Modified human lymphocytes comprising and expressing positive autophagy regulators

Abstract

The present invention relates to modified human lymphocytes comprising and expressing a recombinant nucleic acid or set of recombinant nucleic acids encoding at least one positive autophagy regulator. The modified human lymphocytes according to the present invention may also comprise and express recombinant nucleic acids or groups of recombinant nucleic acids encoding T cell receptors or chimeric antigen receptors. The invention also relates to methods of producing such modified human lymphocytes, pharmaceutical compositions comprising them, and their use in medicine and for methods of treatment of cancer, including immunotherapeutic treatment of cancer.

Description

Technical Field

[0001] This invention relates to modified human lymphocytes that contain and express recombinant nucleic acids or recombinant nucleic acid sequences encoding at least one positive autophagy regulator. The modified human lymphocytes according to the invention may also contain and express recombinant nucleic acids or recombinant nucleic acid sequences encoding a T-cell receptor or a chimeric antigen receptor.

[0002] The present invention also relates to methods for producing such modified human lymphocytes, pharmaceutical compositions comprising such lymphocytes, and their use in medicine and in the treatment of cancer, including methods for immunotherapeutic treatment of cancer. Background Technology

[0003] Over the past few decades, hematology and oncology have made tremendous progress with the development of intensive multimodal therapy, supportive care, and better characterization of tumor biology. However, patients with recurrent or resistant tumors still face unfavorable outcomes. 1,2 For these patients, there is an urgent clinical need to identify new and more effective intervention strategies. It has been clearly demonstrated that tumors are susceptible to targeted and immuno-based therapies, including adoptive cell therapy, which involves harvesting immune cells (T cells, NK cells, and NK-T cells), expanding these immune cells in vitro, and redirecting them to target cancer cells. Although these types of therapy have been studied for decades, they have only recently demonstrated clinical benefit in a broad range of patients. The observation that Epstein-Barr Virus (EBV)-specific cytotoxic T cells (CTLs) from seronegative donors can control EBV-transformed B cells in vitro led to the development of the first antigen-specific T-cell therapy for the treatment of post-transplant lymphoproliferative disorder (PTLD). 3,4Several efforts have also focused on genetically modifying T cells to recognize tumor cells via exogenous T cell receptors (TCRs) or by adding chimeric antigen receptors (CARs). The former can recognize peptide antigens (primarily derived from intracellular proteins) but is limited by the major histocompatibility complex (MHC) and lacks the proper continuation required for optimal T cell activation. In contrast, CARs contain antibody-derived single-chain variable fragments (scFvs) that impart target specificity and fuse with signal transduction domains (such as the CD3ζ chain) capable of activating T cells. This structure allows T cells to recognize antigens (including non-peptide targets such as glycolipids and carbohydrates) without MHC presentation and become activated T cells. 5 Besides α / β-T cells, γδ-T cells and NK cells are also emerging research areas. γδ-T cells, in particular, can recognize unprocessed antigens in a manner independent of the MHC. 6,7 NK cells kill cells by attacking those with reduced or absent MHC-I expression through a "missing-self mechanism". 8 Neither of these two cellular platforms induces graft-versus-host disease (GvHD), making them prime candidates for "off-the-shelf" treatments. 6,9 Recently, several of these advanced approaches have been classified as Advanced Therapy Medicinal Products (ATMPs) and are revolutionizing biomedicine by creating new treatments for currently incurable diseases. 10,11 In particular, CAR immunotherapy is a particularly active area of ​​research, but despite some proposed treatments showing health effects in patients refractory to more conventional therapies, to date, US and European regulatory agencies (FDA and EMA) have approved only five adoptive cell therapies targeting CD19 and B-cell maturation antigens. 12,13 In patients with other malignant diseases (including solid tumors and CD19) neg Achieving a similar response in patients with leukemia / lymphoma remains a significant challenge. 14-16 It has been demonstrated that this limited efficacy is primarily due to the nature of these tumors, the presence of an immunosuppressive tumor microenvironment (TME), and the low persistence and functionality of CAR T cells. 17-20 .

[0004] A recent clinical study (NCT03373097) using 3° generation GD2.CAR T-cell therapy in patients with refractory high-risk neuroblastoma demonstrated that CAR optimization is crucial for improving the safety and efficacy of this technique, with 3-year overall survival and event-free survival observed at the recommended dose of 60% and 36%, respectively. 21 Furthermore, it demonstrates how TME and its components lead to persistence and loss of function in GD2.CART cells, thereby affecting their metabolic and activation states. 20 To address this limitation, several groups, including ours, have investigated novel strategies for metabolic reprogramming of CAR T cells. 22-27 These studies highlight that this reprogramming can be performed using different strategies, including supplementing small lipids during the production of ex vivo CAR T cells, resulting in better performance and memory composition of ATMP. 25 Despite being significantly better, the long-term efficacy of these CAR T cells remains suboptimal in tumors with hostile TMEs and / or those that are difficult to invade. 25,28 .

[0005] Therefore, the inventors further investigated how to improve the function of CAR T cells. Based on their previous research, the inventors observed that the in vitro T cell manipulation required for gene modification could induce important changes, including the regulation of enzymes involved in extracellular membrane degradation. 28 The inventors investigated whether other mechanisms / pathways could be affected by this process. They observed that in vitro manipulation induced metabolic changes in which the strong and prolonged blockade of the autophagy pathway was independent of culture conditions (cytokines used, activation: polyclonal vs. antigen-specific).

[0006] This key finding is new and has not been reported before.

[0007] Autophagy is a self-degradation process that is essential for maintaining metabolic and genetic homeostasis in all eukaryotes. Through autophagy, cellular components are delivered to lysosomes to ensure the basic turnover of cytoplasmic organelles and to provide energy and precursors of macromolecules. 29 Damage to or overactivation of autophagy is associated with several human diseases, ranging from cancer to autoimmune diseases and neurodegenerative diseases. In cancer, autophagy is a Janus-faced process, acting both as a tumor suppressor (clearing cells from damaged organelles) and as a tumor adaptive response (promoting malignant progression and chemoresistance). 30 Notably, recent findings have revealed that autophagy contributes to immunosuppression-related chemoresistance and enhances the ability of tumors to evade immune detection. 31,32 Furthermore, enhanced autophagy in tumor cells and low levels of tumor-infiltrating T lymphocytes have been observed in the late stages of metastatic disease.33 Furthermore, several new studies report that autophagy is also a key pathway for tumor antigen cross-presentation via the major histocompatibility complex and a decisive factor in initiating an effective adaptive immune response. 34 Furthermore, it has been shown that autophagy also plays an important role in protein secretion, regulating the production of both anti-inflammatory and pro-inflammatory cytokines, as well as chemokines. 35 However, the mechanisms by which autophagy manipulates anticancer immunity remain to be elucidated, and controversial results regarding the background-dependent role of autophagy require further investigation. Summary of the Invention

[0008] The inventors have determined to restore the autophagy mechanism in immune effector cells, such as lymphocytes, by forcibly expressing, for example, two exemplary autophagy key proteins in new fourth-generation GD2.CAR T cells, which exhibit non-toxicity and significantly improved anti-tumor activity.

[0009] The inventors have particularly discovered this reactivation of key autophagy proteins in immune effector cells such as lymphocytes.

[0010] - It is non-toxic and can maintain a more immature CAR-T cell phenotype.

[0011] - Improved mitochondrial fitness.

[0012] - Improved antitumor response in both in vitro 2D and 3D models, even at low effector:target ratios, as well as in in vivo models, which reduced the exhaustion spectrum of autophagy-reprogrammed T cells.

[0013] - Does not induce allogeneic reactions.

[0014] - Increases the production and secretion of extracellular vesicles.

[0015] In addition to autophagy, the inventors have discovered that key autophagy proteins can be reactivated in several other pathways involved in stress response, mitosis, post-protein translation, antigen presentation, and activation of the innate immune system.

[0016] They also found that the blockade of the autophagy pathway was not a unique feature observed during CAR T cell production, but could also be detected in the production of antigen-specific T cells, γ / δ-T cells, and natural killer cells, and was unrelated to gene manipulation and cytokines used for in vitro culture.

[0017] Importantly, this new strategy could revolutionize the efficacy of CAR T cells in solid tumors and other malignancies, providing new and long-term interventions that significantly improve patients’ quality of life.

[0018] Finally, the inventors observed that this autophagy blockade was also present in other cell platforms used for CAR therapy and adoptive cell therapy. Therefore, it has been shown that autophagy blockade is not due to genetic manipulation, but rather to in vitro activation and manipulation processes, which are crucial for the expansion of all these cell products. The proposed strategy can be extended to other cell products to improve their long-term durability and potency.

[0019] Therefore, the present invention relates to the following preferred embodiments:

[0020] 1. Modified human lymphocytes containing recombinant nucleic acids or recombinant nucleic acid groups encoding at least one positive autophagy regulator.

[0021] 2. The modified human lymphocytes according to Project 1, wherein at least one autophagy regulator is one or more human proteins.

[0022] 3. Modified human lymphocytes according to Project 1 or 2, wherein one or more human proteins are selected from: 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 polycystic protein 2, human NFT2, and human ATG7.

[0023] 4. Modified human lymphocytes according to any one of the preceding items, wherein the at least one autophagy regulator is human TFEB, human Beclin-1 and / or human ATG5.

[0024] 5. Modified human lymphocytes according to any one of items 1 to 4, wherein at least one autophagy regulator is human TFEB.

[0025] 6. Modified human lymphocytes according to any one of items 1 to 4, wherein at least one autophagy regulator is human Beclin-1.

[0026] 7. Modified human lymphocytes according to any one of items 1 to 4, wherein at least one autophagy regulator is human ATG5.

[0027] 8. Modified human lymphocytes according to Project 1, wherein the at least one autophagy regulator is one or two peptides selected from the group consisting of: (SEQ ID NO: 1) and (SEQ ID NO: 2).

[0028] 9. The modified human lymphocyte according to any one of the preceding items, wherein the lymphocyte expresses at least one positive autophagy regulator derived from the recombinant nucleic acid or recombinant nucleic acid group.

[0029] 10. Modified human lymphocytes according to Item 9, wherein the expression of at least one autophagy positive regulator derived from the recombinant nucleic acid or recombinant nucleic acid group is constitutive.

[0030] 11. Modified human lymphocytes according to Item 9, wherein the expression of at least one autophagy positive regulator derived from the recombinant nucleic acid or recombinant nucleic acid group is inducible.

[0031] 12. The modified human lymphocytes according to any one of the preceding items, wherein the lymphocytes are T cells, NK cells or NKT cells.

[0032] 13. The modified human lymphocytes according to any one of the preceding items, wherein the lymphocytes are T cells.

[0033] 14. The modified human lymphocytes according to any one of the preceding items, wherein the lymphocytes are αβ T cells.

[0034] 15. Modified human lymphocytes according to any one of items 1 to 13, wherein the lymphocytes are γδT cells.

[0035] 16. Modified human lymphocytes according to any one of the preceding items, wherein the lymphocytes are CD8+ cells. + T cells.

[0036] 17. Modified human lymphocytes according to any one of items 1 to 15, wherein the lymphocytes are CD4+ cells. + T cells.

[0037] 18. Modified human lymphocytes according to any one of items 1 to 12, wherein the lymphocytes are NK cells.

[0038] 19. Modified human lymphocytes according to any one of items 1 to 12, wherein the lymphocytes are NKT cells.

