MPC INHIBITION FOR PRODUCING T-CELLS WITH A MEMORY PHENOTYPE

Abstract

The present invention relates to an in vitro cell culture method comprising a step of contacting T-cells with an MPC inhibitor, and further to a cell population comprising T-cells with a memory phenotype obtained by said method, preferably, wherein the T-cells are human cells. The present invention also relates to a method for generating and/or maintaining T-cells and/or B-cells with a memory phenotype comprising the steps of culturing T-cells and or B-cells in vitro and adding an MPC inhibitor to the culture. The invention furthermore relates to a population of T-cells and/or B-cells obtained by the methods of the invention. Also provided are immunotherapies using the cells of the invention. Furthermore, provided is an MPC inhibitor for use in immunotherapy and/or as a vaccine co-adjuvant.

Claims

1. An in vitro cell culture method comprising a step of contacting T-cells with an inhibitor of the mitochondrial pyruvate carrier (MPC inhibitor).

2. The method of claim 1, wherein T-cells with a memory phenotype are generated and/or maintained.

3. The method of claims 1 or 2 comprising a further step of obtaining the T-cells from the culture, thereby producing a cell population comprising T-cells with a memory phenotype.

4. The method of any one of claims 1 to 3, wherein the T-cells are activated during culture.

5. The method of any one of claims 1 to 4, wherein the T-cells are expanded during culture, for example for 3 to 5 weeks.

6. The method of any one of claims 1 to 5, wherein the T-cells comprise CD8+ T-cells.

7. The method of any one of claims 1 to 6, wherein the T-cells are mammalian cells, preferably human cells.

8. The method of any one of claims 1 to 7, wherein the T-cells are human umbilical cord blood mononuclear cells (CBMC) and/or peripheral blood mononuclear cells (PBMC).

9. The method of any one of claims 1 to 8, wherein the T-cells are autologous cells.

10. The method of any one of claims 1 to 8, wherein the T-cells are allogeneic cells.

11. The method of any one of claims 1 to 10, wherein the T-cells are tumor-infiltrating T-cells and/or obtained from tumor-infiltrating T-cells.

12. The method of any one of claims 1 to 11, wherein the T-cells are tumor-draining lymph node cells and/or obtained from tumor-draining lymph nodes.

13. The method of any one of claims 1 to 12, wherein the T-cells comprise a heterologous antigen receptor, preferably a T-cell receptor (TCR) or a chimeric antigen receptor (CAR).

14. The method of any one of claims 1 to 13, wherein the T-cells are contacted with the MPC inhibitor from the beginning of the culture and/or activation.

15. The method of any one of claims 1 to 14, wherein the T-cells are contacted with the MPC inhibitor at least during activation.

16. The method of any one of claims 1 to 15, wherein the T-cells are contacted with the MPC inhibitor during the entire culture period.

17. The method of any one of claims 1 to 16, wherein the T-cells are activated by contacting them with an antigenic peptide, in particular in the presence of antigen-presenting cells, and/or artificial antigen presenting cells.

18. The method of any one of claims 1 to 17, wherein the T-cells are activated by contacting them with anti-CD3 and anti-CD28 antibodies, wherein said antibodies may be in solution, coupled to beads and/or coupled to artificial antigen presenting cells.

19. The method of any one of claims 1 to 18, wherein the T-cells are further contacted with IL-2.

20. The method of any one of claims 1 to 19, wherein the T-cells are contacted with (i) the MPC inhibitor, (ii) IL-2, and (iii) anti-CD3 and anti-CD28 antibodies and/or an antigenic peptide, in particular wherein the MPC inhibitor (i) is present in the culture medium, and IL-2 (ii) and/or the anti-CD3 and anti-CD28 antibodies and/or antigenic peptide (iii) are present in the culture medium and/or attached to the surface of antigen presenting cells and/or artificial antigen presenting cells.

21. The method of any one of claims 1 to 20, wherein the T-cells are first contacted with (i) the MPC inhibitor, (ii) IL-2, and (iii) anti-CD3 and anti-CD28 antibodies and/or an antigenic peptide, and then with IL-2 and IL-7.

22. The method of any one of claims 1 to 21, wherein the T-cells are cultured in a medium comprising (i) the MPC inhibitor, (ii) IL-2, and (iii) anti-CD3 and anti-CD28 antibodies and/or an antigenic peptide.

