markers including, but limited to, CD56 and the absence of CD3 (CD56+, CD3").

[0068] In accordance with the invention, the term "Natural Killer T (NKT)” cell has the meaning known in the art and is typically understood as a specialized subset of T cells that exhibit characteristics of both natural killer (NK) cells and conventional T cells (e.g., the types of T cells referred to herein). They endogenously express a unique T- cell receptor (TCR) and recognize specific lipid antigens presented by a molecule called CD1d. NKT cells coexpress an op T-cell receptor, but also express a variety of molecular markers that are typically associated with NK cells, such as NK1.1 . NKT cells have the ability to produce immune-regulatory molecules, such as cytokines, upon activation. They are involved in modulating immune responses and play a role in both innate and adaptive immunity. NKT cells are known for their involvement in various immune processes, including antimicrobial defence, tumour surveillance, autoimmune diseases, and allergic reactions.

[0069] The term "vector” is known in the art and encompasses, for instance, a plasmid, a retroviral vector, a lentiviral vector, an adeno-virus vector, an adeno-associated virus vector or a transposon vector. It is understood that a vector as used in connection with the present invention is a vector which is suitable for therapeutic applications in humans.

[0070] The term "expression cassette” has the meaning known in the art. As used in accordance with the invention, an expression cassette typically comprises at least a promoter sequence, at least one open reading frame (e.g., an open reading frame encoding the at least one positive regulator of autophagy and / or an open reading frame encoding the T-cell receptor or chimeric antigen receptor), and a 3' untranslated region that usually contains a polyadenylation site. An expression cassette in accordance with the invention may be monocistronic or polycistronic (e.g., bicistronic). For example, a polycistronic (e.g., bicistronic) expression cassette may comprise an open reading frame encoding the at least one positive regulator of autophagy and an open reading frame encoding the T-cell receptor or chimeric antigen receptor. Polycistronic (e.g., bicistronic) expression cassettes are generally known in the art and may comprise, for example, linkages of the open reading frames by internal ribosome entry sites (IRES).

[0071] In addition, the expression cassette may appropriately comprise additional nucleotide sequences such as an adapter or a linker, an enhancer, a selectable marker (e.g., antibiotic resistance marker), a replication unit, a polyA sequence, a tag for purification (e.g., GST, poly-Arg, FLAG, histidine-tag (His-tag) or c-myc, etc..

[0072] In accordance with the methods for obtaining a modified human lymphocyte of the invention, the step of introducing the recombinant nucleic acid or set of recombinant nucleic acids into a human lymphocyte may be performed by using any appropriate standard techniques as known in the art, for example, electroporation, electro-injection, microinjection, calcium phosphate co-precipitation, a calcium chloride / rubidium chloride method, retroviral and lentiviral infection, DEAE-dextran, a cationic liposome method, polyethylene glycol- mediated uptake, gene guns, etc., but is not limited thereto. The nucleic acid or set of recombinant nucleic acids encoding the positive regulator of autophagy may be introduced prior to, simultaneously with, or after introduction of the nucleic acid or set of recombinant nucleic acids encoding and expressing the T-cell receptor or chimeric antigen receptor.

[0073] In accordance with the invention, the term "constitutive” in relation to the expression of recombinant nucleic acids has the meaning known in the art. It is understood that it encompasses the continuous and uniform expression of the recombinant nucleic acid. A constitutive expression is one that is active under normal or standard conditions in the modified human lymphocytes of the invention, regardless of environmental factors or specific cellular signals.

[0074] In accordance with the invention, the term "inducible” in relation to the expression of recombinant nucleic acids refers to expression that can be regulated or controlled by specific signals or factors, e.g., by drugs which are not toxic to humans and can be administered to humans. It is well known how inducible expression can be achieved. For example, the recombinant nucleic acids may contain inducible promoters to ensure the inducible expression. It is understood that such inducible promoters may form part of the expression cassettes referred to herein.

[0075] The term "selection marker” has the meaning known in the art and typically refers to a recombinant nucleic acid sequence to facilitate the identification and selection of cells or organisms that have successfully incorporated a desired genetic modification. For example, the selection marker can be one that confers a selectable phenotype, such as resistance to a specific antibiotic or the ability to grow in a selective medium. By including the selection marker in the same expression cassette, a selective pressure can be applied, such as exposure to the antibiotic or selective medium, to identify and isolate the cells or organisms that have taken up the desired genetic modification. In accordance with the invention, an expression cassette containing selection marker can be used to facilitate the identification and selection of modified lymphocytes of the invention.

[0076] In accordance with all other embodiments of the present invention, a recombinant nucleic acid according to the invention can preferably be a recombinant DNA. Thus, in a preferred embodiment, all recombinant nucleic acids according to the invention are recombinant DNAs.

[0077] In accordance with the invention, the term "cancer cell antigen” has the meaning known in the art. In those embodiments of the invention where the lymphocyte contains and expresses a recombinant nucleic acid or a set of recombinant nucleic acids encoding a chimeric antigen receptor, the cancer cell antigen in accordance with the invention to which the chimeric antigen receptor binds is preferably a cancer cell surface antigen. In those embodiments of the invention where the lymphocyte contains and expresses a recombinant nucleic acid or a set of recombinant nucleic acids encoding a T-cell receptor, the cancer cell antigen in accordance with the invention to which the T-cell receptor binds is preferably an intracellular cancer cell antigen presented by a HLA molecule.

[0078] In accordance with the invention, the cancer can be any known cancer but is preferably solid cancers, a brain cancer, neuroblastoma, sarcoma but also other paediatric or adult cancers.

[0079] Methods of Treatments and Uses of Lymphocytes in such Treatments

[0080] Terms such as "treatment of cancer” or "treating cancer” according to the present invention refer to a therapeutic treatment. An assessment of whether or not a therapeutic treatment works can, for instance, be made by assessing whether the treatment inhibits cancer growth in the treated patient or patients. Preferably, the inhibition is statistically significant as assessed by appropriate statistical tests which are known in the art. Inhibition of cancer growth may be assessed by comparing cancer growth in a group of patients treated in accordance with the present invention to a control group of untreated patients, or by comparing a group of patients that receive a standard cancer treatment of the art plus a treatment according to the invention with a control group of patients that only receive a standard cancer treatment of the art. Such studies for assessing the inhibition of cancer growth are designed in accordance with accepted standards for clinical studies, e.g. double-blinded, randomized studies with sufficient statistical power. The term "treating cancer” includes an inhibition of cancer growth where the cancer growth is inhibited partially (i.e. where the cancer growth in the patient is delayed compared to the control group of patients), an inhibition where the cancer growth is inhibited completely (i.e. where the cancer growth in the patient is stopped), and an inhibition where cancer growth is reversed (i.e. the cancer shrinks). An assessment of whether or not a therapeutic treatment works can be made based on known clinical indicators of cancer progression. A treatment of cancer according to the present invention does not exclude that additional or secondary therapeutic benefits also occur in patients. However, it is understood that the primary treatment for which protection is sought is for treating the cancer itself, and any secondary or additional effects only reflect optional, additional advantages of the treatment of cancer growth.

[0081] The treatment of cancer according to the invention can be a first-line therapy, a second-line therapy, a third-line therapy, or a fourth-line therapy. The treatment can also be a therapy that is beyond fourth-line therapy. The meaning of these terms is known in the art and in accordance with the terminology that is commonly used by the US National Cancer Institute.