[0039] 20. The modified human lymphocyte according to any one of the preceding items, wherein the lymphocyte further comprises and expresses a recombinant nucleic acid or recombinant nucleic acid group encoding a T cell receptor or a chimeric antigen receptor.

[0040] 21. The modified human lymphocytes according to Item 20, wherein the lymphocytes contain and express recombinant nucleic acids or recombinant nucleic acid groups encoding T cell receptors.

[0041] 22. The modified human lymphocytes according to Item 20, wherein the lymphocytes contain and express recombinant nucleic acids or recombinant nucleic acid groups encoding chimeric antigen receptors.

[0042] 23. Modified human lymphocytes according to any one of items 20 to 22, wherein the T cell receptor or chimeric antigen receptor binds to cancer cell antigens.

[0043] 24. Modified human lymphocytes according to Item 23, wherein the cancer cell antigen is an antigen of solid tumor.

[0044] 25. Modified human lymphocytes as described in Item 24, wherein the solid tumor is a brain cancer.

[0045] 26. Modified human lymphocytes according to item 25, wherein the brain cancer is neuroblastoma.

[0046] 27. Modified human lymphocytes according to item 25, wherein the brain cancer is medulloblastoma.

[0047] 28. Modified human lymphocytes according to any one of items 23 to 27, wherein the cancer cell antigen is an antigen of pediatric cancer.

[0048] 29. The modified human lymphocyte according to any one of the preceding items, wherein a recombinant nucleic acid or recombinant nucleic acid group encoding the at least one autophagy positive regulator and / or a recombinant nucleic acid or recombinant nucleic acid group encoding the T cell receptor or chimeric antigen receptor is integrated into the genome of the lymphocyte, preferably into the chromosomal genome.

[0049] 30. The modified human lymphocytes according to any one of the preceding items, wherein the recombinant nucleic acid or recombinant nucleic acid group encoding the at least one autophagy positive regulator and / or the recombinant nucleic acid or recombinant nucleic acid group encoding the T cell receptor or chimeric antigen receptor is an expression cassette.

[0050] 31. Modified human lymphocytes according to item 30, wherein the expression cassette is derived from a retroviral vector.

[0051] 32. Modified human lymphocytes according to item 30 or 31, wherein the expression cassette contains selection markers.

[0052] 33. Modified human lymphocytes according to item 32, wherein the selection marker is ΔCD19.

[0053] 34. A method for obtaining modified human lymphocytes according to any one of the preceding items, the method comprising the step of introducing a recombinant nucleic acid or a group of recombinant nucleic acids encoding the at least one autophagy positive regulator into human lymphocytes to obtain the modified human lymphocytes.

[0054] 35. The method of claim 34, further comprising the step of introducing a recombinant nucleic acid or a group 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 to 33.

[0055] 36. The method according to item 34 or 35, wherein the method includes the step of in vitro amplification of the modified human lymphocytes.

[0056] 37. The method according to any one of items 34 to 36, wherein the method is performed in vitro.

[0057] 38. A pharmaceutical composition comprising modified human lymphocytes according to any one of items 1 to 33 or modified human lymphocytes obtainable by any one of items 34 to 37.

[0058] 39. Modified human lymphocytes according to any one of items 1 to 33, for use in medicine.

[0059] 40. A method of treating cancer in a human patient with modified human lymphocytes according to any one of items 1 to 33 or a pharmaceutical composition according to item 38, wherein the modified human lymphocytes are the modified human lymphocytes according to item 23.

[0060] 41. The modified human lymphocytes or pharmaceutical composition used according to item 40, wherein the treatment of said cancer is an immunotherapeutic treatment of cancer.

[0061] 42. The modified human lymphocytes or pharmaceutical composition used according to item 41, wherein the cancer is a solid cancer, and the modified human lymphocytes are the modified human lymphocytes according to item 24.

[0062] 43. The modified human lymphocytes or pharmaceutical composition used according to item 42, wherein the solid tumor is a brain cancer, and the modified human lymphocytes are the modified human lymphocytes according to item 25.

[0063] 44. The modified human lymphocytes or pharmaceutical composition used according to item 43, wherein the brain cancer is neuroblastoma, and the modified human lymphocytes are the modified human lymphocytes according to item 26.

[0064] 45. The modified human lymphocytes or pharmaceutical composition used according to item 44, wherein the brain cancer is medulloblastoma, and the modified human lymphocytes are the modified human lymphocytes according to item 27.

[0065] 46. ​​The modified human lymphocytes or pharmaceutical composition for use according to any one of items 41 to 45, wherein the cancer cell antigen is an antigen of pediatric cancer, and the modified human lymphocytes are the modified human lymphocytes according to item 28. Attached Figure Description

[0066] Figure 1 : Blocking of autophagy mechanism in primary human T cells manipulated in vitro.

[0067] A) and T0 (CD3 immediately after separation) + Compared to T cells, polyclonal activated CD3+ cells isolated from human peripheral blood at different time points... + Quantitative real-time PCR (qPCR) analysis of fold changes in RNA levels of four key autophagy genes (LC3, AMBRA1, p62, ULK1) in primary human T cells; n=4. B) Volcano plot showing the regulation of 85 autophagy-related mRNAs. C) Polyclonal activated CD4+ cells at different time points after isolation from human peripheral blood compared to T0 (freshly isolated T cells). + qPCR analysis of fold changes in RNA levels of four key autophagy genes (LC3, AMBRA1, p62, ULK1) in primary human T cells; n=3. D) Comparison of polyclonal activated CD8+ cells at different time points after isolation from human peripheral blood with T0 (freshly isolated T cells). + qPCR analysis of fold changes in RNA levels of four key autophagy genes (LC3, AMBRA1, p62, ULK1) in primary human T cells; n=3. E) Compared with T0 (freshly isolated T cells), polyclonal activated CD3 + qPCR analysis of fold changes in RNA levels of four key autophagy genes (LC3, AMBRA1, p62, ULK1) in primary human T cells (NT) and CAR-T cells; n=3. F) CAR-T cells cultured with IL2 and polyclonal activated CD3 cells compared to T0 (freshly isolated T cells). + Representative Western blot analysis of protein levels of ACTIN, p62, LC3, mTOR, ULK1, and AMPK in primary human T cells (NT); n=3. (G) Compared to T0, CD4+ levels on day 14 after IL-2 culture. + and CD8 +Representative Western blot analysis of protein levels of ACTIN, p62, LC3, mTOR, and ULK1 in CAR+ T cells; n=2. (H) Compared to T0, polyclonal activated CD4+ cells cultured with IL7 / 15 on day 14. + and CD8 + CAR + Representative Western blot analysis of protein levels of ACTIN, p62, LC3, and ULK1 in T cells; n=2. I) Representative Western blot analysis of 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); n=2. J) Western blot analysis of T cells cultured for 6 hours or 24 hours with or without the mTOR inhibitor (Torin1) with or without IL2 (UT). K) Summary table of descriptions of autophagic fluxes shown in the Western blot plots. Data are shown as mean ± SEM. L) qPCR analysis of the fold change in RNA levels of five key autophagy genes (LC3, AMBRA, p62, ULK, Beclin1, ATG5) in NK and γδ-T cells compared to T0 (freshly isolated NK and γδ-T cells); n=3. M) Western blot analysis of representative protein levels of ACTIN, ULK1, ATG14, pULK1, pATG14, and LC3 in NK cells after 14 days of culture compared to T0; n=3.

[0068] Figure 2 HEK293T cells were transduced using retroviral vectors encoding ΔCD19, TFEB+ΔCD19, and Beclin1+ΔCD19.

[0069] A) Transduction efficiency of HEK 293T cells at different time points after transduction, monitored by FACS; n=1. B) GFP MFI of HEK 293T cells at different time points after transduction, monitored by FACS; n=1. C) qPCR analysis of fold changes in the RNA levels of Beclin1 and TFEB in wild-type and transduced HEK293T cells; n=3. D) Representative Western blot analysis of the protein levels of TFEB, ACTIN, and Beclin1 in wild-type and transduced HEK 293T cells (ΔCD19: #116, TFEB+ΔCD19: #276, Beclin1+ΔCD19: #277). Data are shown as mean ± SEM. p-values ​​shown are from two-way ANOVA.

[0070] Figure 3 Primary human T cells were transduced using retroviral vectors encoding ΔCD19, TFEB+ΔCD19, and Beclin1+ΔCD19.

[0071] A) In vitro expansion of T cells from day 0 (transduction) to day 11. B) Transduction efficiency of primary human T cells at different time points after transduction, monitored by FACS; n=4. C) ΔCD19 MFI of primary human T cells at different time points after transduction, monitored by FACS; n=4. D) qPCR analysis of fold changes in the RNA levels of Beclin1 and TFEB in primary human T cells; n=3. E) Representative Western blot analysis of the protein levels of TFEB, ACTIN, and Beclin1 in primary human T cells (ΔCD19: #116, TFEB+ΔCD19: #276, Beclin1+ΔCD19: #277). F) CD4+ in primary human T cells monitored by FACS. + With CD8 + Cell distribution; n=4. G) FACS-based analysis of fold changes in mitochondrial potential MFI in primary human T cells; n=4. H) Representative Western blot analysis of starvation experiments using control ΔCD19 and Beclin1+ΔCD19 primary human T cells cultured in growth media (GM) or nutrient-free EBS with or without the addition of p62, LC3II, ACTIN, and TFEB antibodies, with or without the addition of chloroquine (CQ). I) Quantitative analysis of autophagic flux in untransduced and TFEB-modified primary human T cells based on Western blot intensity (determined by intensity-quotient of LC3II and ACTIN); n=4. J) Percentage tumor kill of ΔCD19, TFEB+ΔCD19, and Beclin1+ΔCD19 against the CHLA255 neuroblastoma cell line at a 1:1 ratio in a 24-hour kill assay. Data are shown as mean ± SEM (A to C, I) or mean ± SD (D, F, G).

[0072] Figure 4 CAR-T cells were generated from primary human T cells using retroviral vectors encoding GD2.CAR, GD2.CAR+TFEB, and GD2.CAR+Beclin1.

[0073] A) Fold-up expansion of primary human T cells at different time points during culture, based on cell count; n=4. B) Transduction efficiency of primary human T cells at different time points after transduction, monitored by FACS; n=5. C) CAR MFI of primary human T cells at different time points after transduction, monitored by FACS; n=5. D) qPCR analysis of fold-up changes in Beclin1 and TFEB RNA levels in primary human T cells; n=3. E) CD4+ in primary human T cells, monitored by FACS. + With CD8 + Cell distribution; n=3. F) Representative Western blot analysis of protein levels of TFEB, ACTIN, and Beclin1 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 Beclin1+GD2.CAR-T cells and #50 GD2.CAR T cells used to monitor autophagic flux. J) Representative Western blot analysis of protein levels of p-ATG14, ULK1, ATG14, and ACTIN in #203 Beclin1+GD2.CAR-T cells and #50 GD2.CAR T cells. K) Representative Western blot analysis of LC3II and ACTIN protein levels in #203 Beclin1+GD2.CAR-T cells and #50 GD2.CAR T cells with or without BafA1, used to monitor autophagy activity. L) Representative Western blot analysis of LC3II and ACTIN protein levels in #202 TFEB+GD2.CAR-T cells and #50 GD2.CAR T cells with or without BafA1, used to monitor autophagy activity. M) Immunofluorescent spot determination and quantification of LC3 accumulation in #203 Beclin1+GD2.CAR-T cells and #50 GD2.CAR T cells in EBS medium or EBS medium + BafA1. Data are shown as mean ± SEM (G, H, M) or mean ± SD (A to EE, F).