23. The method of any one of claims 1 to 22, wherein the T-cells are first cultured in a medium comprising (i) the MPC inhibitor, (ii) IL-2, and (iii) anti-CD3 and anti-CD28 antibodies and/or an antigenic peptide and then in a second medium comprising TL-2 and IL-7.

24. The method of any one of claims 1 to 23, wherein the MPC inhibitor comprises a small molecule, a nucleotide or a precursor thereof which interferes with Mpc1 and/or Mpc2 RNA (siRNA or shRNA), and/or an antibody and/or monobody.

25. The method of any one of claims 1 to 24, wherein the MPC inhibitor comprises at least one small molecule and/or an siRNA, preferably at least one small molecule.

26. The method of claim 25, wherein the at least one small molecule comprises UK5099 Pioglitazone, Rosiglitazone, MSDC-0602, MSDC-0160 and/or Zaprinast, in particular wherein UK5099 is 2-cyano-3-(1-phenyl-1H-indol-3-yl)-2-propenoic acid.

27. The method of any one of claims 1 to 26, wherein the MPC inhibitor comprises UK5099.

28. The method of claims 26 or 27, wherein the concentration of UK5099 is 25, 50 or 75 μM, preferably 25 μM.

29. A cell population comprising T-cells with a memory phenotype obtained by the method of any one of claims 3 to 28, preferably wherein the T-cells are human cells.

30. The cell population of claim 29 or the method of any one of claims 3 to 28, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cells in the population are T-cells with a memory phenotype, in particular wherein said T-cells with a memory phenotype express CD62L, preferably wherein said CD62L expression is at the cell surface.

31. The cell population of claims 29 or 30, wherein at least 90%, 95%, 98% or 99% of the cells in the population are T-cells.

32. The cell population of any one of claims 29 to 31, wherein at least 90%, 95%, 98% or 99% of the cells in the population are CD8+ T-cells.

33. The cell population of any one of claims 29 to 32 or the method of any one of claims 3 to 28, wherein the cell population comprises a higher proportion of T-cells with a memory phenotype and/or shows in average a more pronounced memory phenotype compared to a control cell population, in particular wherein the control cell population is obtained in parallel by the same method except that the control T-cells have not been contacted with an MPC inhibitor.

34. The cell population of any one of claims 29 to 33, or the method of any one of claims 3 to 28, wherein the T-cells maintain a memory phenotype in vivo when administered to a subject.

35. The cell population of any one of claims 29 to 34, or the method of any one of claims 3 to 28, wherein the T-cells efficiently give rise to memory precursor effector T-cells (MPECs) and/or central memory T-cells in vivo, for example in the spleen, when administered to a subject, in particular upon reencounter of the antigen.

36. The cell population of any one of claims 29 to 35, or the method of any one of claims 3 to 35, wherein the T-cells give rise to T-cells in vivo when administered to a subject, which have an unaltered or enhanced capacity of producing IFNγ and/or TNF upon restimulation, in particular upon reencounter of the antigen.

37. The cell population of the method of claim 36, wherein the T-cells are CD8+ T-cells that give rise to tumor-infiltrating cells in vivo when administered to the subject.

38. The cell population of any one of claims 29 to 37, or the method of any one of claims 3 to 28, wherein the T-cells give rise to T-cells, i.e. tumor-infiltrating cells, in vivo when administered to a subject, which have a decreased expression of PD-1.

39. The cell population of any one of claims 29 to 38, or the method of any one of claims 3 to 28, wherein the T-cells are CD8+ T-cells that give rise to memory precursor effector (MPEC) T-cells and/or central memory (CM) T-cells and/or tumor-infiltrating T-cells in vivo when administered to a subject, wherein said tumor-infiltrating T-cells have a decreased expression of PD-1 and/or an enhanced capacity of producing IFNγ and/or TNF upon reencounter of the antigen.

40. The cell population of any one of claims 29 to 39 or the method of any one of claims 1 to 28, wherein the T-cells are not contacted with an AKT inhibitor and/or have not been contacted with an AKT inhibitor.

41. The cell population of any one of claims 29 to 40 or the method of any one of claims 1 to 28, wherein the T-cells are not contacted with an MPC inhibitor and/or have not been contacted with an MPC inhibitor.