[0082] The term "immunotherapeutic treatment of cancer” has the meaning known in the art and generally relates to a treatment of cancer in which the immune system of the patient is used to treat the cancer. Cancer cells harbor genomic mutations which give rise to cancer cell antigens that are specific to the cancer cells and different from the antigens of non-cancerous cells. Thus, in a preferred aspect of an immunotherapeutic treatment of cancer in accordance with the present invention, an immunotherapeutic treatment of cancer is a treatment wherein such cancer cell antigens are recognized by the modified lymphocytes of the invention, and wherein cancer cells expressing these antigens are killed. An immunotherapeutic treatment of cancer can be assessed by immunomonitoring methods known in the art, e.g. by measuring intracellular IFN-y expression (e.g. in CD8+T- cells and / or NK cells) in blood samples, measuring CD107a cell surface expression (e.g. on CD8+T-cells and / or NK cells) in blood samples, measuring intracellular TNF-o expression (e.g. on leukocytes) in blood samples, intracellular lnterleukin-2 expression (e.g. in CD8+T-cells and / or in CD4+T-cells) in blood samples, CD154 cell surface expression (e.g. in CD8+T-cells and / or in CD4+T-cells) in blood samples, tetramer or dextramer staining for tumour antigen- specific T cells in blood samples, CTL activity against autologous tumour cells or presence of T cells against neoantigens derived from tumour-specific mutations. Preferred methods to assess cancer immunotherapy are the methods according to Gouttefangeas C et al.: "Flow Cytometry in Cancer Immunotherapy: Applications, Quality Assurance and Future.” (2015) In: Cancer Immunology: Translational Medicine from Bench to Bedside (N. Rezaei editor). Springer. Chapter 25: pages 471-486; and the methods according to Van der Burg SH, et al.: "Immunoguiding, the final frontier in the immunotherapy of cancer.” (2014) In Cancer Immunotherapy meets oncology (CM Britten, S Kreiter, M. Diken & HG Rammensee eds). Springer International Publishing Switzerland p37-51 ISBN: 978-3-319-05103-1.

[0083] In accordance with the present invention, each occurrence of the term "comprising” may optionally be substituted with the term "consisting of'.

[0084] Methods for obtaining a modified human lymphocyte The methods for obtaining a modified human lymphocyte according to the invention are as defined herein, including the claims. They may be performed ex vivo, e.g., using isolated human lymphocytes as starting material. That is, preferably, the method for obtaining a modified human lymphocyte according to the invention is not a method for treatment of the human or animal body by surgery or therapy and is not a diagnostic method practiced on the human or animal body.

[0085] Preparation of pharmaceutical compositions of the Invention

[0086] Pharmaceutical compositions of the present invention are prepared in accordance with known standards for the preparation of pharmaceutical compositions.

[0087] For instance, the pharmaceutical compositions are prepared in a way that they can be stored and administered appropriately. The pharmaceutical compositions of the invention may therefore comprise pharmaceutically acceptable components such as carriers, excipients and / or stabilizers.

[0088] Such pharmaceutically acceptable components are not toxic in the amounts used when administering the pharmaceutical composition to a human patient. The pharmaceutical acceptable components added to the pharmaceutical compositions may depend on the chemical nature of the active ingredients present in the composition, the particular intended use of the pharmaceutical compositions and the route of administration.

[0089] In general, the pharmaceutically acceptable components used in connection with the present invention are used in accordance with knowledge available in the art.

[0090] Positive regulators of autophagy

[0091] Preferred positive regulators of autophagy are as defined in the preferred embodiments and in the claims.

[0092] In accordance with the invention, preferred positive regulators of autophagy are selected from a list comprising the following human proteins: human TFEB, human Beclin-1 , human AMBRA1, human ULK1, human ATG14, human STYK1 (also known as WIPI 1), human WIPI2, human SQSTM1 (also known as p62), human PIK3C3 (also known as VP34), human ATG4A, human ANXA2 (also known as Annexin A2), human PKD2 (also known as Polycystin 2), human GABP (also known as NFT2), human ATG7, and human ATG5.

[0093] The regulators human TFEB and human Beclin-1 were experimentally tested in the non-limiting examples below.

[0094] The proteins AMBRA1 , ULK1 , ATG14, STYK1, WIPI2, SQSTM1 , PIK3C3, ATG4A, ANXA2, PKD2, GABP, ATG7, and ATG5 are positive regulators of autophagy and have been shown to induce autophagy when overexpressed in cells. They are involved in the upstream regulation of the autophagic process and regulate the formation of autophagosomes (see references 56-77 included in the references section). According to the teaching of the present invention, these proteins can also be used as positive regulators of autophagy in the modified lymphocytes of the invention and are useful to improve the treatment of cancer in this context.

[0095] Similarly, the peptides TNVFNATFHIWHSGQFGT (SEQ ID NO: 1) and TNVFNATFEIWHDGEFGT (SEQ ID NO: 2) were shown to induce autophagy. This has been reported in Shoji-Kawata et al., Nature. 2013 Feb 14;494(7436):201 -6. According to the teaching of the present invention, these peptides can also be used as positive regulators of autophagy in the modified lymphocytes of the invention and are useful to improve the treatment of cancer in this context.

[0096] The present invention is further illustrated by the following non-limiting examples:

[0097] EXAMPLES

[0098] Methods and Techniques

[0099] Cell lines. Neuroblastoma (NB)-derived cell line SHSY5Y and the immortalized human embryonic kidney 293T were obtained from DSMZ (Braunschweig, Germany). The D341-MED Medulloblastoma and A673 (Sarcoma) cell lines were purchased from ATCC (Washington DC, USA), the U373.MG from ECACC (Salisbury, United Kingdom). The CHLA 255 was kindly provided by Prof. Malcolm Brenner from Baylor College of Medicine). SHSy5Y, CHLA255, U373.MG, CT1036and A673 were maintained in culture with DMEM medium (Merck - Darmstadt, Germany) supplemented with 10% foetal bovine serum (FBS) (Thermo Scientific, Pittsburgh, PA - USA) and 2 mM GlutaMax (Gibco, Thermo Scientific) while the D341-MED were cultured in MEM (Thermo Scientific) supplemented with 20% FBS, 1 % sodium pyruvate, 1 % non-essential amino acids and 2 mM GlutaMax. Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C. All cell lines were routinely tested for mycoplasma and for surface expression of target antigens as well as authenticated by STR analysis (Eurofins Genomics, Ebersberg - Germany).

[0100] Isolationand transduction of CAR T cells. Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats obtained from healthy donors (University Hospital Wurzburg, Wurzburg, Germany) who signed a written informed consent, in accordance with the rules set by the Institutional Review Board (Approval number 250 / 20-am), using Lymphocyte separation medium Ficoll (Ge Healthcare, Uppsala - Sweden). T lymphocytes were activated with immobilized OKT3 (1 mg / ml, Thermo Scientific) and anti-CD28 (1 mg / ml, BD Biosciences, Europe) monoclonal antibody (mAb) in complete medium consisting of GTS OpTmizer T Cell Expansion medium (Thermo Scientific) supplemented with 2.5% human AB serum (AnProtec, Bruckberg - Germany), 2 mM GlutaMax in a humidified atmosphere containing 5% CO2 at 37°C. The day after activation, T cells were fed with recombinant human interleukin 2 (IL2, 100U / ml; Miltenyi Biotec, Germany) or IL7 (500 U / ml; Miltenyi Biotec) and IL15 (50 U / ml; Miltenyi Biotec). Activated T cells were transduced on day 3 in 24-well plates precoated with recombinant human RetroNectin (Takara-Bio. Kyoto - Japan) using a specific retroviral supernatant and the above-described cytokines. On day 5 after transduction, T cells were removed from RetroNectin and expanded in complete media17’28.