[0074] Figure 5 Immunophenotypic characterization of autophagy-modified CAR-T cells.

[0075] A) CD4 +FACS-based analysis of primary human T-cell memory phenotypes (CM central memory, EM effect memory, ET terminal effect); n=3. B) CD8 + FACS-based analysis of primary human T cell memory phenotypes (CM central memory, EM effect memory, ET terminal effect); n=3. C) CD4 + and CD8 + FACS-based analysis of the SCMT (stem cell-like memory T cell) population in primary human T cells; n=3. D) CD4 + FACS-based analysis of exhaustion marker expression (Lag3, PD1, CD95, Tim3) in primary human T cells and exhaustion cells (expressing all four markers); n=3. E)CD4 + FACS-based analysis of exhaustion markers MFI (Lag3, PD1, CD95, Tim3) in primary human T cells; n=3. (F)CD8 + FACS-based analysis of exhaustion marker expression (Lag3, PD1, CD95, Tim3) in primary human T cells and exhaustion cells (expressing all four markers); n=3. + FACS-based analysis of exhaustion markers MFI (Lag3, PD1, CD95, Tim3) in primary human T cells; n=3. H)CD4 + FACS-based analysis of activation-related marker expression (CD28, CD25, HLA-DR) in primary human T cells; n=3. I) CD4 + FACS-based analysis of activation-related markers MFI (CD28, CD25, HLA-DR) in primary human T cells; n=3. J)CD8 + FACS-based analysis of activation-related marker expression (CD28, CD25, HLA-DR) in primary human T cells; n=3. + FACS-based analysis of activation-related markers MFI (CD28, CD25, HLA-DR) in primary human T cells; n=3. Data are shown as mean ± SD.

[0076] Figure 6 Functional 2D killing assay of autophagy-modified CAR-T cells.

[0077] After 12 days (A to C) and 24 days (D to F) of in vitro culture using SHSy5Y (A, D), CHLA255 (B, E) and D341 (C, F) tumor cell lines at different E:T ratios, the percentage improvement in tumor killing of GD2.CAR+TFEB and GD2.CAR+Beclin1 relative to the GD2.CAR control was measured in a short-term killing assay (24 hours) (A to F).

[0078] After 12 days (G to I) and 24 days (J to L) of in vitro culture using SHSy5Y (G, J), CHLA255 (H, K) and D341 (I, L) tumor cell lines at different E:T ratios, the percentage improvement in tumor killing of GD2.CAR+TFEB and GD2.CAR+Beclin1 relative to the GD2.CAR control (G to L) in long-term killing assays (4 days) was evaluated (n=3).

[0079] Figure 7 Functional 3D spheroid killing assay using autophagy-modified CAR-T cells.

[0080] A) Monitoring at different time points using #50 and #203 GD2-specific CAR-T cells targeting GFP at an E:T ratio of 1:3. + A) Representative fluorescence-based 3D spheroid killing assay performed on SH-SY5Y neuroblastoma cells; B) Monitoring of GFP-targeting using #50 and #203 GD2-specific CAR-T cells at a 1:3 E:T ratio at different time points. + Mean GFP intensity in a fluorescence-based 3D spheroid killing assay performed on SH-SY5Y neuroblastoma cells; n=2. C) Monitoring of GFP targeting using #50 and #203 GD2-specific CAR-T cells at a 1:3 E:T ratio at different time points. + A representative fluorescence-based 3D spheroid killing assay was performed on D341 medulloblastoma cells. D) Monitoring was conducted at different time points using #50 and #203 GD2-specific CAR-T cells against GFP at an E:T ratio of 1:3. + The mean GFP intensity of a fluorescence-based 3D spheroid killing assay was performed on D341 medulloblastoma cells; n=2.

[0081] Figure 8 Validation of autophagy recovery using ATG5 and Beclin1 peptides or by applying different CARs targeting the B7-H3 antigen.

[0082] A) Fold-over-fold expansion of primary human T cells at different time points during culture; cells transduced with GD2.CAR or GD2.CAR+ATG5 or GD2.CAR+Beclin1_peptide; n=3. B) Transduction efficiency of primary human T cells on day 10 post-transduction, as monitored by FACS; n=3. C through D) Percentage improvement in tumor killing of GD2.CAR+ATG5 and GD2.CAR+Beclin1_peptide relative to the GD2.CAR control at different E:T ratios using D341 (C) or SHSy5Y (D) in a long-term killing assay (4 days); n=3. E) Fold-over-fold expansion of primary human T cells at different time points during culture, based on cell count; cells transduced 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 to G) Percentage improvement in tumor killing relative to B7-H3.CAR control at different E:T ratios using A673 (F, Ewing Sarcoma) and CT-10 (G, rhabdomyosarcoma) cell lines in a long-term killing assay (4 days), n=1.

[0083] Figure 9 Proteomics, transcriptomics, and metabolomics profiles of autophagy-reprogrammed CAR T cells.

[0084] A) A heatmap showing the different proteomic profiles of cells transduced with GD2.CAR, GD2.CAR+TFEB (#202), and GD2.CAR+Beclin1 (#203) 48 hours after antigen stimulation via CAR activation (1A7, anti-idiotype). B) A Venn diagram showing the distribution of proteins regulated by TFEB and Beclin1 48 hours after antigen stimulation via CAR activation (1A7, anti-idiotype). C) Pathway enrichment analysis of regulated proteins detected only in GD2.CAR+TFEB, compared with GD2.CAR T cells. D) Pathway enrichment analysis of regulated proteins detected only in GD2.CAR+Beclin1, compared with GD2.CAR T cells. E) Pathway enrichment analysis of regulated proteins detected in both GD2.CAR+TFEB and GD2.CAR+Beclin1, compared with GD2.CAR T cells. F) Pathway enrichment analysis of regulated mRNAs detected in GD2.CAR+TFEB or GD2.CAR+Beclin1, compared with GD2.CAR T cells, 96 hours after antigen stimulation via CAR activation (1A7, anti-idiotype). G) Significantly upregulated or downregulated metabolites found in GD2.CAR, GD2.CAR+TFEB, GD2.CAR+Beclin1, GD2.CAR+ATG5, and GD2.CAR+Beclin1 peptides, 96 hours after antigen stimulation via CAR activation (1A7, anti-idiotype).

[0085] Figure 10 Extracellular detection of GD2.CAR T cells.

[0086] A) Electron microscopy analysis of GD2.CAR, GD2.CAR+TFEB, and GD2.CAR+Beclin1. B) Quantification of extracellular vesicles / μl in the culture supernatant of GD2.CAR, GD2.CAR+TFEB, GD2.CAR+Beclin1, and GD2.CAR+ATG5 without antigen stimulation. C) Quantification of extracellular vesicles / μl in the culture supernatant of GD2.CAR, GD2.CAR+TFEB, GD2.CAR+Beclin1, and GD2.CAR+ATG5 with antigen stimulation via 1A7.

[0087] Figure 11 In vivo validation of autophagy-recovered GD2.CAR T cells.

[0088] A) In vivo bioluminescence imaging of NSG mice carrying SHSY5Y-FF-Luc.GFP cells treated with NT, GD2.CAR, GD2.CAR+TFEB, and 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, and GD2.CAR+Beclin1 T cells 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, and GD2.CAR+Beclin1 T cells 12 days after CAR T cell treatment. D) Resting CD4 and CD8 T cells (CD27) in the peripheral blood of mice treated with GD2.CAR, GD2.CAR+TFEB, and GD2.CAR+Beclin1 T cells 12 days after CAR T cell treatment. neg / CD28 neg The frequency of ). The median fluorescence intensity of CAR, CXCR3, PD1, Lag3 and HLA-DR in both CD8 chamber (E) and CD4 chamber (F). Detailed Implementation

[0089] Unless otherwise defined below, the terms used in this invention should be understood in accordance with their ordinary meaning as known to those skilled in the art. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. Publications mentioned herein may be cited by specifying their full references in the text or by specifying reference numbers and adding the corresponding full references in the references section at the end of the specification.

[0090] definition

[0091] According to the present invention, the term "autophagy" has its known meaning. It should be understood that the meaning of this term encompasses cellular processes involving the degradation and recycling of cellular components (e.g., proteins, organelles, and macromolecules) through highly regulated mechanisms. It is a fundamental process for maintaining cellular homeostasis and is involved in a variety of physiological and pathological states.

[0092] According to the present invention, "modified human lymphocytes" refers to human lymphocytes that have been modified (e.g., altered or engineered) for example to achieve a specific purpose. Modification includes modifications according to claims 1, namely, the presence of a recombinant nucleic acid genome encoding at least one positive autophagy regulator according to claim 1, and optionally further includes the presence of a recombinant nucleic acid or recombinant nucleic acid genome encoding a T-cell receptor or chimeric antigen receptor. Modification may also include, but is not limited to, genetic manipulation, the introduction of specific receptors or proteins, and / or other modifications, such as modifications to lymphocyte function or targeting ability.

[0093] It should be understood that the modified human lymphocytes of the present invention are preferably suitable for human patients. Methods for preparing modified human lymphocytes for human patient use are known in the art. The modified human lymphocytes of the present invention are preferably suitable for the treatment of cancer in human patients, such as immunotherapy for cancer in human patients. According to the present invention, human lymphocytes isolated from healthy donors or patients with cancer can be modified by introducing the recombinant nucleic acid or recombinant nucleic acid group as defined in the claims and administered to patients to treat said cancer. The human lymphocytes are preferably selected from NK cells, NKT cells, and T cells. T cells are preferably CD8. + T cells, CD4 + T cells, αβ T cells, and / or γδ T cells.

[0094] According to the present invention, the immune receptor is a transmembrane receptor that mediates an immune response when expressed by immune cells. The immune receptor can be an endogenous immune receptor or a non-natural immune receptor, i.e., a genetically modified immune receptor. According to the present invention, some exemplary immune receptors are T-cell receptors (TCRs) and chimeric antigen receptors (CARs), such as recombinant T-cell receptors (TCRs) and recombinant chimeric antigen receptors (CARs). The CARs and / or TCRs according to the present invention, in their monomeric form, can consist of a single molecule containing all of its domains, or a heterodimer containing all of its domains. The CARs and / or TCRs can bind directly to their antigens, or they can bind indirectly through an adaptor.

[0095] It should be understood that the CAR and / or TCR according to the present invention bind to antigens, preferably cancer cell antigens. CARs typically bind to the extracellular domains of cancer cell antigens (i.e., cancer cell surface antigens), while TCRs typically bind to intracellular cancer cell antigens presented by HLA molecules.

[0096] When used in conjunction with this invention, the term "binding" and its variations refer to specific binding with a target antigen. It should be understood that when referring to the term "binding" and its variations, they refer to the inherent ability of a CAR or TCR to specifically bind to an antigen without further modification, but they do not require the actual presence of the antigen.