42. The cell population of any one of claims 29 to 41 or the method of any one of claims 1 to 28, wherein the T-cells are further contacted with an MPC inhibitor and/or have been contacted with an MPC inhibitor, preferably wherein said MPC inhibitor comprises AG221 and/or AGI6780, in particular wherein AG221 is 2-methyl-1-[[4-[6-(trifluoromethyl)pyridin-2-yl]-6-[[2-(trifluoromethyl)pyridin-4-yl]amino]-1,3,5-triazin-2-yl]amino]propan-2-ol and AGI6780 is 1-[5-(cyclopropylsulfamoyl)-2-thiophen-3-ylphenyl]-3-[3-(trifluoromethyl)phenyl]urea.

43. The cell population of any one of claims 29 to 42, or the method of any one of claims 3 to 28, wherein at least 40%, 50% 60%, 70%, 80%, or 90% are human central memory T-cells and/or human T-cells that co-express CD45RO and CCR7 and preferably CD62L, and preferably have no or low expression of CD45RA.

44. The cell population of any one of claims 29 to 43, wherein at least 40%, 50% 60%, 70%, 80%, or 90%, preferably at least 60%, of the cells in the population are human CD8+ T-cells with a memory phenotype that express CD62L, wherein said CD8+ T-cells have not been contacted with an AKT inhibitor.

45. The cell population of any one of claims 29 to 44, wherein said cell population is comprised in an in vitro cell culture.

46. The cell population of claim 45, wherein the cell culture comprises an MPC inhibitor.

47. The method of any one of claims 2 to 28 or the cell population of any one of claims 29 to 46, wherein the memory phenotype comprises expression of at least one memory marker selected from the group consisting of: CD62L (Sell), TCF1 (TCF7), CD27, CD127, CCR7 and CD28.

48. The method of any one of the preceding claims or the cell population of any one of claims 29 to 47, wherein the memory phenotype comprises absence of detectable expression of the non-memory marker KLRG1.

49. The method of any one of the preceding claims or the cell population of any one of claims 29 to 48, wherein the memory phenotype comprises expression of CD45RO, CCR7, CD27, CD28 and no or low expression of CD45RA, in particular wherein the T-cells with a memory phenotype are human cells.

50. The method of any one of the preceding claims or the cell population of any one of claims 29 to 49, wherein the memory phenotype comprises expression of the memory marker(s) CD62L and/or TCF1, in particular CD62L.

51. The method of any one of the preceding claims or the cell population of any one of claims 29 to 50, wherein the memory phenotype comprises surface expression of the memory marker CD62L.

52. The method of any one of any one of the preceding claims or the cell population of any one of claims 29 to 51, wherein the T-cells with a memory phenotype, i.e. the in vivo progeny thereof, (i) express CD62L and CD44 and/or (ii) express CD127 and lack KLRG1 expression, in particular wherein said cells in (i) are central memory T-cells, and the cells in (ii) are memory precursor effector T-cells.

53. The method of any one of the preceding claims or the cell population of any one of claims 29 to 52, wherein the memory phenotype comprises an increased basal oxygen consumption, maximal respiratory capacity and/or spare respiratory capacity, i.e. compared to the respective parameters in a control cell population, wherein the control T-cells have not been contacted with an MPC inhibitor.

54. The method of any one of the preceding claims or the cell population of any one of claims 29 to 53, wherein the memory phenotype comprises an open chromatin configuration, in particular wherein the open chromatin configuration is characterized by an increased trimethylation on the lysine 4 residue of histone 3 (H3K4-3Me), an increased acetylation on lysine 27 residue of histone 3 (H3K27-Ac) and/or more accessible chromatin regions, i.e. compared to the respective parameters in a control cell population, wherein the control T-cells have not been contacted with an MPC inhibitor.

55. The method of any one of the preceding claims or the cell population of any one of claims 29 to 54, wherein the memory phenotype comprises an open chromatin configuration at one or more, preferably at least 2, 3, 4 or 5, regulatory regions of at least one gene selected from the group consisting of: Sell (CD62L), Tcf7 (Tcf1), and Ccr7.

56. The method of any one of the preceding claims or the cell population of any one of claims 29 to 55, wherein the T-cells express at least one activation marker, in particular wherein said at least one activation marker is selected from the group consisting of: CD25, CD44, CD71 and CD98.

57. The method of any one of the preceding claims or the cell population of any one of claims 29 to 56, wherein the T-cells with a memory phenotype have a higher concentration of Acetyl-CoA.