[0101] Isolation and expansion of NK cells. NK cells were isolated from buffy coats obtained from healthy donors (University Hospital Wurzburg) who signed a written informed consent, in accordance with the rules set by the Institutional Review Board (Approval number 250 / 20-am), using first Lymphocyte separation medium Ficoll (Ge Healthcare,) and then Rosette kit (Stem Cells, Cologne - Germany). NK cells were then cultured in NK media (Miltenyi) supplemented with 5% human AB serum either in presence of feeder cells and IL-2 (1 OOU / ml) or with IL-2 / IL-15 (500 U / ml and 150U / ml, respectively). Cells were then fed every 3-4 day and kept in a humidified atmosphere containing 5% CO2 at 37°C.

[0102] Isolation and expansion of y / 5-T cells. y / 5-T cells were isolated from buffy coats obtained from healthy donors (University Hospital Wurzburg) who signed a written informed consent, in accordance with the rules set by the Institutional Review Board (Approval number 250 / 20-am), using first Lymphocyte separation medium Ficoll (Ge Healthcare,) and then Rosette kit (Stem Cells). y / 5-T cells were then cultured in complete medium consisting of CTS OpTmizer T Cell Expansion medium (Thermo Scientific) supplemented with 2.5% human AB serum (AnProtec), 2 mM GlutaMax in a humidified atmosphere containing 5% CO2 at 37°C in presence of IL-2 / IL-15 (100 U / ml and 150U / ml, respectively) and zoledronic acid (10 pM). Cells were then fed every 3-4 days.

[0103] Retroviral constructs, y-retrovirus constructs have been generated using a SFG backbone37and encode for: Beclin-1 or TFEB in frame with an internal ribosome entry site (IRES) and a truncated CD 19 (ACD19) selectable marker or the enhanced green fluorescence protein (eGFP); a control vector encoding only for IRES.ACD19 or IRES.eGFP; a third-generation GD2.CAR with the CD28.4-1 BB as costimulatory molecules and the zeta-chain (0 as the signal endo-domain27and the above-mentioned third-generation GD2.CAR in frame with I RES. Bed in- 1 or IRES.TFEB or IRES. Beclin-1 peptide38(TNVFNATFHIWHSGQFGT) or IRES.ATG5. An additional retroviral vector encoding eGFP-Firefly-Luciferase (eGFP / FFLuc) was used in selected experiments to label tumour cells for in vitro co-culture and in vivo studies as previously described39. In selective experiments, also the retrovirus vectors encoding a third generation CD30.CAR and B7-H3.CAR with the CD28.4-1 BB were used40.

[0104] Generation of cytomegalovirus specific T cells. To generate cytomegalovirus specific T lymphocytes (CMV CTLs), PBMCs from CMV seropositive donors were stimulated with dendritic cells (DCs) at a ratio of 20: 1 loaded with the CMV-pp65 pepmix (HCMVA, JPT, Berlin, Germany) at 5 mmol / L for 2 hours at 37°C in 5% CO2. Cells were then plated in complete T cell media. After 10 days, T cells were restimulated with DCs loaded with the same pepmix. After the second round of stimulation, cells were expanded and fed with IL7 (500 U / ml) and IL15 (50 U / ml)41.

[0105] Phenotypic analysis. Expression of cell surface molecules was determined by flow-cytometry using standard methodology. The following mAbs were used: CD3, CD4, CD8, CD19, CD25, CD27, CD28, CD30, CD45RA, CD45RO, CD56, CD57, CD62L, CD95, CD127, CD197, CD223 (Lag3), CD279 (PD1), TIM3 and GD2 (Miltenyi and BioLegend, San Diego, CA - USA). The expression of GD2.CAR on T cells was detected using a specific anti-idiotype antibody (1A7)42. For the quantification of mitochondria mass and mitochondria potential, 100 nM of MitoTRK Green and 250 nM of MitoTRK Orange (Thermo Scientific) has been used following the manufactory procedure25. Samples were acquired with a BD FACS Canto II and analysed using the Flowlogic software (I nival, Melbourne - Australia). For each sample, a minimum of 25,000 events were analysed.

[0106] Quantitative RT-PCR (qPCR) analysis. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands) and 10Ong reversely transcribed with the cDNA SuperScript™ VILO™ cDNA Synthesis Kit (Applied Biosystems, Foster City, CA, USA). RT-PCR was performed using SYBR Green Master mix (Bio-RAD, Helculaes, California, USA) following the manufacturer's indications using QuantStudio 6. Relative expression values were normalized to the housekeeping gene GAPDH. The following primers were used: AMBRA1 Forward 5'-CTCACAGCAATTGTTAACCCC-3', AMBRA1 Reverse 5'-CAGGAAATTGTGGAGGAGAGG-3', LC3 Forward 5'-GATGTCCGACTTATTCGAGAGC-3', LC3 Reverse 5'-TTGTTTTATCCAGAACAGGAAAGC-3', p62 Forward 5'-GGAGCAGATGAGGAAGATCG-3', p62 Reverse 5'-TGGGTCCAGTCATCATCTCC-3', GAPDH Forward 5'-CCCCTTCATTGACCTCAACTACAT-3' and GAPDH Reverse 5'-CGCTCCTGGAAGATGGTGA-3'. In selected experiments pre-coated autophagy pathway assay from Bio-RAD was used.

[0107] Western blot analysis. Proteins were extracted from 5x10® cells, using RIPA lysing buffer (Cell Signaling Technology, Danvers, Massachusetts, USA) supplemented with a protease inhibitor cocktail (Merck, Darmstadt, Germany). Fifty mg of protein were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Bio-RAD), and blocked with 5% (WA / ) non-fat dry milk in Tris-buffered saline with 0.1 % (VAX) Tween-20. Blots were incubated with primary antibodies in 5% non-fat dry milk in PBS plus 0.1 % Tween20 overnight at 4°C. Detection was achieved using horseradish peroxidase-conjugate secondary antibody (Bio-RAD) and visualized with ECL plus (Amersham Bioscience, Slough, Buckinghamshire, UK).

[0108] Immunofluorescence. Cells were cultured in complete T cell medium (or starved in Earle's Balanced Salt Solution 1X (Merck) and subsequently seeded at a density of 1x10^ / L on chamber slides coated with fibronectin 10ng / ml for 1 h at room temperature in presence or not of autophagy inhibitors Bafilomycin. Primary antibodies used for staining LC3 puncta were diluted 1 :300 and conjugated with secondary antibodies diluted 1 :300. Images were then acquired with Olympus confocal microscope and analyzed by Imaged.