[0097] According to the present invention, the term "T cell receptor (TCR)" has the meaning known in the art. Generally, TCR is understood as a heterodimeric cell surface protein of the immunoglobulin superfamily that participates in the activation of T cells that respond to antigen binding. The TCR complex may consist of TCRα / β chains and CD3γ / δ / ε / ζ subunits that can associate via hydrophobic interactions. Somatic VDJ recombination allows for the generation of different TCRα and TCRβ chains, and the TCRαβ heterodimer is typically responsible for antigen recognition by binding to the peptide-MHC complex. CD3 transmits TCR-triggered signals via an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic tail region, but it is not typically directly involved in antigen recognition. An ITAM is a tandem repeat of a tyrosine-containing sequence (YXXL / I), with each CD3γ / δ / ε chain containing one ITAM and the CD3ζ chain containing three ITAMs. As a result of TCR binding, ITAM phosphorylation can be induced by protein tyrosine kinases (PTKs), allowing other effector molecules to interact with the TCR complex. TCRs can be present on the cell surface or in a soluble form. TCRs can be full-length or intact, including but not limited to αβ or γδ forms such as dimer TCRs (dTCRs) and single-chain TCRs (scTCRs). A TCR is an antigen-binding moiety smaller than the full-length TCR but binding to a specific peptide in an MHC molecule, such as an MHC-peptide complex. In some cases, the antigen-binding moiety or fragment of a TCR may contain only a portion of the structural domains of a full-length or intact TCR, but can still bind to peptide epitopes bound to a full-length TCR, such as an MHC-peptide complex. In some cases, the antigen-binding moiety contains variable domains of the TCR sufficient to form a binding site for binding to a specific MHC-peptide complex, such as variable α-chains and variable β-chains of the TCR. Typically, the variable chain of a TCR contains a complementarity-determining region involved in recognizing peptides, MHC, and / or MHC-peptide complexes. A TCR may contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail. Each chain of a TCR may have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. TCRs can associate with invariant proteins of the CD3 complex involved in mediating signal transduction.

[0098] According to the present invention, the term "chimeric antigen receptor (CAR)" has the meaning known in the art. Generally, CAR is understood as a receptor protein modified to endow T cells with a new ability to target specific antigens. This receptor is chimeric because it combines antigen-binding function and T cell activation function into a single receptor. CAR T cells can be derived from T cells in the patient's own blood (autologous) or from T cells from another healthy donor (allogeneic). Once isolated from a human, these T cells can be genetically modified to express a specific CAR, thereby programming them to target antigens, such as antigens present on the surface of cancer cells.

[0099] CARs are designed to enhance the recognition and targeting of cancer cells or other diseased cells. CARs according to the present invention typically comprise, but are not limited to, three main components: an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular domain is typically derived from an antibody fragment and provides specificity for the target antigen. The transmembrane domain anchors the receptor to the lymphocyte (e.g., T cell) membrane, while the intracellular signaling domain initiates activation signals upon antigen binding. When expressed in lymphocytes (e.g., T cells), the CAR enables modified lymphocytes (e.g., T cells) to recognize and bind to the target antigen, thereby activating and killing target cells expressing the target antigen.

[0100] It should also be understood that a CAR can be any known type of CAR, such as a first-generation CAR, second-generation CAR, third-generation CAR, fourth-generation CAR, or fifth-generation CAR. First-generation CARs typically have an intracellular signaling domain containing an intracellular signaling domain of CD3ζ, FcγRI, or other ITAM-containing activation domain to provide T cell activation signals. Second- and third-generation CARs also contain a co-stimulatory signaling domain (e.g., a co-stimulatory signaling domain derived from endogenous T cell co-stimulatory receptors such as CD28, 4-1BB, or ICOS) or two co-stimulatory signaling domains, respectively. Conversely, fourth-generation CARs may express one or two co-stimulatory molecules, as well as a constitutive or inducible expression cassette containing a transgenic protein (e.g., a cytokine or enzyme). Fifth-generation CARs are known in the art and may contain additional intracellular domains compared to first- to fourth-generation CARs. CARs may contain, but are not limited to, truncated intracellular domains of cytokine receptors (e.g., fragments of the IL-2R chain) having motifs for binding transcription factors such as STAT-3 / 5.

[0101] According to the present invention, the term "natural killer (NK) cells" has the meaning known in the art and is generally understood to refer to a type of cytotoxic lymphocyte, a subset of white blood cells that plays a key role in the innate immune system. NK cells are known for their ability to recognize and eliminate target cells (e.g., virus-infected cells and tumor cells) without prior sensitization or the need for specific antigen recognition. This can be achieved through the presence of markers (including, but not limited to, CD56) and the absence of CD3 (CD56). + CD3 － To identify human NK cells.

[0102] According to the present invention, the term "natural killer T (NKT) cells" has the meaning known in the art and is generally understood to be a specific subset of T cells that exhibit characteristics of both natural killer (NK) cells and conventional T cells (e.g., T cell types referred to herein). They endogenously express unique T cell receptors (TCRs) and recognize specific lipid antigens presented by a molecule called CD1d. NKT cells co-express αβ T cell receptors but also express a variety of molecular markers commonly associated with NK cells, such as NK1.1. NKT cells have the ability to produce immunomodulatory molecules such as cytokines upon activation. They participate in the regulation of immune responses and play a role in both innate and adaptive immunity. NKT cells are known for their involvement in a variety of immune processes, including antimicrobial defense, tumor surveillance, autoimmune diseases, and allergic reactions.

[0103] The term "vector" is known in the art and encompasses, for example, plasmids, retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, or transposon vectors. It should be understood that the vectors used in conjunction with this invention are vectors suitable for therapeutic use in humans.

[0104] The term "expression cassette" has the meaning known in the art. Expression cassettes used according to the invention typically comprise at least a promoter sequence, at least one open reading frame (e.g., an open reading frame encoding at least one positive regulator of autophagy and / or an open reading frame encoding a T cell receptor or chimeric antigen receptor), and a 3' untranslated region typically containing a polyadenylation site. Expression cassettes according to the invention can be monocistronic or polycistronic (e.g., bicistronic). For example, a polycistronic (e.g., bicistronic) expression cassette may comprise an open reading frame encoding at least one positive regulator of autophagy and an open reading frame encoding a T cell receptor or chimeric antigen receptor. Polycistronic (e.g., bicistronic) expression cassettes are generally known in the art and may include links to open reading frames, for example, via internal ribosome entry sites (IRES).

[0105] In addition, the expression cassette may appropriately contain other nucleotide sequences, such as adaptors or linkers, enhancers, selection markers (e.g., antibiotic resistance markers), replication units, polyA sequences, and tags for purification (e.g., GST, poly-Arg, FLAG, histidine tags (His-tag), or c-myc, etc.).

[0106] According to the method for obtaining modified human lymphocytes of the present invention, the step of introducing recombinant nucleic acids or recombinant nucleic acid groups into human lymphocytes can be performed using any suitable standard technique known in the art, such as electroporation, electroinjection, microinjection, calcium phosphate coprecipitation, calcium chloride / rubidium chloride method, retroviral and lentiviral infection, DEAE-dextran, cationic liposome method, polyethylene glycol-mediated uptake, gene gun, etc., but not limited thereto. Nucleic acids or recombinant nucleic acid groups encoding positive autophagy regulators can be introduced before, simultaneously with, or after the introduction of nucleic acids or recombinant nucleic acid groups encoding and expressing T-cell receptors or chimeric antigen receptors.

[0107] According to the present invention, the term "constitutive" in relation to recombinant nucleic acid expression has the meaning known in the art. It should be understood that it encompasses continuous and uniform expression of recombinant nucleic acids. Constitutive expression is active expression in the modified human lymphocytes of the present invention under normal or standard conditions, regardless of environmental factors or specific cellular signals.

[0108] According to the present invention, the term "inducible" in relation to recombinant nucleic acid expression refers to expression that can be regulated or controlled by a specific signal or factor (e.g., a drug that is non-toxic to humans and can be administered to humans). How to achieve inducible expression is well known. For example, recombinant nucleic acids may contain inducible promoters to ensure inducible expression. It should be understood that such inducible promoters may form part of the expression cassette described herein.

[0109] The term "selection marker" has the meaning known in the art and generally refers to a recombinant nucleic acid sequence that has been successfully incorporated into a cell or organism with a desired genetic modification. For example, a selection marker can be a marker that confers a selectable phenotype, such as resistance to a specific antibiotic or the ability to grow in a selective culture medium. By including the selection marker in the same expression cassette, selective pressure (e.g., exposure to antibiotics or selective culture media) can be applied to identify and isolate cells or organisms with the desired genetic modification. According to the invention, an expression cassette containing a selection marker can be used to facilitate the identification and selection of modified lymphocytes of the invention.

[0110] According to all other embodiments of the invention, the recombinant nucleic acid according to the invention may preferably be recombinant DNA. Therefore, in a preferred embodiment, all recombinant nucleic acids according to the invention are recombinant DNA.

[0111] According to the present invention, the term "cancer cell antigen" has the meaning known in the art. In those embodiments of the invention, wherein the lymphocytes contain and express recombinant nucleic acids or recombinant nucleic acid groups encoding a chimeric antigen receptor, the cancer cell antigen according to the invention that binds to the chimeric antigen receptor is preferably a cancer cell surface antigen. In those embodiments of the invention, wherein the lymphocytes contain and express recombinant nucleic acids or recombinant nucleic acid groups encoding a T cell receptor, the cancer cell antigen according to the invention that binds to the T cell receptor is preferably an intracellular cancer cell antigen presented by an HLA molecule.

[0112] According to the present invention, the cancer can be any known cancer, but is preferably solid cancer, brain cancer, neuroblastoma, sarcoma, but can also be other pediatric or adult cancers.

[0113] Treatment methods and the use of lymphocytes in such treatments

[0114] The terms "treatment of cancer" or "treating cancer" according to the invention refer to therapeutic treatment. For example, the effectiveness of a therapeutic treatment can be assessed by evaluating whether the treatment inhibits cancer growth in the treated patient. Preferably, the inhibition is statistically significant, as assessed by suitable statistical tests known in the art. Inhibition of cancer growth can be assessed by comparing cancer growth in a group of patients treated according to the invention with a control group of untreated patients, or by comparing a group of patients receiving standard cancer treatment plus treatment according to the invention with a control group of patients receiving only standard cancer treatment. Such studies for assessing cancer growth inhibition are designed according to acceptable criteria for clinical research, such as double-blind randomized studies with sufficient statistical power. The term "treating cancer" includes inhibition of cancer growth in which cancer growth is partially inhibited (i.e., cancer growth is delayed in patients compared to control patients), inhibition in which cancer growth is completely inhibited (i.e., cancer growth stops in patients), and inhibition in which cancer growth is reversed (i.e., cancer shrinks). The effectiveness of a therapeutic treatment can be assessed based on known clinical indicators of cancer progression.

[0115] The treatment of cancer according to the present invention does not preclude additional or secondary therapeutic benefits in patients. However, it should be understood that the primary treatment sought to protect is for treating the cancer itself, and any secondary or additional effects merely reflect optional additional advantages in treating cancer growth.

[0116] The cancer treatment according to the present invention can be first-line, second-line, third-line, or fourth-line treatment. Treatment can also extend beyond fourth-line therapy. The meanings of these terms are known in the art and conform to the terminology commonly used by the National Cancer Institute of the United States.