58. The method of any one of the preceding claims or the cell population of any one of claims 29 to 57, wherein the T-cells with a memory phenotype incorporate carbon atoms from glutamine more efficiently into Acetyl-CoA than carbon atoms from glucose.

59. The method of any one of the preceding claims or the cell population of any one of claims 29 to 58, wherein more Acetyl-CoA in the T-cells with a memory phenotype is derived from glutamine than from glucose, in particular wherein more than 20%, 30%, 40%, 50% or 60% of the Acetyl-CoA is derived from glutamine and/or less than 20% of the Acetyl-CoA is derived from glucose.

60. The cell population of any one of claims 29 to 59 for use in immunotherapy, in particular wherein the cell population or the T-cells comprised in said cell population is/are administered to a subject.

61. An MPC inhibitor for use in immunotherapy.

62. The MPC inhibitor for use according to claim 61, wherein the immunotherapy comprises administering the MPC inhibitor to a subject.

63. The MPC inhibitor for use according to claims 61 or 62, wherein the immunotherapy comprises administering T-cells to a subject, i.e. by adoptive cell transfer, wherein said T-cells have been contacted with the MPC inhibitor during in vitro culture according to the method of any of the preceding claims, in particular wherein said T-cells have thereby acquired a memory phenotype in vitro.

64. The cell population for use according to claim 60 or the MPC inhibitor for use according to any one of claims 61 to 63, wherein the subject is a mammal, preferably a human, a domestic animal, or a pet, more preferably a human, most preferably a human patient in need for therapy.

65. The cell population or the MPC inhibitor for use according to the preceding claims, wherein the immunotherapy is a therapy for treating cancer, a chronic viral infection or an autoimmune disease.

66. The cell population or the MPC inhibitor for use according to claim 65, wherein the immunotherapy is a therapy for treating cancer, in particular an advanced cancer, preferably wherein the cancer, e.g the advanced cancer, is resistant to chemotherapy, therapy with an immune checkpoint inhibitor, targeted therapy and/or antibody-mediated immunotherapy and/or wherein the cancer comprises metastases.

67. The cell population or the MPC inhibitor for use according to claims 65 or 66, wherein the cancer is a hematological malignancy and/or a solid tumor, wherein said solid tumor is resistant to therapy with an immune checkpoint inhibitor (primary immune checkpoint blockade resistance) and/or acquires resistance to such a therapy; wherein the chronic viral infection is HIV or SARS-CoV-2, preferably HIV; and/or wherein the autoimmune disease is caused by and/or associated with autoreactive and/or pathogenic T-cells.

68. The cell population or the MPC inhibitor for use according to any one of the preceding claims, wherein the T-cells comprise CD8+ T-cells, wherein the T-cells are autologous cells, and/or wherein the T-cells are obtained from tumor-infiltrating T-cells.

69. The cell population or the MPC inhibitor for use according to any one of the preceding claims, wherein the T-cells comprise a heterologous antigen receptor, preferably a T-cell receptor (TCR) or a chimeric antigen receptor (CAR).

70. The cell population or the MPC inhibitor for use according to any one of the preceding claims, wherein an additional anti-cancer drug, preferably a checkpoint inhibitor, is administered to the patient.

71. An MPC inhibitor for use as a vaccine adjuvant.

72. A composition, in particular a pharmaceutical composition, comprising a vaccine and an MPC inhibitor.

73. The composition of claim 72, wherein said composition promotes the formation of T-cells with a memory phenotype in vivo when administered to a subject, in particular wherein said T-cells are activated.

74. The composition of claims 72 or 73, wherein said composition increases the number of T-cells expressing CD127 and having no or low expression of KLRG1, i.e. memory precursor effector T-cells, in vivo when administered to a subject compared to the administration of the respective vaccine without an MPC inhibitor.

75. A kit comprising (i) a vaccine and an MPC inhibitor, and/or (ii) the composition of any one of claims claim 72 to 74.

76. The MPC inhibitor for use according to claim 71, the composition of any one of claims 72 to 74 or the kit of claim 75, wherein the vaccine is a subunit vaccine.

77. The MPC inhibitor for use according to claims 71 or 76, the composition of any one of claims 72 to 74 or 76 or the kit of claims 75 or 76, wherein the vaccine comprises a further adjuvant such as an aluminium salt, AS01, AS04, MF59, a TLR agonist, and/or a STING agonist.