[0109] Seahorse extracellular flux analysis. Seahorse experiments were performed using XF Cell Mito Stress kit (Seahorse Bioscience). OCR and ECAR were measured with XF96 Extracellular Flux Analyzers (Seahorse Bioscience). Briefly, cells were plated on poly-D-lysine-coated 96-well polystyrene Seahorse plates (200,000 T cells / well), equilibrated for 1 h at 37°C and assayed for OCR (pmol / min) and ECAR (mpH / min) in basal conditions and after addition of oligomycin (1 pM), carbonyl cyanide-4-phenylhydrazone(1.5 pM) and antimycin A / rotenone (1 pM / 0.1 pM).

[0110] Co-culture assay. For co-culture experiments, untransduced T cells (NT), Beclin-1 .IRES.ACD19, TFEB.IRES.ACD19, GD2.CAR. I RES. Beclin-1, GD2.CAR.IRES.TFEB, GD2.CAR.IRES.Beclin1 peptide or GD2.CAR.IRES.ATG5 T lymphocytes plated in 24-well plates at the indicated E:T ratios with eGFP modified tumour cell lines (SHSy5Y, CHLA255, U373.MG, D341.MED, CT10 and A673). Following 5 days of incubation at 37°C, co-cultures were collected and analysed by FACS to detect residual tumour cells (GFP+) and T cells based on CD3 expression6.

[0111] 3D spheroid co-culture experiment. Tumour spheroids were generated using 96-well ultra-low attachment plates. Neuroblastoma cells were seeded at a low concentration of 1 ,000 cells per well in a total volume of 10Oul of the specific medium and 0.24% methylcellulose per well. Medulloblastoma cells were plated at a low concentration of 250 cells per well and cultured in 100pil MEM supplemented with 20% FBS and 0.24% methylcellulose per well. Then, the plates were centrifuged at 50xg for 1 min without acceleration and without brake at room temperature and afterwards placed in the incubator for 72h. Then, plates were subjected to automated imaging using a Celigo Image Cytometer to assess the pre-co-culture fluorescence intensity. At this point, NT T cells and CAR-T cells were added at different E:T ratios and the plates cultured for other 10 days at 37°C while being subjected to day-by-day automated imaging.

[0112] Extra-cellular vesicle detection. Cell supernatants were collected 24-48h after medium change. Afterwards, 100 pl of cell supernatants were stained with Lipophilic Cationic Dye (LCD) and Phalloidin (FITC) for 45 minutes in the dark at room temperature (RT). Samples were then properly diluted with PBS and acquired using a CytoFLEX flow cytometer (Beckman Coulter, Sacramento, CA, USA) equipped with a volumetric count. The threshold was set to the APC channel, the flow rate was adjusted to acquire max 7,000 events / second and at least 1 million events per sample were recorded.

[0113] Next generation sequencing. Next generation sequencing experiments were performed by Genomix4life (Baronissi, Salerno, Italy). RNA concentration in each sample was assayed with a ND-1000 spectrophotometer (NanoDrop) and its quality assessed with the Agilent Tapestation 4200 (Agilent Technologies). Indexed libraries were prepared from 1 pg / ea purified RNA with TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to the manufacturer's instructions. Libraries were quantified using the Agilent TapeStation 4200 (Agilent Technologies) and pooled such that each index-tagged sample was present in equimolar amounts, with final concentration of the pooled samples of 2 nM. The pooled samples were subjected to cluster generation and sequencing using an Illumina Nextseq 500 (Illumina) in a 2A~ 75 paired-end format at a final concentration of 1 .8 pmol. The raw sequence files generated (.fastq files) underwent quality control analysis using FastQC Fastq underwent Quality Control using FastQC tool (http: / / www. bioin forma tics, babra ham. ac. uk / proje cts / astqc / ). The mapping of paired-end reads was performed using STAR (version 2.5.2a)43on reference genome assembly hg19 obtained from Ensembl (GRCh37.p13)44(https: / / grch37. ensem bl. org / Homo_ sapie ns / Info / Index). The quantification of transcripts expressed for each sample was performed using FeatureCount45, algorithm. DESeq246was used to normalize the data and then to perform the differential expression analysis.

[0114] Xenograft mouse model for in vivo studies. To investigate the in vivo antitumour activity of autophagy reprogrammed CAR T cells, 0.75x106SHSY5Y-FF-Luc.GFP were intraperitoneally injected in presence of matrigel, in 5 weeks old NOD.Cg-Prkdcscid Il2rgtm1 Wjl / SzJ male mice (Charles River). After engraftment, mice received an intravenous injection (i.v.) of 10x106of NT or genetically modified T cells. Tumour growth was evaluated using I VIS imaging system (Perkin Elmer). All in vivo experiments were conducted in compliance with the ethical international, EU and national requirements and were approved by the Italian Health Ministry (N 195 / 2021 -PR).

[0115] Statistical analysis. Data are summarized as mean ± standard deviation (SD). Student t-test (two-sided) was used to determine statistically significant differences between samples, with p<0.05 indicating a significant difference. When multiple comparison analyses were required, statistical significance was evaluated by a repeated measures ANOVA followed by a Log-rank (Mantel-Cox) test. Graph generation and statistical analyses were performed using Prism v9 software (GraphPad, La Jolla, CA, USA). Significant p values have been shown in the graphs using the following reference: * is used for p values of 0.01-0.05; ** for p values of 0.001-0.009; *** for p values of 0.0001-0.0009; **** for p values <0.0001.

[0116] Results

[0117] Example 1 : Autophagy pathway is blocked in ex-vivo manipulated T lymphocytes.

[0118] In order to solve the unexplored biological question regarding how autophagy is regulated in ex-vivo manipulated T lymphocytes, first freshly isolated primary T cells from healthy donors and activate them polyclonally with OCD3 / CD28 mAb using a protocol normally applied for the generation of gene-modified T lymphocytes. The T cells were then analysed at different time points after activation (Day+1 , +2, +3, +7) until day 14, the average day for the ex-vivo culture of ATMP. As reported in Figure 1 A, immediately after activation LC3, Ambral , p62 and ULK1 autophagy related mRNA are significantly downregulated in all the analysed time points (p<0.0001). This down-regulation was still observed also when expanded T cells were restimulated polyclonally (day 15). The data were then verified using a commercially available assay for the autophagy monitor which revealed the magnitude of the autophagy block underlined the downregulation of other autophagy related transcripts as well as the upregulation of BNIP3 and Caspase-3 mRNA reported to have a negative impact on the autophagy activation47’48(Figure 1 B). It was then proofed that this phenomenon of down regulation of the autophagic pathway involves both the CD4 and CD8 populations with the same magnitude and significance observed in the total CD3 population (p<0.0001) (Figure 1C-D). Based on this observation, 3 different CAR redirected T cells (GD2.CAR, CD30.CAR and B7-H3.CAR) were tested on day 14 after transduction, each of one generated from a different healthy donor and the related non-genetically modified T cells (NT) in real time PCR for the expression of the above-mentioned autophagy related mRNAs (LC3, Ambral, p62 and ULK1). Figure 1 E shows how also in this case both NT and CAR-T cells, regardless of the construct, show a strong and significant downregulation of the 4 autophagic transcripts compared to the corresponding freshly isolated and non-cultured T cells (p<0.0001). The data were then validated also at protein level through western blot analysis which revealed in different prospective a clear block of the autophagic process as demonstrated by the accumulation of p62, the reduction / absence of LC3II, an increase of p-mTOR, the significant accumulation of p-ULK1 and the downregulation of p-AMPK (Figure 1 F and 1 K). Further data analysis of both the CD4+and CD8+CAR T cell population at day 14 of culture confirmed the observation on the total CAR T cell population (Figure 1 G). It was proved that this phenomenon is not due to the use of a specific cytokine (IL2) or the strategy used to activate the T cells (oCD3 / CD28 mAb). In fact, as reported in Figure 1 H, the autophagy block was detectable also when CAR T cells were activated and expanded with IL7 / 1549and when T cells were activated with professional antigen presenting cells pulsed with the pp65-CMV peptide in order to generate CMV specific CTLs37 50. Having at this point clearly demonstrated how the ex-vivo manipulation of T lymphocytes can induce a significant and lasting block of the autophagic process, the inventors tried to verify if it was possible to pharmacologically re-induce the activation of the autophagic pathway in CAR T cells. Therefore, GD2.CAR at day 14 of culture were treated with Torinl (250nM)51, an effective inducer of autophagy capable to block phosphorylation of mTOR, with or without IL2 for 6h and 24h. It was found that, at least pharmacologically, autophagy can be restored in ex-vivo expanded T cells also when strong stimulus (cytokine) is provided. It was observed in fact that overall level of ULK1 remains unchanged, its phosphorylation is reduced already after 6h of treatment while a reduction of p62 can be observed only after 24h from the addition of the drug (Figure 1 J).