[0117] The term "immunotherapy for cancer" has a meaning known in the art and generally refers to cancer treatment that uses a patient's immune system to treat cancer. Cancer cells carry genomic mutations that produce cancer cell antigens that are specific to cancer cells and distinct from non-cancer cell antigens. Therefore, in a preferred aspect of the immunotherapy for cancer according to the invention, the immunotherapy for cancer is a treatment in which such cancer cell antigens are recognized by the modified lymphocytes of the invention, and in which cancer cells expressing these antigens are killed. The immunotherapy for cancer can be evaluated using immune monitoring methods known in the art, for example by measuring intracellular IFN-γ expression (e.g., in CD8) in a blood sample. + (T cells and / or NK cells), measuring CD107a cell surface expression (e.g., on CD8) in blood samples. + On T cells and / or NK cells), intracellular TNF-α expression in blood samples (e.g. on leukocytes) and intracellular interleukin-2 expression in blood samples (e.g. on CD8 cells) were measured. + T cells and / or CD4 + CD154 is expressed on the surface of cells (e.g., in T cells) and blood samples (e.g., in CD8 cells). + T cells and / or CD4 +T cells), tetramers or dextramer staining of tumor antigen-specific T cells in blood samples, CTL activity against autologous tumor cells, or the presence of T cells against neoantigens derived from tumor-specific mutations. The preferred methods for evaluating cancer immunotherapy are based on the methods of Gouttefangeas C et al.: “FlowCytometry in Cancer Immunotherapy: Applications, Quality Assurance and Future.” (2015) In: Cancer Immunology: Translational Medicine from Bench to Bedside (N. Rezaei, ed.). Springer. Chapter 25: pp. 471-486; and on the methods of Van der Burg SH et al.: “Immunoguiding, the final frontier in the immunotherapy of cancer.” (2014) In Cancer Immunotherapy meets oncology (CM Britten, SKreiter, M. Diken & HG Rammensee, eds.). Springer International Publishing, Switzerland, pp. 37-51, ISBN: 978-3-319-05103-1.

[0118] According to the present invention, each occurrence of the term "comprising / including" may optionally be replaced by the term "consisting of".

[0119] Methods for obtaining modified human lymphocytes

[0120] Methods for obtaining modified human lymphocytes according to the invention are as defined herein, including the claims. They can be performed in vitro, for example using isolated human lymphocytes as starting material. In other words, preferably, the methods for obtaining modified human lymphocytes according to the invention are not methods for treating a human or animal body by surgery or treatment, and are not diagnostic methods performed on a human or animal body.

[0121] Preparation of the pharmaceutical composition of the present invention

[0122] The pharmaceutical compositions of the present invention are prepared according to known standards for preparing pharmaceutical compositions.

[0123] For example, pharmaceutical compositions are prepared in a manner that allows them to be appropriately stored and administered. Therefore, pharmaceutical compositions of the present invention may contain pharmaceutically acceptable components, such as carriers, excipients, and / or stabilizers.

[0124] When the pharmaceutical composition is administered to a human patient, such a pharmaceutically acceptable component is non-toxic at the amount used. The pharmaceutically acceptable component added to a pharmaceutical composition may depend on the chemical properties of the active ingredient present in the composition, the specific intended use of the pharmaceutical composition, and the route of administration. Generally, the pharmaceutically acceptable components used in conjunction with this invention are used based on knowledge available in the art.

[0125] Autophagy positive regulator

[0126] Preferred autophagy positive regulators are as defined in some preferred embodiments and claims.

[0127] According to the present invention, preferred autophagy positive regulators are selected from a list of human proteins including: human TFEB, human Beclin-1, human AMBRA1, human ULK1, human ATG14, human STYK1 (also known as WIPI1), 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 polycystic protein 2), human GABP (also known as NFT2), human ATG7, and human ATG5.

[0128] The regulators human TFEB and human Beclin-1 were experimentally tested in the following non-limiting embodiments.

[0129] 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 autophagy process and regulate the formation of autophagosomes (see references 56-77 included in the References section). Based on the teachings of the present invention, these proteins can also be used as positive regulators of autophagy in modified lymphocytes according to the present invention, and in this context, can be used to improve cancer treatment.

[0130] Similarly, peptides are shown. (SEQ ID NO: 1) and (SEQ ID NO: 2) Induces autophagy. This was reported in Shoji-Kawata et al., Nature. 2013 Feb 14;494(7436):201-6. Based on the teachings of the present invention, these peptides can also be used as positive regulators of autophagy in modified lymphocytes according to the present invention, and in this context, can be used to improve cancer treatment.

[0131] The invention is further illustrated by the following non-limiting embodiments:

[0132] Example

[0133] Methods and Techniques

[0134] Cell lines. The neuroblastoma (NB) cell line SHSY5Y and 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), and U373.MG was purchased from ECACC (Salisbury, United Kingdom). CHLA 255 was kindly provided by Professor Malcolm Brenner of Baylor College of Medicine. SHSY5Y, CHLA255, U373.MG, CT10 36 A673 cells were maintained in DMEM medium (Merck-Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS) (Thermo Scientific, Pittsburgh, PA-USA) and 2 mM GlutaMax (Gibco, Thermo Scientific), while D341-MED cells were cultured in MEM medium (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 at 37°C with 5% CO2. Routine tests for mycoplasma and target antigen surface expression were performed on all cell lines, and validation was performed using STR analysis (Eurofins Genomics, Ebersberg, Germany).

[0135] Isolation and transduction of CAR T cells. Peripheral blood mononuclear cells (PBMCs) were isolated from the erythrocyte sedimentation rate (ESR) amber layer of a healthy donor (University Hospital Würzburg, Germany) with signed informed consent using Ficoll lymphocyte isolation medium (Ge Healthcare, Uppsala-Sweden). T lymphocytes were activated in a complete medium consisting of CTSOpTmizer T cell expansion medium (Thermo Scientific) supplemented with 2.5% human AB serum (AnProtec, Bruckberg-Germany) and 2 mM GlutaMax supplemented with 2.5% human AB serum (AnProtec, Bruckberg-Germany) and transduced according to the rules established by the Institutional Review Board (Approval No. 250 / 20-am), using Ficoll lymphocyte isolation medium (Ge Healthcare, Uppsala-Sweden), and TMCs from a healthy donor (University Hospital Würzburg, Würzburg, Germany) who had obtained written informed consent. On the day following activation, T cells were fed with recombinant human interleukin-2 (IL2, 100 U / 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 pre-coated with recombinant human RetroNectin (Takara-Bio.Kyoto-Japan) using specific retroviral supernatant and the aforementioned cytokines. On day 5 post-transduction, T cells were removed from RetroNectin and expanded in complete culture medium. 17,28 .

[0136] Isolation and expansion of NK cells. Following the rules established by the Institutional Review Board (Approval No. 250 / 20-am), NK cells were first isolated from the erythrocyte sedimentation rate (ESR) amber layer from healthy donors (University Hospital Würzburg) with signed informed consent using Ficoll lymphocyte isolation medium (Ge Healthcare) followed by the Rosette kit (Stem Cells, Cologne-Germany). NK cells were then cultured in NK medium supplemented with 5% human AB serum (Miltenyi) in the presence of feeder cells and IL-2 (100 U / ml) or with IL-2 / IL-15 (500 U / ml and 150 U / ml, respectively). Cells were then fed every 3 to 4 days and maintained at 37°C in a humidified atmosphere containing 5% CO2.

[0137] Isolation and expansion of γ / δ-T cells. Following the rules established by the Institutional Review Board (Approval No. 250 / 20-am), γ / δ-T cells were first isolated from the erythrocyte sedimentation rate (ESR) amber layer of a healthy donor (University Hospital Würzburg) with signed informed consent using Ficoll lymphocyte isolation medium (Ge Healthcare) followed by the Rosette kit (StemCells). γ / δ-T cells were then cultured in a complete medium consisting of CTS OpTmizer T cell expansion medium (Thermo Scientific) supplemented with 2.5% human AB serum (AnProtec) and 2 mM GlutaMax in the presence of IL-2 / IL-15 (100 U / ml and 150 U / ml, respectively) and zoledronic acid (10 μM), under a humid atmosphere of 5% CO2 at 37°C. Cells were then fed every 3 to 4 days.

[0138] Retroviral construct. SFG backbone used. 37 Generate γ-retroviral constructs encoding: Beclin-1 or TFEB with the internal ribosome entry site (IRES) and a truncated CD19 (ΔCD19) selector or enhanced green fluorescence protein (eGFP); a control vector encoding only IRES.ΔCD19 or IRES.eGFP; and an intracellular signaling domain with CD28.4-1BB as a co-stimulatory molecule and the ζ chain (ζ) as the signaling domain. 27 The third-generation GD2.CAR, and the IRES.Beclin-1 or IRES.TFEB or IRES.Beclin-1 peptide38 ( The aforementioned third-generation GD2.CAR, along with IRES.ATG5, was used. In the selected experiments, additional retroviral vectors encoding eGFP-firefly luciferase (eGFP / FFLuc) were used to label tumor cells for in vitro co-culture and in vivo studies as described above. 39 In selectivity experiments, retroviral vectors encoding B7-H3.CAR with CD28.4-1BBζ and third-generation CD30.CAR were also used. 40 .

[0139] Cytomegalovirus-specific T cell generation. To generate cytomegalovirus-specific T lymphocytes (CMVCTLs), PBMCs from CMV-positive donors were stimulated for 2 hours at 37°C in 5% CO2 with dendritic cells (DCs) loaded with CMV-pp65 pepmix (HCMVA, JPT, Berlin, Germany) at a ratio of 20:1, 5 mmol / L. Cell plates were then seeded in complete T cell culture medium. After 10 days, T cells were restimulated with DCs loaded with the same pepmix. Following the second stimulation, cells were expanded and fed with IL-7 (500 U / ml) and IL-15 (50 U / ml). 41 .

[0140] Phenotypic analysis. The expression of cell surface molecules was determined by flow cytometry using standard methods. 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-idiotypic antibody (1A7). 42 To quantify mitochondrial mass and mitochondrial potential, 100 nM MitoTRK Green and 250 nM MitoTRK Orange (Thermo Scientific) were used according to the manufacturer's procedures. 25 Samples were collected using BD FACS Canto II and analyzed using Flowlogic software (Inivai, Melbourne - Australia). At least 25,000 events were analyzed for each sample.

[0141] Quantitative RT-PCR (qPCR) analysis. Total RNA was extracted using the RNeasy mini kit (Qiagen, Venlo, The Netherlands) and 100 ng was reverse transcribed using the cDNA SuperScript™ VILO™ cDNA Synthesis Kit (Applied Biosystems, Foster City, CA, USA). RT-PCR was performed using QuantStudio6 with SYBR Green Master mix (Bio-RAD, Helculaes, California, USA) according to the manufacturer's instructions. Relative expression values ​​are normalized relative to the housekeeping gene GAPDH. The following primers were used: In the selected experiments, the pre-coated autophagy pathway of Bio-RAD was used for assays.

[0142] Western blot analysis was performed using RIPA lysis buffer (Cell Signaling Technology, Danvers, Massachusetts, USA) supplemented with a protease inhibitor mixture (Merck, Darmstadt, Germany) from 5 × 10⁻⁶ blots. 6 Proteins were extracted from cells. 50 mg of protein was isolated by SDS-PAGE, transferred to a polyvinylidene fluoride membrane (Bio-RAD), and blocked with 5% (w / v) skim milk powder in Tris-buffered saline containing 0.1% (v / v) Tween 20. The blot was incubated overnight at 4°C with primary antibody in PBS containing 0.1% Tween 20 and 5% skim milk powder. Detection was performed using horseradish peroxidase-conjugated secondary antibody (Bio-RAD), and visualization was performed using ECL plus (Amersham Bioscience, Slough, Buckinghamshire, UK).