78. The MPC inhibitor for use according to any one of claims 71, 76 or 77, the composition of any one of claims 72 to 74, 76 or 77 or the kit of any one of claims 75 to 77, wherein the vaccine comprises an antigenic peptide, a nucleic acid encoding an antigenic peptide, a polysaccharide, a glycoprotein, a proteoglycane and/or a viral or bacterial vector comprising a nucleic acid encoding an antigenic peptide and/or the protein part of a glycoprotein and/or a proteoglycane, preferably wherein said vector encodes an antigenic peptide.

79. The MPC inhibitor for use according to any one of claims 71 or 76 to 78, the composition of any one of claims 72 to 74 or 76 to 78 or the kit of any one of claims 75 to 78, wherein the vaccine without an MPC inhibitor must be administered to a patient more than once to achieve the desired therapeutic and/or prophylactic effect, i.e. the required immunity.

80. The MPC inhibitor for use according to any one of claims 71 or 76 to 79, the composition of any one of claims 72 to 74 or 76 to 79 or the kit of any one of claims 75 to 79, wherein the vaccine is against cancer.

81. The MPC inhibitor for use according to any one of claims 71 or 76 to 80, the composition of any one of claims 72 to 74 or 76 to 80 or the kit of any one of claims 75 to 80, wherein the MPC inhibitor comprises at least one small molecule such as UK5099, Pioglitazone, Rosiglitazone, MSDC-0602, MSDC-0160 and/or Zaprinast, preferably at least 2-cyano-3-(1-phenyl-1H-indol-3-yl)-2-propenoic acid (UK5099).

82. The composition of any one of claims 72 to 74 or 76 to 81 or the kit of any one of claims 75 to 81 for use in treating and/or preventing a disease.

83. The composition or the kit for use according to claim 82, wherein the disease is cancer, or a chronic viral infection.

84. The composition or the kit for use according to claim 83, wherein the cancer is an advanced cancer, resistant to chemotherapy, therapy with an immune checkpoint inhibitor, targeted therapy and/or antibody-mediated immunotherapy and/or comprises metastases.

85. The composition or the kit for use according to claim 83 which is used for preventing the development of a preneoplastic lesion into a cancer, in particular wherein the preneoplastic lesion is Barretts's esophageous, cervical intraepithelial neoplasia, or a familial carcinoma such as familial melanoma, and/or characterized by germ line BRCA mutations associated with and/or leading to breast and ovarian carcinomas in women. In general, preneoplastic lesions refractory to standard of care and which invariable lead to tumor formation.

86. The composition or the kit for use according to claim 83 which is used for preventing a viral disease such as AIDS, or manifestation of a viral infection such as HIV.

87. The MPC inhibitor for use according to any one of claims 71 or 76 to 81, or the kit for use according to any one of claims 83 or 86, wherein the MPC inhibitor is administered to the subject prior to the vaccine, simultaneously with the vaccine, and/or subsequent to the vaccine, and/or more than once.

88. The MPC inhibitor for use according to any one of claims 71 or 76 to 81 or 87, or the kit for use according to any one of claims 83 to 87, wherein the MPC inhibitor is first administered to the patient together with the vaccine, and then at least once without the vaccine.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0224] FIG. 1: Treating CD8 T cells with a small molecule inhibiting the mitochondrial pyruvate carrier (UK5099, MPCi) results in enhanced memory characteristics that are maintained during Listeria infection in vivo.

[0225] (A) Schematic representation of the in vitro mouse CD8 T cell activation and treatment. (B) FACS analysis at day 7 showing CD62L histograms of OT1 cells activated in the presence of MPCi (UK5099) or the solvent DMSO. (C) RNAseq data revealing the fold change in gene expression in mouse CD8 T cells upon UK5099 treatment at day 7 as compared to DMSO (D) Quantification of the percentage of mouse CD8 T cells expressing low or high levels of CD62L as measured by flow cytometry. (E) Schematic representation of the ex vivo activation and treatment of human cord blood PBMCs. (F-G) Representative FACS histogram (F) and analysis (G) at day 11 showing CD62L expression in MPCi-treated human CD8 T-cells versus DMSO control-treated cells. (H) Schematic representation of Listeria-Ova infection. 100,000 OT-1 cells cultured in the presence of MPCi or DMSO were transferred into naïve recipients followed by 2000 cfu Listeria-Ova infection. (I) Percentage of transferred cells (CD45.1+) out of the total CD8+ T cell population in the blood upon transfer of MPCi-treated T-cells. (J) Representative plots and quantification of flow cytometry data of KLRG1-negative and CD127-positive cells in the CD8+CD45.1+ population in the blood at day 7 post-infection. (K) Representative plots of flow cytometry data of CD44 and CD62L double positive cells in the CD8+CD45.1+ population in the blood at day 28 post-infection. (L-N) T cell persistence (L), MPEC at d7 (M) and central memory T cells at d28 post infection (N) in mice receiving adoptively transferred WT or MPC1 KO T cells. *p<0.05 versus DMSO or WT. Graphs show mean±Standar Error of Mean (SEM).