[0119] Based on these results and in order to investigate if autophagy block is a general phenomenon due to the ex- vivo culture, we analysed other two platforms used for adoptive cell therapy, natural killer (NK) and y5-T cells. As performed for the conventional T cells (a / p), NK and y5-T cells were isolated form heathy donors and ex- vivo expanded. Also in this scenario, we observed that in both the cellular products after 14 days of culture a strong autophagy block can be detected both at mRNA and protein level (Figure 1L-M).

[0120] Example 2: The constitutive reactivation of the autophagic process is not harmful to ex vivo expanded T cells. The immediate translational implication of this findings is that T cells ex-vivo manipulated and cultured for adoptive immunotherapy lack a well-functioning autophagic process that could help them survive in hostile environments and react to stressful situations ensuring their longer persistence and better cellular fitness, features demonstrated to be crucial for a lasting anti-tumour response. Based on these data and on the fact that the autophagic process could be re-activated, the inventors went to verify if the autophagic pathway could be constitutively re-activated through the overexpression of key proteins involved in the process. They selected two autophagic proteins, TFEB and Beclinl , and generated retroviral constructs. Since these two proteins are intracellular molecules, a selection marker was inserted, ACD19, into the constructs, which allows to monitor the transduced cells with an analytical method such as flow cytometry. The constructs on immortalized tumour cells, Hek293T, was first validated by observing that the expression of the surface marker, ACD19, remained constant over time after transduction at both the percentage (Figure 2A) and intensity level (MFI) (Figure 2B). It was confirmed that TFEB and Beclinl were then upregulated accordingly at both the transcript (p<0.0001) (Figure 2C) and protein level (Figure 2D).

[0121] Therefore, the constructs were tested on primary T cells. In all cases tested, T lymphocytes transduced with the constructs ACD19+control, TFEB+ACD19 and Beclinl +ACD19 showed no signs of toxicity and reported a level of expansion comparable to that of the respective non-genetically modified T cells (NT) (Figure 3A). Transduction levels were greater than 60% and remained unchanged over time both as absolute percentage level (Figure 3B) and the signal intensity level of the ACD19 marker (Figure 3C). Although TFEB expression was always lower compared to the other two conditions, this difference was not significant.

[0122] As demonstrated previously, cells genetically modified with TFEB and Beclinl were able to constitutively overexpress both the corresponding transcripts (Figure 3D) (p<0.0001) and the proteins (Figure 3E). No differences were observed in terms of the distribution of CD4 and CD8 cell subpopulations under any of the conditions tested. Interestingly, an increase in mitochondrial potential (Mitrotracker) was observed in the T cell population genetically modified with the retroviral construct encoding Beclinl . Furthermore, the analysis of the autophagic flux reveals that through the overexpression of the autophagic genes it is possible to modulate the pathway by reactivating it (Figure 3H-I). Figure 31 demonstrates how autophagy is active in autophagy gene- modified T cells in normal culture condition (basal level) (p<0.05) and above all when the cells are exposed to stress conditions such as those caused by nutrient starvation (EBS) (p<0.001). Lastly, it was demonstrated that the overexpression of TFEB and Beclinl is not associated with an increase of alloreactivity since they are not able to kill the CHLA255 neuroblastoma cell line at the ratio 1 : 1 in a short-term 24h killing assay (Figure 3J).

[0123] Example 3: The overexpression of TFEB and Beclinl in GD2.CAR T cells restores autophagy pathway and improves their phenotype and cytotoxic activity.

[0124] So far, the inventors were able to demonstrate that it was possible to restore in ex-vivo expanded T cells the autophagic pathway without any toxic effects by the overexpression of the 2 autophagic proteins TFEB and Beclinl . They decided then to translate these results into a CAR platform. They therefore generated new retroviral bicistronic constructs encoding a third generation GD2.CAR and the TFEB or Beclinl proteins. The two expression cassettes were separated by an IRES sequence. As demonstrated in Figure 4A, no differences in terms of proliferation were observed between the GD2.CAR+TFEB and GD2.CAR+Beclin1 constructs compared to the GD2.CAR control. However, all the constructs encoding the third generation GD2.CAR showed, as already reported by the inventors in a previous study27, a significant increase in proliferation on day 10 of culture compared to the non-genetically modified control cells (NT) (p<0.05). Transduction levels, monitored by flow cytometry with a GD2.CAR anti-idiotype mAb (1A7)52, were greater than 75% with no statistical difference between groups. Furthermore, the levels of transduction remained constant over time, confirming the absence of toxicity following the overexpression of autophagic proteins. These data were confirmed both as absolute transduction percentage (Figure 4B) and CAR expression intensity (MFI) (Figure 4C). At mRNA level, the two transcripts were confirmed to be significantly overexpressed in the corresponding transduced cells (TFEB: p<0.0001 ; Beclinl : p<0.01). However, a discrepancy in terms of expression was observed between TFEB and Beclinl expressing cells, as the first one showed expression levels 3 times higher than the others (Figure 4D). Furthermore, the expression levels of both autophagic proteins were markedly lower than that observed in HEK293T cells (Figure 2C). These findings were also observed at the protein level (Figure 4E). It is also important to underline that they did not observe any difference in the expression and distribution of the transgenic molecules between the various conditions analysed in both CD4 and CD8 T cell subpopulations.

[0125] The inventors then characterized the metabolic state of the autophagy restored GD2.CAR T cells both on basal levels and in response to stress with the use of the extracellular flux analyser SeaHorse. The energetic map revealed both the autophagy restored GD2.CAR T cells as more energetic under both basal (Time: 0 to 18 minutes) and stressed (Time: 38 to 58 minutes; Uncoupler of mitochondrial oxidative phosphorylation - FCCP: 1 ,5 piM) conditions (Figure 4G). Indeed, both the autophagy restored GD2.CAR T cells displayed a higher 02 Consumption Rate (OCR) on the basal state in comparison to normal GD2.CAR, which was indicative of their augmented OXPHOS. Most importantly, upon disruption of mitochondria complexes with FCCP, both the autophagy restored GD2.CAR T cells showed a significant increase in maximal respiration levels, suggesting that both the autophagy restored GD2.CAR T cells may respond better to increased energetic demands and adapt to stress conditions. Here it is important to note that the response to the stress induced by FCCP is more marked in cells expressing TFEB, but this could be due to the unequal expression of the two proteins in the two constructs.