[0143] Immunofluorescence. Cells were cultured in complete T-cell medium (or starved in Earle balanced salt solution 1× (Merck)) and then inoculated with 1×10⁻⁶ cells. 5 Inoculations were performed at a density of / μL on compartment slides coated with 10 ng / ml fibronectin and incubated at room temperature for 1 hour in the presence or absence of the autophagy inhibitor bafilomycin. Primary antibody for staining LC3 spots was diluted 1:300 and conjugated with a 1:300 diluted secondary antibody. Images were then acquired using an Olympus confocal microscope and analyzed via ImageJ.

[0144] Seahorse extracellular flux analysis. Seahorse experiments were performed using the XF Cell Mito Stress kit (SeahorseBioscience). OCR and ECAR were measured using the XF96 extracellular flux analyzer (Seahorse Bioscience). Briefly, cell plates were seeded onto poly-D-lysine-coated 96-well polystyrene Seahorse plates (200,000 T cells / well), equilibrated at 37°C for 1 h, and OCR (pmol / min) and ECAR (mpH / min) were measured under basal conditions with the addition of oligomycin (1 μM), carbonyl cyanide-4-phenylhydrazone (1.5 μM), and antimycin A / rotenone (1 μM / 0.1 μM).

[0145] Co-culture assays. For co-culture experiments, untransduced T cells (NT), Beclin-1.IRES.ΔCD19, TFEB.IRES.ΔCD19, GD2.CAR.IRES.Beclin-1, GD2.CAR.IRES.TFEB, GD2.CAR.IRES.Beclin1 peptide, or GD2.CAR.IRES.ATG5 T lymphocytes were seeded together with eGFP-modified tumor cell lines (SHSy5Y, CHLA255, U373.MG, D341.MED, CT10, and A673) at a specified E:T ratio into 24-well plates. After incubation at 37°C for 5 days, the co-cultures were collected and analyzed by FACS to detect residual tumor cells (GFP). + ) and CD3-based T cells 6 .

[0146] 3D spheroid co-culture assay. Tumor spheroids were generated using 96-well ultra-low adhesion plates. Neuroblastoma cells were seeded at a low concentration of 1,000 cells / well in 100 μL of specific medium and 0.24% methylcellulose per well. Medulloblastoma cells were seeded at a low concentration of 250 cells / well and cultured in 100 µL of MEM supplemented with 20% FBS and 0.24% methylcellulose per well. The plates were then centrifuged at 50 x g for 1 min at room temperature without acceleration or obstruction and subsequently incubated for 72 h. The plates were then automatically imaged using a Celigo imaging cytometer to assess fluorescence intensity before co-culture. NTT and CAR-T cells were added at this point with different E:T ratios, and the plates were cultured at 37°C for another 10 days with daily automatic imaging.

[0147] Extracellular vesicle detection. Cell supernatant was collected 24 to 48 hours after culture medium replacement. 100 μl of the cell supernatant was then stained in the dark at room temperature (RT) with a lipophilic cationic dye (LCD) and phalloidin (FITC) for 45 minutes. Samples were then appropriately diluted with PBS and acquired using a CytoFLEX flow cytometer (Beckman Coulter, Sacramento, CA, USA) equipped with volume counting. The threshold was set to the APC channel, the flow rate was adjusted to obtain a maximum of 7,000 events / second, and at least 1 million events were recorded per sample.

[0148] Next-generation sequencing. Next-generation sequencing experiments were performed using Genomix4life (Baronissi, Salerno, Italy). RNA concentration in each sample was determined using an ND-1000 spectrophotometer (NanoDrop), and its quality was assessed using an Agilent TapeStation 4200 (Agilent Technologies). Index libraries were prepared from RNA purified at 1 μg / ea using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina) according to the manufacturer's instructions. The libraries were quantified and pooled using an Agilent TapeStation 4200 (Agilent Technologies) so that each indexed sample was present in an equimolar amount, with a final pooled sample concentration of 2 nM. Cluster generation and sequencing of the pooled samples were performed using an IlluminaNextseq 500 (Illumina) at a final concentration of 1.8 pmol in 2 Å–75 pM paired-ends. The generated raw sequence files (.fastq files) were analyzed using the FastQC tool (http: / / www.bioinformation.babraham.ac.uk / projects / astqc / ) with FastQC Fastq Underwent Quality Control. STAR (version 2.5.2a) was used. 43 Assembly of hg19 (GRCh37.p13) based on reference genome obtained from Ensembl. 44 (https: / / grch37.ensembl.org / Homo_sapians / Info / Index) A map is constructed on the double-ended readout. FeatureCount is used.45 The algorithm quantifies the expressed transcripts for each sample. DESeq2 46 Used to normalize the data, which is then used for differential expression analysis.

[0149] A xenograft mouse model was used for in vivo studies. To investigate the in vivo antitumor activity of autophagy-reprogrammed CAR T cells, 0.75 × 10⁻⁶ cells were injected intraperitoneally into 5-week-old NOD.Cg-Prkdcscid II2rgtm1Wjl / SzJ male mice (Charles River) in the presence of matrix gel. 6 SHSY5Y-FF-Luc.GFP. Following transplantation, mice received an intravenous injection (iv) of 10 × 10⁻⁶ dextrose. 6 NT or genetically modified T cells. Tumor growth was evaluated using the IVIS imaging system (PerkinElmer). All in vivo experiments were conducted in accordance with international ethical, EU and national requirements and were approved by the Italian Ministry of Health (N 195 / 2021-PR).

[0150] Statistical analysis. Data were summarized as mean ± standard deviation (SD). The Student's t-test (two-tailed) was used to determine statistical significance between samples, where p < 0.05 was considered statistically significant. When multiple comparisons were required, statistical significance was assessed by repeated measures ANOVA followed by a Mantel-Cox test. Graph generation and statistical analysis were performed using Prism v9 software (GraphPad, La Jolla, CA, USA). The following references were used to display significance p-values ​​in the graphs: For p-values ​​from 0.01 to 0.05; p-values ​​from 0.001 to 0.009; p-values ​​for the range of 0.0001 to 0.0009; For p-values ​​< 0.0001.

[0151] result

[0152] Example 1: The autophagy pathway was blocked in isolated T lymphocytes.

[0153] To address unexplored biological questions regarding the regulation of autophagy in ex vivo manipulated T lymphocytes, primary T cells were first isolated fresh from healthy donors and polyclonal activated using αCD3 / CD28 mAb according to protocols typically used to generate genetically modified T lymphocytes. The T cells were then analyzed at different time points after activation (days +1, +2, +3, and +7) up to day 14 (mean days of ATMP ex vivo culture). Figure 1 As reported by Institute A, autophagy-related mRNAs of LC3, Ambra1, p62, and ULK1 were immediately and significantly downregulated at all analyzed time points after activation (p < 0.0001). This downregulation was still observed when expanded T cells were restimulated polyclonally (day 15). The data were then validated using a commercially available autophagy monitoring assay, revealing the scale of autophagy blockade, highlighting the downregulation of other autophagy-related transcripts and the upregulation of BNIP3 and cysteine-3 mRNAs, which have been reported to have a negative impact on autophagy activation. 47,48 ( Figure 1 B). It was then demonstrated that this downregulation of the autophagy pathway involved both the CD4 and CD8 populations, with the same scale and significance observed in the total CD3 population (p<0.0001). Figure 1 Based on this observation, three different CAR-redirected T cells (GD2.CAR, CD30.CAR, and B7-H3.CAR) were tested on day 14 post-transduction, and real-time PCR was performed on the expression of the aforementioned autophagy-related mRNAs (LC3, Ambra1, p62, and ULK1) in each of the related unmodified T cells (NTs) generated from different healthy donors. Figure 1 E showed that, under these conditions, both NT and CAR-T cells (regardless of construct) exhibited strong and significant downregulation of four autophagy transcripts compared to the corresponding freshly isolated and uncultured T cells (p<0.0001). The data were then validated at the protein level by Western blot analysis, revealing a clear blockage of autophagy processes in various prospective studies, as indicated by: accumulation of p62, reduction / loss of LC3II, increase of p-mTOR, significant accumulation of p-ULK1, and downregulation of p-AMPK. Figure 1 F and 1K). Regarding CD4 count on day 14 of culture. + and CD8 + Further data analysis of both CAR T cell populations confirmed the observation of the total CAR T cell population. Figure 1 G). It has been shown that this phenomenon is not due to the use of specific cytokines (IL2) or strategies for activating T cells (αCD3 / CD28 mAb). In fact, as... Figure 1As reported by H, when CAR T cells are activated and expanded by IL7 / 15... 49 And when T cells are activated by professional antigen-presenting cells pulsed with pp65-CMV peptide to produce CMV-specific CTLs. 37,50 Autophagy inhibition could also be detected at this time. At this point, it has been clearly demonstrated how in vitro manipulation of T lymphocytes can induce a significant and durable blockade of the autophagy process. The inventors sought to verify whether it was possible to pharmacologically re-induce activation of the autophagy pathway in CAR T cells. Therefore, in the presence or absence of IL2, GD2.CAR cells were cultured on day 14 with Torin1 (250 nM), a potent autophagy inducer capable of blocking mTOR phosphorylation. 51 Treatment was conducted at 6 and 24 hours. It was found, at least pharmacologically, that autophagy was restored in ex vivo expanded T cells upon strong stimulation (cytokines). In fact, total ULK1 levels were observed to remain unchanged, while its phosphorylation decreased after 6 hours of treatment, whereas a decrease in p62 was only observed after 24 hours of drug addition. Figure 1 J).

[0154] Based on these results, and to investigate whether autophagy inhibition is a generalized phenomenon caused by in vitro culture, we analyzed two other platforms used for adoptive cell therapy: natural killer (NK) and γδ-T cells. As with conventional T cells (α / β), NK and γδ-T cells were isolated from healthy donors and expanded in vitro. Again, under these conditions, we observed strong autophagy inhibition at both the mRNA and protein levels in both cell products after 14 days of culture. Figure 1 L to M).

[0155] Example 2: Constitutive reactivation of autophagy is harmless to in vitro expanded T cells.

[0156] The direct translational significance of this discovery is that ex vivo manipulated and cultured T cells used for adoptive immunotherapy lack a well-functioning autophagy process, which helps them survive in harsh environments and respond to stress, thus ensuring long persistence and good cellular adaptability—characteristics that have been shown to be crucial for a durable antitumor response. Based on these data and the fact that autophagy can be reactivated, the inventors set out to verify whether the autophagy pathway can be constitutively reactivated by overexpression of key proteins involved in this process. They selected two autophagy proteins, TFEB and Beclin1, and generated a retroviral construct. Since these two proteins are intracellular molecules, the selection marker ΔCD19 was inserted into the construct, allowing for monitoring of transduced cells using analytical methods such as flow cytometry. By observing the percentage of ΔCD19 expression after transduction (…), the results were obtained. Figure 2 A) and intensity level (MFI) Figure 2 B) Both remained constant over time, and the construct was validated for the first time on immortalized tumor cells Hek293T. Then, the levels of TFEB and Beclin1 in the transcript were determined (p<0.0001). Figure 2 C) and protein levels ( Figure 2 D) Both will be adjusted upwards accordingly.

[0157] Therefore, the construct was tested on primary T cells. In all test cases, T lymphocytes transduced with the construct ΔCD19+ control, TFEB+ΔCD19, and Beclin1+ΔCD19 showed no signs of cytotoxicity and reported expansion levels comparable to the corresponding unmodified T cells (NT). Figure 3 A). Transduction level greater than 60%, and at the absolute percentage level ( Figure 3 B) and the signal strength level marked by ΔCD19 (B) Figure 3 C) Both aspects remained constant over time. Although TFEB expression was consistently lower compared to the other two conditions, this difference was not significant.