[0226] FIG. 2: MPCi-treated CD8 T cells show enhanced anti-tumoral activity. (A) Schematic representation of the tumor experiment. 1 day after a low dose (5Gy) whole body irradiation, 100,000 OT-1 cells, either cultured with the small molecule or DMSO, were transferred into B16-OVA tumor-bearing mice. Mice were subsequently vaccinated s.c. with CpG and OVA peptide. (B) B16-OVA tumor growth in mice upon transfer of MPCi- or DMSO treated cells, or in untreated mice (PBS). (C) Percentage of CD44 and CD62L double positive T cells out of the transferred CD8+CD45.1+ population in the spleen. (D) Percentage of PD-1 positive T cells out of the transferred CD8+CD45.1+ population in the tumor. (E) The single-cell suspension of tumors was re-stimulated with SIINFEKL peptide for 4 h. IFNγ and TNFα production was measured by flow cytometry. *p<0.05 versus DMSO. Graphs show mean±SEM.

[0227] FIG. 3: Epigenetic mechanisms underlie the enhanced memory characteristics of MPCi-treated CD8 T cells. (A) Total acetyl-CoA levels measured by mass spectrometry. (B) Percentage of acetyl incorporation by carbons derived from 13C-glucose or 13C-glutamine into the total acetyl-CoA pool. (C) Western blot showing trimethylation of lysine 4 (H3K4-3Me) and acetylation of lysine 27 (H3K27-Ac) on histone 3 upon UK5099 treatment. (D) Visualization of the number of genes that are associated with more open or less open chromatin regions upon MPCi treatment. (E) Example of ATAC-seq traces in the gene loci of Sell, Tcf7 and Ccr7 of OT1 T cells treated with UK5099 or DMSO. *p<0.05 versus DMSO. Graphs show mean±SEM.

[0228] FIG. 4: UK5099 as adjuvant drives a memory phenotype in T cells upon vaccination and protects against tumor growth. (A) Schematic representation of the vaccination experiment. UK5099 (MPCi) was given s.c. at 0.5 mg/mouse at d0 and d2. (B) Quantification of flow cytometry data of KLRG1-negative and CD127-positive MPECs in the CD8+CD45.1+ population in the blood at day 14 post-vaccination. (C-D) B16-OVA tumor growth (C) and weight (D). *p<0.05 versus DMSO. Graphs show mean±SEM.

[0229] FIG. 5: Phenotypic analysis of mouse and human CD4 T cell activation upon MPCi treatment. (A) Schematic representation of mouse CD4 T cell activation and treatment. (B and C) Histogram representing flow cytometry data on CD62L expression in CD4 T cells upon treatment with DMSO or MPC inhibitor (UK5099) (B), and the quantification thereof (C). (D) Quantification of flow cytometry analysis at day 11 post-activation of human cord blood PBMCs showing the percentage of CD62L+CD4 T cells in the CD3-positive/CD8-negative T cell population. *p<0.05, compared to DMSO. Graph shows mean±SEM.

[0230] FIG. 6: MPC inhibition during the production of murine CAR T cells improves their memory phenotype and antitumor function upon adoptive cell transfer therapy. (A) Experimental design. (B) Tumor growth curve. (C) Number of Her2-CAR T cells per tumor-draining lymph node. (D) Number of Her2-CAR T cells per spleen. (E) Percentage of TCF1-positive cells out of Her2-CAR T-cells in the spleen. (F) Number of Her2-CAR T cells per mg of tumor. (G) Percentage of TCF1-positive cells out of Her2-CAR T cells in the tumor. (H and I) Percentages of progenitor-exhausted T cells (H) and terminally exhausted T cells (I) out of Her2-CAR T cells in the tumor. (J) Percentage of exhaustion marker (PD1 and TIM3)-expressing Her2-CAR T cells in the tumor. Pooled data from 2 independent experiments. ns: non significant, *p<0.05, **p>0.01, ***p<0.001. Data represents mean±s.e.m.