[0126] Furthermore, the metabolic profile analysing glycolysis were extended, which has been reported essential for the exertion of effector functions under stress53. The glycolytic activity was therefore evaluated by analysing the extracellular acidification rate (ECAR) observing a higher metabolic activity of GD2.CAR+Beclin in basal condition compared to the GD2.CAR+TFEB and normal GD2.CAR even in the presence of the unfavourable Beclinl amount (Figure 4H).

[0127] However, both GD2.CAR expressing autophagic proteins have a better response under stress conditions induced by treatment with Oligomycin (1 pi M) indicating their better activity than normal GD2.CAR T cells (Figure 4H).

[0128] These data corroborate the reactivation of the autophagic pathway shown in Figure 4I, where an accumulation of the LC3II protein was demonstrated when autophagy restored GD2.CAR T cells are exposed to a drug capable of blocking the autophagic process by blocking the fusion of autophagosomes with lysosomes (chloroquine). The phenomenon is even more accentuated when the cells are subjected to severe stress such as that induced by nutrient deprivation. The reactivation of the pathway is then also demonstrated by the accumulation of other proteins involved in the process such as ATG14, p-ATG14 and ULK1 (Figure 4J). All these molecular data were then reconfirmed by a second inhibitor of the autophagic pathway, Bafolomycin A1 (Figure 4K-L). The results were then confirmed using a further method which allows the visualization and quantification of the LC3 protein per cell (Figure 4M). Also here, the inventors were able to demonstrate that under nutritional stress conditions, cells expressing autophagic genes have a greater quantity of autophagosomes (LC3 per cell) (p<0.05).

[0129] Example 4: Immunocharacterization of autophagy reprogrammed CAR T cells.

[0130] Several pre-clinical and clinical studies have clearly demonstrated the importance of some immunophenotypic aspects to ensure a long-lasting anti-tumour response of T and NK-based ATMPs including an undifferentiated T cell phenotype, the expression of activation markers and the absence of exhaustion. Therefore, a deep immunophenotypic characterization of the GD2.CAR T cells was performed, observing that Beclinl overexpression is able to significantly increase both in the CD4+CAR+and CD8+CAR+populations the amount of naive cells (CD45RA+CD62L+) and reduce that of effector memory cells (EM) (CD45RO+CD62L-) compared to conventional GD2.CAR T cells (CD4 naive: p<0.001 ; CD8 naive: p<0.05; CD4 EM: p<0.0001 ; CD8 EM: p<0.001). A statistical difference was also observed between GD2.CAR+Beclin1 and GD2.CAR+TFEB T cells especially in CD4+CAR+subpopulation where the percentage of naive and EM cells were higher and lower respectively in CAR T cells expressing Beclinl (naive: p <0.01 ; EM: p<0.01). A statistical difference was observed only in the CD8+CAR+EM subpopulation between GD2.CAR+Beclin1 and GD2.CAR+TFEB where Beclinl-expressing cells showed lower levels than TFEB-expressing cells (p<0.01) (Figure 5A-B). Although not statistically significant, an increase in the stem cell memory like (SCMT) subpopulation in cells where the autophagy pathway was reactivated was also observed. This cell population has been reported to be extremely important for the long-term response of CAR-T therapy54 55. This increase was similar in the CD4+CAR+ subpopulation between TFEB and Beclinl but more pronounced in Beclinl+T cells in the CD8+CAR+subpopulation (Figure 5C). In none of the analysed conditions an exhausted phenotype was found (Figure 5D- G). However, regardless of the construct used, the CAR T cells show a good level of activation. A significantly increased percentage of the activation marker Lag3 was observed in GD2.CAR+Beclin1 T cells compared to both GD2.CAR and GD2.CAR+TFEB T cells in both CD4+and CD8+CAR+subpopulations (p<0.05 and p<0.001 , respectively) (Figure 5D-G). Also two other activation markers, HLA-DR and CD25 (only in the CD8+CAR+subpopulation), were found to be increased in terms of expression intensity (MFI) in the GD2.CAR+Beclin1 T population compared to GD2.CAR+TFEB (HLA-DR_CD4+CAR+: p<0.05; HLA- DR_CD8 CAR+: p<0.01 ; CD25_CD8+CAR+: p<0.05) and GD2.CAR T cells (CD25_CD8+CAR+: p<0.01) (Figure 5H-K). Lastly, the characterization of GD2.CAR+Beclin1 / TFEB was completed by performing short- (24h) and longterm (4 days) cytotoxicity studies using 12 days or 24 days ex-vivo expanded T cells and co-cultivating them with 3 different tumour cell lines: SHSy5Y (neuroblastoma), CHLA255 (neuroblastoma) and D341 (medulloblastoma) at different effectortarget ratios. As reported in Figure 6, in general a better trend of antitumour activity was detected for both constructs, even if more accentuated with the GD2.CAR+Beclin1 construct, when compared to the GD2.CAR control. Better results are usually observed at unfavourable effectontarget ratios (more tumour cells than T cells - a condition closest to that present in patients). In shortterm co-cultures with T cells at day 12, GD2.CAR+TFEB cells often showed an improvement in tumour cell killing without achieving significant improvement. Furthermore, GD2.CAR+Beclin1 T cells always showed a significant improvement compared to GD2.CAR+TFEB T cells (p value between 0.05 and 0.01) (Figure 6A-C; A: SHSy5Y, B: CHLA255; C: D341). However, when the co-cultures were conducted with T cells at day+24 (more exhausted), the results showed a greater and significant increase in the anti-tumour activity of TFEB and Beclin 1 restored T cells even if the data were, once again, better with cells overexpressing Bed in 1 (Figure 6D- F; D: SHSy5Y, E: CHLA255; F: D341). The same results were also reproduced in long-term (4 days) co-culture assays with T cells at day +12 (Figure 6G-I) and day +24 (Figure 6J-L).

[0131] Finally, since these data underlined so far, the superiority of the GD2.CAR+Beclin1 construct, the functional efficacy of these cells versus the conventional GD2.CAR T cells was further validated using a more sophisticated model where T cells are placed in co-culture with tumour cells that have been previously organized as spheroids in order to more accurately mimic the tumour mass present in the patient. As shown in Figure 7, GD2.CAR+Beclin1 T cells showed faster and more marked anti-tumour activity than GD2.CAR T cells in two different tumour models: neurobalstoma_SHSy5Y (Figure 7A-B; p<0.05) and medulloblastoma_D341 (Figure 7C-D; p<0.05). This feature is extremely important in the treatment of refractory patients as it prevents tumour adaptation following the immunological pressure exerted by the T cells.