[0158] As previously demonstrated, cells genetically modified with TFEB and Beclin1 are able to constitutively overexpress the corresponding transcripts ( Figure 3 D)(p<0.0001) and protein ( Figure 3 E) Both. No differences in the distribution of CD4 and CD8 cell subsets were observed under any test conditions. Interestingly, an increase in mitochondrial potential (Mitrotracker) was observed in T cell populations genetically modified with a retroviral construct encoding Beclin1. Furthermore, autophagy flux analysis revealed that pathways could be regulated by reactivating autophagy genes through overexpression. Figure 3 H to I). Figure 3 I demonstrated how autophagy is activated in autophagy-modified T cells under normal culture conditions (basal level) (p<0.05) and especially when cells are exposed to stress conditions such as those induced by nutritional starvation (EBS) (p<0.001). Finally, it was shown that overexpression of TFEB and Beclin1 was not associated with increased allogeneic reactivity, as they failed to kill the CHLA255 neuroblastoma cell line at a 1:1 ratio in a short-term 24-hour killing assay. Figure 3 J).

[0159] Example 3: Overexpression of TFEB and Beclin1 in GD2.CAR T cells restores autophagy pathway and improves its phenotype and cytotoxic activity.

[0160] To date, the inventors have demonstrated that it is possible to restore the autophagy pathway in ex vivo expanded T cells without any toxicity through overexpression of two autophagy proteins, TFEB and Beclin1. They then decided to translate these results to a CAR platform. Therefore, they generated a novel retroviral bicistronic construct encoding a third-generation GD2.CAR and either TFEB or Beclin1 protein. The two expression cassettes are separated by an IRES sequence. Figure 4 As indicated by A, no difference in proliferation was observed between the GD2.CAR+TFEB and GD2.CAR+Beclin1 constructs compared to the GD2.CAR control. However, as the inventors have previously studied... 27 All constructs encoding third-generation GD2.CAR, as previously reported, showed significantly increased proliferation on day 10 of culture compared to unmodified control cells (NT) (p<0.05). GD2.CAR anti-idiotypic mAb (1A7) was also used by flow cytometry. 52 The monitored transduction levels were greater than 75%, and there were no statistically significant differences between groups. Furthermore, the transduction levels remained constant over time, confirming the absence of toxicity following autophagy protein overexpression. These data were determined as absolute transduction percentages (%). Figure 4 B) and CAR expression intensity (MFI) Figure 4 C) Both. At the mRNA level, both transcripts were found to be significantly overexpressed in their respective transduced cells (TFEB: p < 0.0001; Beclin1: p < 0.01). However, differences in expression were observed between TFEB and Beclin1 expressing cells, as the expression level of the first was shown to be 3 times higher than that of the other. Figure 4 D). Furthermore, the expression levels of both autophagy proteins were significantly lower than those observed in HEK293T cells (D). Figure 2 C). These findings were also observed at the protein level. Figure 4 E). It is equally important to emphasize that they did not observe any differences in the expression and distribution of transgenic molecules between the CD4 and CD8 T cell subsets across the various conditions analyzed.

[0161] Subsequently, the inventors used the SeaHorse extracellular flux analyzer to characterize the metabolic state of autophagy-recovered GD2.CAR T cells under both basal and stress-responsive conditions. Energy maps revealed that both types of autophagy-recovered GD2.CAR T cells were more viable under both basal (time: 0–18 min) and stress (time: 38–58 min; mitochondrial oxidative phosphorylation uncoupling agent - FCCP: 1.5 μM) conditions. Figure 4(G). In fact, under basal conditions, both autophagy-recovered GD2.CAR T cells exhibited a higher O2 Consumption Rate (OCR) compared to normal GD2.CAR, indicating enhanced OXPHOS. Most importantly, after disruption of the mitochondrial complex with FCCP, both autophagy-recovered GD2.CAR T cells showed a significant increase in maximal respiration, indicating that both autophagy-recovered GD2.CAR T cells can better respond to increased energy demands and adapt to stress conditions. It is important to note here that the response to FCCP-induced stress was more pronounced in cells expressing TFEB, but this may be due to unequal expression of the two proteins in the two constructs.

[0162] Furthermore, the metabolic profile of glycolysis has been expanded, which has been reported to be crucial for its effector function under stress. 53 Therefore, glycolytic activity was evaluated by analyzing the extracellular acidification rate (ECAR). It was observed that under basal conditions, GD2.CAR+Beclin exhibited higher metabolic activity compared to GD2.CAR+TFEB and normal GD2.CAR, even in the presence of unfavorable Beclin1 levels. Figure 4 H).

[0163] However, under stress induced by treatment with oligomycin (1 μM), both GD2.CARs expressing autophagy proteins showed a better response, indicating superior activity compared to normal GD2.CAR T cells. Figure 4 H).

[0164] These data confirm Figure 4 The reactivation of the autophagy pathway is illustrated in Figure I, showing the accumulation of LC3II protein when autophagy-recovered GD2.CAR T cells are exposed to a drug (chloroquine) that can block the autophagy process by inhibiting the fusion of autophagosomes with lysosomes. This phenomenon is even more pronounced when cells are subjected to severe stress, such as nutrient deprivation-induced stress. The reactivation of this pathway is also indicated by the accumulation of other proteins involved in the process, such as ATG14, p-ATG14, and ULK1. Figure 4 J). Then, all these molecular data were reconfirmed using the second inhibitor of the autophagy pathway (Bafolomycin A1). Figure 4 K to L). This result was then confirmed using an additional method that allows for visualization and quantification of LC3 protein in each cell. Figure 4M). Similarly, the inventors were able to demonstrate that, under nutritional stress conditions, cells expressing autophagy genes have a larger number of autophagosomes (LC3 per cell) (p<0.05).

[0165] Example 4: Immune characterization of autophagy-reprogrammed CAR T cells.

[0166] Several preclinical and clinical studies have clearly demonstrated the importance of certain immunophenotypic aspects in ensuring long-term antitumor responses to T and NK-based ATMPs, including undifferentiated T cell phenotype, activation marker expression, and exhaustion-free response. Therefore, deep immunophenotypic characterization of GD2.CAR T cells was performed, and Beclin1 overexpression was observed to significantly increase CD4 counts compared to conventional GD2.CAR T cells. + CAR + and CD8 + CAR + The initial cells (CD45RA) in both populations + CD62L + The amount of ) and the reduction of effector memory cells (EM) (CD45RO) + The amount of CD62L- was observed. (CD4 initial: p<0.001; CD8 initial: p<0.05; CD4·EM: p<0.0001; CD8 EM: p<0.001). This was particularly evident in CD4 α-cells between GD2.CAR+Beclin1 and GD2.CAR+TFEB T cells. + CAR + Statistically significant differences existed within the subpopulations, with higher and lower percentages of naïve and EM cells in CAR T cells expressing Beclin1 (naïve: p<0.01; EM: p<0.01). Statistical differences were observed only between GD2.CAR+Beclin1 and GD2.CAR+TFEB subpopulations on CD8. + CAR + Statistically significant differences were observed in the EM subsets, with Beclin1-expressing cells showing lower levels than TFEB-expressing cells (p<0.01). Figure 5 (A to B). Although not statistically significant, an increase in the stem cell memory-like (SCMT) subset was observed in cells where the autophagy pathway was reactivated. This cell population has been reported to be extremely important for long-term responses to CAR-T therapy. 54,55 This increase occurs between TFEB and Beclin1 on CD4. + The CAR+ subgroup is similar, but the Beclin1 subgroup is not. + T cells in CD8 + CAR + More pronounced in subgroups ( Figure 5C). No exhaustion phenotype was observed under any of the analytical conditions. Figure 5 (D to G). However, regardless of the construct used, CAR T cells showed good activation levels. Compared to both GD2.CAR and GD2.CAR+TFEB T cells, GD2.CAR+Beclin1 T cells showed significantly higher activation levels in CD4+ cells. + and CD8 + CAR + The percentage of the activated marker Lag3 was significantly increased in both subgroups (p<0.05 and p<0.001, respectively). Figure 5 D to G). Furthermore, it was also found to be related 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 + Compared to two other activating markers, HLA-DR and CD25 (only on CD8), p < 0.01, the two other activating markers were significantly different. + CAR + The expression intensity (MFI) of the subpopulation in the GD2.CAR+Beclin1 T population was increased. Figure 5 H to K).

[0167] Finally, the characterization of GD2.CAR+Beclin1 / TFEB was completed by: expanding T cells in vitro for 12 or 24 days and co-culturing them with three different tumor cell lines: SHSy5Y (neuroblastoma), CHLA255 (neuroblastoma), and D341 (medulloblastoma) at different effector:target ratios to conduct short-term (24-hour) and long-term (4-day) cytotoxicity studies. Figure 6 The report indicates that, compared to the GD2.CAR control, both constructs generally showed a better trend in antitumor activity, even more pronounced in the GD2.CAR+Beclin1 construct. Better results were typically observed at unfavorable effector:target ratios (tumor cells outnumbering T cells – the state closest to that found in patients). In short-term co-culture with T cells at day 12, GD2.CAR+TFEB cells generally showed improved tumor cell killing, but not a significant improvement. Furthermore, GD2.CAR+Beclin1 T cells consistently showed a significant improvement compared to GD2.CAR+TFEB T cells (p values ​​ranging from 0.05 to 0.01). Figure 6A to C; A: SHSy5Y, B: CHLA255; C: D341). However, when co-cultured with T cells on day +24 (more exhausted), the results showed a greater and more significant increase in the antitumor activity of T cells restored by TFEB and Beclin1, and the data were even better again in cells overexpressing Beclin1. Figure 6 D to F; D: SHSy5Y, E: CHLA255; F: D341). On the +12th day ( Figure 6 G to I) and +24 days ( Figure 6 The same results were also reproduced in the long-term (4 days) co-culture assay of J to L cells with T cells.

[0168] Ultimately, given that these data highlight the advantages of the GD2.CAR+Beclin1 construct, the functional potency of these cells relative to conventional GD2.CAR T cells was further validated using a more sophisticated model in which T cells were co-cultured with tumor cells that had previously been organized into spheroids to more accurately mimic tumor masses present in patients. Figure 7 As shown, in the following two different tumor models, GD2.CAR+Beclin1 T cells exhibited faster and more significant anti-tumor activity than GD2.CAR T cells: Neuroblastoma_SHSy5Y ( Figure 7 A to B; p<0.05) and medulloblastoma_D341 ( Figure 7 C to D; p<0.05). This feature is extremely important in the treatment of refractory patients because it prevents tumor adaptation following immunological pressure exerted by T cells.

[0169] To demonstrate that autophagy recovery can also be achieved using other autophagy proteins and autophagy-inducing peptides, the effects of ATG5 and Beclin1 peptides were investigated. Figure 8 Reports A through B indicate that overexpression of ATG5 and Beclin1 peptides on primary T cells is feasible and does not affect in vitro cell growth. In both cases, when GD2.CAR T cells are equipped with autophagy proteins / peptides, these cells exhibit improved cell growth against medulloblastoma (D341). Figure 8 C) and neuroblastoma (SHSY5Y, Figure 8 D) It mainly showed superior antitumor activity in long-term co-culture at low E:T ratios. This also demonstrated that this phenomenon was not caused by GD2.CAR, but could be applied to any CAR. Therefore, the above data were validated using a novel CAR targeting the B7-H3 antigen. Figure 8 (E to G), and tested its functionality against the tested sarcoma cell lines (Ewing sarcoma and rhabdomyosarcoma).