[0132] In order to prove that the restoration of autophagy could be performed also with other autophagy proteins as well as with autophagy-inducing peptides, the impact of ATG5 and Beclin 1 peptide was investigate. As reported in Figure 8A-B, the overexpression of ATG5 and Beclin 1 peptide on primary T cells were feasible and did not affect cell growth in vitro. In both the condition, when GD2.CAR T cells were equipped with autophagy protein / peptide, these cells showed superior anti-tumour activity in long-term co-culture with a medulloblastoma (D341 , Figure 8C) and neuroblastoma (SHSY5Y, Figure 8D) mainly at low E:T ratio. It was also proved that this phenomenon is not due to the GD2.CAR but can be applied to any CAR. Therefore, the above-mentioned data were validated using a new CAR targeting the B7-H3 antigen (Figure 8E-G) and their functionality against sarcoma cell lines (Ewing and Rhabdomyosarcoma) tested was tested.

[0133] Example 5: Molecular characterization of autophagy reprogrammed CAR T cells. Then, based on the data produced, the autophagy restored CAR T cells were further in deep molecular characterized using omics platforms.

[0134] First, the impact of autophagy recovering on GD2.CAR using un-targeting proteomic approach was investigated. After 48h from antigen stimulation via CAR, by anti-CAR idiotype (1A7), the analysis revealed that the overexpression of TFEB and Beclin 1 is able to induce a change in protein content within the genetically modified lymphocytes (Figure 9A). This change in protein profile has common characteristics between the two groups but also unique specific patterns. It is important to note that the modifications induced by TFEB are significantly greater than those triggered by Beclin 1 (Figure 9B). This phenomenon is presumably due to the fact that TFEB is a transcriptional factor while Beclin 1 is not. In particular from this analysis, it was observed that TFEB upregulation was able to induce specifically a significant improvement in several pathway including: the eukaryotic translation elongation / termination, response to aminoacidic deficiency, metabolism of RNA / aminoacidic / protei n, cytokine signalling and positive regulation of the immune response (Figure 9C). While the Beclinl up-regulation was able specifically to significantly enrich several other pathways including ER-phagosome pathway, antigen processing-cell presentation, activation of innate and adaptive system, TCR signalling, endosomal / vacuole pathway and extracellular exosome (Figure 9D). However, both TFEB and Beclinl demonstrated being able to significantly promote migration, cellular response to starvation / stress / stimuli, apoptosis regulation, downstream TCR signalling, IL1 signalling, intracellular signalling by second massagers, transport of small molecules, CD28 dependent Vavi pathway as well as other pathways (Figure 9E).

[0135] The long effect of TFEB and Beclinl expression on GD2.CAR T cells was then investigated by analysing their mRNA profiles. A RNAseq study after 96h from a CAR antigen stimulation was therefore performed, which revealed that TFEB is able to continuously modulate the metabolism on different levels, probably due to the fact that it is a transcription factor, regulating pathway involved in the lysosome degradation, mTOR signalling, glycosaminoglycan degradation, amino sugar and nucleotide sugar metabolism, inositol phosphate metabolism and ferroptosis. On the other hand, Beclinl seems attenuate its metabolic influence and provide a better T cell fitness regulating cell cycle, DNA replication, JAK-STAT signalling, cytokine-cytokine receptor interaction, NOD and RIG-l-like receptor signalling, p53 signalling, apoptosis, TNF and NF-kappa B signalling (Figure 9F).

[0136] Lastly, by an un-targeted metabolic approach we explored the metabolite profile on the different autophagy reprogrammed GD2.CAR T cells (GD2.CAR, GD2.CAR+TFEB, GD2.CAR+Beclin1 , GD2.CAR+ATG5 and GD2.CAR+Beclin1 peptide) after 96h from a CAR antigen stimulation. As reported in Figure 9G, in all autophagy edited condition, a significant increase or reduction of amino acid derived proteins, lipids or metabolites and a general increase of deoxynucleotides crucial for DNA replication and autophagy regulation was discovered (Figure 9G).

[0137] Example 6: Quantification of extracellular vesicle produced by autophagy restored GD2.CAR T cells. Based on the data generated and the beneficial effect observed so far on Beclin 1 over-expression, one of the pathways identified in the proteomic analysis that was detectable in Beclin 1 modified T cells but not in TFEB was validated. Therefore, the extracellular exosome profile was examined by quantifying the amount of extracellular vesicle. The results demonstrated that with the autophagy modulation induced an increase in vesicle / vacuole formation within the gene-modified T lymphocytes (Figure 10A). However, only in a Beclin 1 condition a significant increase of extracellular vesicles on culture supernatant already in the resting condition was observed (Figure 10B). This phenomenon is conserved after antigen stimulation where also the ATG5 reprogrammed GD2.CAR T cells are able to produce more extracellular vesicles compared to GD2.CAR T cells (Figure 10C).

[0138] Example 7: Evaluation of autophagy restored GD2.CAR T cells in xenograft murine model.

[0139] In order to validate the in vitro data in a more sophisticate model, a xenograft murine model was performed. These experiments were carried out on service in a blind fashion using a facility located in Rome - Italy (Plaisant). Since the inventors were aware of the limit that they could observe in the animal model with the GD2.CAR - in the preliminary study that led to the phase l / ll clinical study (NCT03373097), the gene-modified T cells completely eradicated the tumour after few weeks unlike what was then observed in the human study, it was decided to challenge the system mimicking a cellular exhaustion. Lymphocytes grown in vitro for at least three weeks were therefore used.

[0140] In this condition, the GD2.CAR treated mice showed incapacity to control tumour growth after 20 days from CAR infusion with evident sign of CAR toxicities. However, both the groups treated with GD2.CAR+TFEB and GD2.CAR+Beclin1 clearly demonstrated strong and durable anti-tumour activities until the end of the experiment on day +90 (Figure 11 A). The peripheral blood of all treated animals was then analysed by flow cytometry (facs) on day 12 after T cell infusion. From this analysis no statistical differences were observed in the distribution of human T lymphocytes and CAR, however a lower frequency of these subsets was observed in groups modified with autophagic proteins (Figure 11 B). Also, the analysis of the CD4 / CD8 distribution did not reveal any statistical difference between the groups however a slight reduction of CD4 and slight increase of CD8 were observed in autophagy reprogrammed T cells, mainly in the Beclinl treated mice (Figure 11C). To validate the functionality of the circulated T lymphocytes, an immune characterization was carried out (Figure 11D-F). The percentage of resting CD4 and CD8 CAR T cells were not significant different between the cohorts however lower in the Beclinl treated group (Figure 11D). The analysis of exhaustion markers in both CD8 and CD4 subpopulation like PD1 and Lag3, instead, underlined a significant reduction of the expression of these markers in the GD2.CAR+Beclin1 treated group and only a slight reduction on GD2.CAR+TFEB group. Lastly, the analysis of the chemochine receptor 3 important for migration and memory phenotype even though no significant between the groups, its expression was higher on autophagy reprogrammed T cells in particular in GD2.CAR+Beclin1 group (Figure 11 E-F). INDUSTRIAL APPLICABILITY

[0141] The pharmaceutical compositions, recombinant nucleic acids, modified lymphocytes, and products for use in the invention are industrially applicable. For example, they can be used in the manufacture of, or as, pharmaceutical products.

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Claims

CLAIMS1. A modified human lymphocyte containing a recombinant nucleic acid or set of recombinant nucleic acids encoding at least one positive regulator of autophagy.

2. The modified human lymphocyte according to claim 1, wherein the at least one positive regulator of autophagy is one or more human protein(s).

3. The modified human lymphocyte according to claim 1 or 2, wherein the one or more human protein(s) are selected from the group consisting of: human TFEB, human Beclin-1, human ATG5, human AMBRA1, human ULK1, human ATG14, human WIPI1, human WIPI2, human p62, human VP34, human ATG4A, human Annexin A2, human Polycystin 2, human NFT2, and human ATG7.

4. The modified human lymphocyte according to any one of the preceding claims, wherein the at least one positive regulator of autophagy is human TFEB, human Beclin-1, and / or human ATG5.

5. The modified human lymphocyte according to any one of claims 1-4, wherein the at least one positive regulator of autophagy is human TFEB.

6. The modified human lymphocyte according to any one of claims 1-4, wherein the at least one positive regulator of autophagy is human Beclin-1 .

7. The modified human lymphocyte according to any one of claims 1-4, wherein the at least one positive regulator of autophagy is human ATG5.

8. The modified human lymphocyte according to claim 1, wherein the at least one positive regulator of autophagy is one or two peptides selected from the following group: TNVFNATFHIWHSGQFGT (SEQ ID NO: 1) and TNVFNATFEIWHDGEFGT (SEQ ID NO: 2).

9. The modified human lymphocyte according to any one of the preceding claims, wherein the lymphocyte expresses said at least one positive regulator of autophagy from said recombinant nucleic acid or set of recombinant nucleic acids.

10. The modified human lymphocyte according to claim 9, wherein the expression of said at least one positive regulator of autophagy from said recombinant nucleic acid or set of recombinant nucleic acids is constitutive.11 . The modified human lymphocyte according to claim 9, wherein the expression of said at least one positive regulator of autophagy from said recombinant nucleic acid or set of recombinant nucleic acids is inducible.

12. The modified human lymphocyte according to any one of the preceding claims, wherein the lymphocyte is a T-cell, an NK cell, or an NKT cell.

13. The modified human lymphocyte according to any one of the preceding claims, wherein the lymphocyte is a T-cell.

14. The modified human lymphocyte according to any one of the preceding claims, wherein the lymphocyte is an op T-cell.

15. The modified human lymphocyte according to any one of claims 1-13, wherein the lymphocyte is an y5 T- cell.

16. The modified human lymphocyte according to any one of the preceding claims, wherein the lymphocyte is a CD8+T-cell.

17. The modified human lymphocyte according to any one of claims 1-15, wherein the lymphocyte is a CD4+T-cell.

18. The modified human lymphocyte according to any one of claims 1-12, wherein the lymphocyte is an NK cell.

19. The modified human lymphocyte according to any one of claims 1-12, wherein the lymphocyte is a NKT cell.

20. The modified human lymphocyte according to any one of the preceding claims, wherein the lymphocyte further contains and expresses a recombinant nucleic acid or a set of recombinant nucleic acids encoding a T-cell receptor or a chimeric antigen receptor.21 . The modified human lymphocyte according to claim 20, wherein the lymphocyte contains and expresses a recombinant nucleic acid or a set of recombinant nucleic acids encoding a T-cell receptor.

22. The modified human lymphocyte according to claim 20, wherein the lymphocyte contains and expresses a recombinant nucleic acid or a set of recombinant nucleic acids encoding a chimeric antigen receptor.

23. The modified human lymphocyte according to any one of claims 20-22, wherein the T-cell receptor or chimeric antigen receptor binds to a cancer cell antigen.

24. The modified human lymphocyte according to claim 23, wherein the cancer cell antigen is an antigen of a solid cancer.

25. The modified human lymphocyte according to claim 24, wherein the solid cancer is a brain cancer.

26. The modified human lymphocyte according to claim 25, wherein the brain cancer is a neuroblastoma.

27. The modified human lymphocyte according to claim 25, wherein the brain cancer is a medulloblastoma.

28. The modified human lymphocyte according to any one of claims 23-27, wherein the cancer cell antigen is an antigen of a pediatric cancer.

29. The modified human lymphocyte according to any one of the preceding claims, wherein the recombinant nucleic acid or set of recombinant nucleic acids encoding said at least one positive regulator of autophagy and / or the recombinant nucleic acid or set of recombinant nucleic acids encoding said T-cell receptor or chimeric antigen receptor is integrated into the genome, preferably the chromosomal genome, of the lymphocyte.

30. The modified human lymphocyte according to any one of the preceding claims, wherein the recombinant nucleic acid or set of recombinant nucleic acids encoding said at least one positive regulator of autophagy and / or the recombinant nucleic acid or set of recombinant nucleic acids encoding said T-cell receptor or chimeric antigen receptor is an expression cassette.31 . The modified human lymphocyte according to claim 30, wherein the expression cassette is from a retroviral vector.

32. The modified human lymphocyte according to claim 30 or 31, wherein the expression cassette contains a selection marker.

33. The modified human lymphocyte according to claim 32, wherein the selection marker is ACD19.

34. A method for obtaining a modified human lymphocyte according to any one of the preceding claims, the method comprising a step of introducing the recombinant nucleic acid or set of recombinant nucleic acids encoding said at least one positive regulator of autophagy into a human lymphocyte, in order to obtain the modified human lymphocyte.

35. The method according to claim 34, further comprising a step of introducing the recombinant nucleic acid or set of recombinant nucleic acids encoding the T-cell receptor or chimeric antigen receptor into the human lymphocyte, wherein the modified human lymphocyte is a modified human lymphocyte according to any one of claims 20-33.

36. The method according to claim 34 or 35, wherein the method comprises a step of expanding said modified human lymphocyte ex vivo.

37. The method according to any one of claims 34-36, wherein the method is carried out in vitro.

38. A pharmaceutical composition comprising a modified human lymphocyte according to any one of claims 1- 33 or a modified human lymphocyte obtainable by the method of any one of claims 34-37.

39. A modified human lymphocyte according to any one of claims 1-33, for use in medicine.

40. A modified human lymphocyte according any one of claims 1-33 or a pharmaceutical composition according to claim 38, for use in a method for the treatment of cancer in a human patient, wherein the modified human lymphocyte is a modified human lymphocyte according to claim 23.41 . The modified human lymphocyte or pharmaceutical composition for use according to claim 40, wherein the treatment of cancer is an immunotherapeutic treatment of cancer.

42. The modified human lymphocyte or pharmaceutical composition for use according to claim 41, wherein the cancer is a solid cancer and the modified human lymphocyte is a modified human lymphocyte according to claim 24.

43. The modified human lymphocyte or pharmaceutical composition for use according to claim 42, wherein the solid cancer is a brain cancer and the modified human lymphocyte is a modified human lymphocyte according to claim 25.

44. The modified human lymphocyte or pharmaceutical composition for use according to claim 43, wherein the brain cancer is a neuroblastoma and the modified human lymphocyte is a modified human lymphocyte according to claim 26.

45. The modified human lymphocyte or pharmaceutical composition for use according to claim 44, wherein the brain cancer is a medulloblastoma and the modified human lymphocyte is a modified human lymphocyte according to claim 27.

46. The modified human lymphocyte or pharmaceutical composition for use according to any one of claims 41- 45, wherein the cancer cell antigen is an antigen of a pediatric cancer and the modified human lymphocyte is a modified human lymphocyte according to claim 28.

Images