[0170] Example 5: Molecular characterization of CAR T cells reprogrammed by autophagy.

[0171] Then, based on the generated data, the autophagy-recovered CAR T cells were further characterized in depth using an omics platform.

[0172] First, the effects of autophagy recovery on GD2.CAR were investigated using non-targeted proteomics. After 48 hours of antigen stimulation with CAR via an anti-CAR idiotype (1A7), analysis revealed that overexpression of TFEB and Beclin1 induced changes in protein content within genetically modified lymphocytes. Figure 9 A). These changes in the proteomic profiles share common characteristics between the two groups, but also exhibit unique specific patterns. It is important to note that the modifications induced by TFEB are significantly stronger than those triggered by Beclin1. Figure 9 B). This phenomenon may be due to the fact that TFEB is a transcription factor while Beclin1 is not. Specifically, this analysis showed that TFEB upregulation specifically induced significant improvements in several pathways, including: eukaryotic translation elongation / termination, response to amino acid deficiency, RNA / amino acid / protein metabolism, cytokine signaling, and positive regulation of immune responses. Figure 9 C). Beclin1 upregulation specifically and significantly enriches several other pathways, including: the ER-phagosome pathway, antigen processing-cell presentation, activation of intrinsic and adaptive systems, TCR signaling, endosome / vacuole pathway, and extracellular efflux bodies (C). Figure 9 D). However, it has been shown that both TFEB and Beclin1 can significantly promote migration, cellular responses to starvation / stress / stimulation, apoptosis regulation, downstream TCR signaling, IL1 signaling, intracellular signaling via second messengers, small molecule transport, the CD28-dependent Vavi pathway, and other pathways (D). Figure 9 E).

[0173] The long-term effects of TFEB and Beclin1 expression on GD2.CAR T cells were then investigated by analyzing their mRNA profiles. RNA-seq studies were performed 96 hours after CAR antigen stimulation, revealing that TFEB could sustainably regulate metabolism at different levels, likely due to its status as a transcription factor involved in lysosomal degradation, mTOR signaling, glycosaminoglycan degradation, amino and nucleotide sugar metabolism, phosphoinositol metabolism, and ferroptosis pathways. On the other hand, Beclin1 appeared to attenuate its metabolic effects and provide better T cell adaptation, regulating cell cycle, DNA replication, JAK-STAT signaling, cytokine-cytokine receptor interactions, NOD and RIG-I-like receptor signaling, p53 signaling, apoptosis, TNF and NF-κB signaling. Figure 9 F).

[0174] Finally, using a non-targeted metabolic approach, we explored the metabolite profiles of different autophagy-reprogrammed GD2.CAR T cells (GD2.CAR, GD2.CAR+TFEB, GD2.CAR+Beclin1, GD2.CAR+ATG5, and GD2.CAR+Beclin1 peptide) 96 hours after CAR antigen stimulation. Figure 9 G reported that, under all autophagy editing conditions, significant increases or decreases in amino acid-derived proteins, lipids, or metabolites were observed, along with a general increase in deoxynucleotides, which are crucial for DNA replication and autophagy regulation. Figure 9 G).

[0175] Example 6: Quantification of extracellular vesicles produced by GD2.CAR T cells recovered from autophagy.

[0176] Based on the generated data and the beneficial effects of Beclin1 overexpression observed to date, one of the identification pathways in proteomics analysis was validated, which is detectable in Beclin1-modified T cells but not in TFEB-modified T cells. Therefore, the extracellular efflux profile was examined by quantifying the amount of extracellular vesicles. The results showed that autophagy regulation induced an increase in vesicle / vacuolar formation in genetically modified T lymphocytes ( Figure 10 A). However, it was observed that, only under Beclin1 conditions, the number of extracellular vesicles in the culture supernatant that was already in a resting state significantly increased ( Figure 10 B). This phenomenon persists after antigen stimulation, and ATG5-reprogrammed GD2.CAR T cells also produce more extracellular vesicles compared to GD2.CAR T cells. Figure 10 C).

[0177] Example 7: Evaluation of autophagy-recovered GD2.CAR T cells in a xenograft mouse model.

[0178] To validate the in vitro data in more complex models, xenograft mouse models were used. These experiments were conducted in a blinded manner using an institution (Plaisant) in Rome, Italy. The inventors were aware of the limitations observed in animal models using GD2.CAR—in a preliminary study leading to a Phase I / II clinical trial (NCT03373097), the genetically modified T cells completely eradicated the tumor after several weeks, unlike what was subsequently observed in human studies—leading them to challenge a system simulating cell exhaustion. Therefore, lymphocytes grown in vitro for at least three weeks were used.

[0179] Under these conditions, mice treated with GD2.CAR showed no control of tumor growth and exhibited significant signs of CAR toxicity 20 days after CAR infusion. However, the two groups treated with GD2.CAR+TFEB and GD2.CAR+Beclin1 clearly demonstrated strong and durable antitumor activity until the end of the experiment at +90 days. Figure 11 A). Peripheral blood from all treated animals was then analyzed by flow cytometry (FACs) on day 12 following T-cell infusion. No statistically significant differences were observed in the distribution of human T lymphocytes and CARs from this analysis; however, a lower frequency of these subsets was observed in the group modified with autophagy proteins. Figure 11 B). Furthermore, analysis of CD4 / CD8 distribution did not reveal any statistically significant differences between groups; however, a slight decrease in CD4 and a slight increase in CD8 were observed in autophagy-reprogrammed T cells, primarily in Beclin1-treated mice. Figure 11 C). To verify the function of circulating T lymphocytes, immunocharacterization was performed ( Figure 11 D to F). There was no significant difference in the percentage of resting CD4 and CD8 CAR T cells between groups, however, it was lower in the Beclin1-treated group ( Figure 11 D). Alternatively, analysis of depletion markers (such as PD1 and Lag3) in both CD8 and CD4 subsets highlighted that these markers were significantly reduced in the GD2.CAR+Beclin1 treatment group and only slightly reduced in the GD2.CAR+TFEB group. Finally, analysis of chemochine receptor 3, which is important for migration and memory phenotypes, showed that although there was no significant difference between groups, its expression was higher on autophagy-reprogrammed T cells, particularly in the GD2.CAR+Beclin1 group. Figure 11 E to F).

[0180] Industrial applicability

[0181] The pharmaceutical compositions, recombinant nucleic acids, modified lymphocytes, and products used in this invention are industrially applicable. For example, they can be used to manufacture or as pharmaceutical products.

[0182] References

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Claims

1. Modified human lymphocytes containing recombinant nucleic acids or recombinant nucleic acid groups encoding at least one autophagy positive regulator.

2. The modified human lymphocytes according to claim 1, wherein the at least one autophagy regulator is one or more human proteins.

3. The modified human lymphocytes according to claim 1 or 2, wherein the one or more human proteins are selected from: 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 polycystic protein 2, human NFT2, and human ATG7.

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

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

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

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

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

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

10. The modified human lymphocytes of claim 9, wherein the expression of at least one autophagy positive regulator derived from the recombinant nucleic acid or recombinant nucleic acid group is constitutive.

11. The modified human lymphocytes of claim 9, wherein the expression of at least one autophagy positive regulator derived from the recombinant nucleic acid or recombinant nucleic acid group is inducible.

12. The modified human lymphocytes according to any one of the preceding claims, wherein the lymphocytes are T cells, NK cells, or NKT cells.

13. The modified human lymphocytes according to any one of the preceding claims, wherein the lymphocytes are T cells.

14. The modified human lymphocytes according to any one of the preceding claims, wherein the lymphocytes are αβ T cells.

15. The modified human lymphocytes according to any one of claims 1 to 13, wherein the lymphocytes are γδT cells.

16. The modified human lymphocytes according to any one of the preceding claims, wherein the lymphocytes are CD8+. + T cells.

17. The modified human lymphocytes according to any one of claims 1 to 15, wherein the lymphocytes are CD4+. + T cells.

18. The modified human lymphocytes according to any one of claims 1 to 12, wherein the lymphocytes are NK cells.

19. The modified human lymphocytes according to any one of claims 1 to 12, wherein the lymphocytes are NKT cells.

20. The modified human lymphocytes according to any one of the preceding claims, wherein the lymphocytes further comprise and express recombinant nucleic acids or recombinant nucleic acid groups encoding T cell receptors or chimeric antigen receptors.

21. The modified human lymphocyte of claim 20, wherein the lymphocyte comprises and expresses a recombinant nucleic acid or recombinant nucleic acid group encoding a T cell receptor.

22. The modified human lymphocyte of claim 20, wherein the lymphocyte comprises and expresses a recombinant nucleic acid or recombinant nucleic acid group encoding a chimeric antigen receptor.

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

24. The modified human lymphocytes of claim 23, wherein the cancer cell antigen is an antigen of solid tumor.

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

26. The modified human lymphocytes of claim 25, wherein the brain cancer is neuroblastoma.

27. The modified human lymphocytes of claim 25, wherein the brain cancer is medulloblastoma.

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

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

30. The modified human lymphocytes according to any one of the preceding claims, wherein the recombinant nucleic acid or recombinant nucleic acid group encoding the at least one autophagy positive regulator and / or the recombinant nucleic acid or recombinant nucleic acid group encoding the T cell receptor or chimeric antigen receptor is an expression cassette.

31. The modified human lymphocytes of claim 30, wherein the expression cassette is derived from a retroviral vector.

32. The modified human lymphocytes of claim 30 or 31, wherein the expression cassette comprises a selection marker.

33. The modified human lymphocytes of claim 32, wherein the selection marker is ΔCD19.

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

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

36. The method of claim 34 or 35, wherein the method comprises the step of in vitro amplification of the modified human lymphocytes.

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

38. A pharmaceutical composition comprising modified human lymphocytes according to any one of claims 1 to 33 or modified human lymphocytes obtainable by any one of claims 34 to 37.

39. The modified human lymphocytes according to any one of claims 1 to 33, for use in medicine.

40. The modified human lymphocytes according to any one of claims 1 to 33 or the pharmaceutical composition according to claim 38, in a method of treating cancer in a human patient, wherein the modified human lymphocytes are the modified human lymphocytes according to claim 23.

41. The modified human lymphocytes or pharmaceutical composition of claim 40, wherein the treatment of the cancer is an immunotherapeutic treatment of cancer.

42. The modified human lymphocytes or pharmaceutical composition of claim 41, wherein the cancer is a solid cancer, and the modified human lymphocytes are the modified human lymphocytes of claim 24.

43. The modified human lymphocytes or pharmaceutical composition of claim 42, wherein the solid tumor is a brain cancer, and the modified human lymphocytes are the modified human lymphocytes of claim 25.

44. The modified human lymphocytes or pharmaceutical composition of claim 43, wherein the brain cancer is neuroblastoma, and the modified human lymphocytes are the modified human lymphocytes of claim 26.

45. The modified human lymphocytes or pharmaceutical composition of claim 44, wherein the brain cancer is medulloblastoma, and the modified human lymphocytes are the modified human lymphocytes of claim 27.

46. ​​The modified human lymphocytes or pharmaceutical composition according to any one of claims 41 to 45, wherein the cancer cell antigen is an antigen of pediatric cancer, and the modified human lymphocytes are the modified human lymphocytes according to claim 28.

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