[0231] FIG. 7: MPC inhibition during the activation and culture of adult human T cells induces a memory phenotype. (A) Experimental design. PBMCs were isolated from adult healthy volunteers and activated with anti-CD3/CD28 beads in the presence of DMSO or UK5099 (25 μM). Beads were removed at day 5 and T cell phenotype was analysed at day 9. (B) Percentage of CD8 T cells expressing the memory marker CD62L. (C) The mean fluorescent intensity of CD62L in the respective positive population. (D) Percentage of CD8 T cells expressing markers that allows their identification as stem cell memory T cells (TSCM). (E-F) Mitochonrial mass (identified by MitoGreen staining, E) and mitochondrial membrane potential (TMRM, F), measured by flow cytometry and expressed as fold change as compared to DMSO. (G) Mitochondrial membrane potential normalized for the mitochondrial mass, expressed as the ratio of MitoGreen over TMRM, fold change as compared to DMSO. Pooled data from 2 independant experiments. ns: non-significant, *p<0.05, **p>0.01, ***p<0.001. Data represents mean±s.e.m.

EXAMPLES

[0232] To evaluate the effects of MPC inhibition on T cell differentiation, OT1 splenocytes were cultured in the presence of 1 μg/ml ovalbumin-derived N4 (SIINFEKL) peptide, 100 IU/ml recombinant human IL-2 (rhIL-2) and the MPC inhibitor (MPCi) UK5099 at 75 μM or control DMSO for 3 days. The inhibitor or DMSO was then washed away and the cells were cultured for 4 more days in the presence of 100 IU/ml IL-2 and 10 ng/ml rhIL-7 (FIG. 1A). When analyzing the surface expression of the central memory marker CD62L by flow cytometry on day 7, we could observe a strong increase in cells treated with MPCi compared to DMSO (FIG. 1i). Furthermore, RNA sequencing revealed that Sell (CD62L) expression was at least 1.5-fold increased at day 7 in cells treated with MPCi, as well as other memory markers Tcf7 and Ccr7 (FIG. 1C). Thus, the increase in the memory markers upon MPCi in vitro treatment occurs at the transcriptional level. Of note, also continuing MPCi treatment between d3 and d7 induced an upregulation of surface CD62L expression at day 7, even more pronounced than a d0-d3 treatment only (FIG. 1D). This indicates that the memory induction induced between d0 and d3 is stable until day 7, but a continuous treatment with MPCi is inducing the highest CD62L expression.

[0233] Important for the clinical translation is to prove whether an improved memory T cell differentiation can also be obtained in human CD8 T cells. To most closely mimic the experimental conditions that were successful by using naïve mouse OT1 CD8 T cells, it was tested whether the MPCi on the most naïve human CD8+ T cells can be obtained, i.e. from umbilical cord blood (FIG. 1E). Importantly, in 3 different donors a significant increase in CD62L expression with MPCi was observed (FIGS. 1F and 1G).

[0234] In order to evaluate whether this memory phenotype is maintained in vivo, 10.sup.5 MPCi- or DMSO-treated OT1 cells were intravenously injected in healthy mice. Twenty-four hours after cell transfer, the mice were infected with a sub-lethal dose of ovalbumine-expressing Listeria monocytogenes (LM-OVA, 2000 CFU i.v.) (FIG. 1H). Weekly bleeding of the mice did not reveal significant differences in the frequency of the transferred cells during the peak of the immune response, nor during the contraction phase (FIG. 1I). At the peak of the immune response, day 7 post-LM-OVA, the frequency of memory precursor cells (CD127+, KLRG1low) was significantly increased in OT1 cells pretreated with the MPCi (FIG. 1J). Consequently, 28 days after LM-OVA infection, during the memory phase of the immune reaction, the proportion of central memory T cells (CD44+CD62L+) among the transferred cells in the blood was larger in mice that received cells pre-treated with MPCi (FIG. 1K). Importantly, the effects of the MPC inhibitor were target specific, since an increase in memory T cell differentiation could also be observed when MPC1 KO T cells were transferred in this Listeria model (FIG. 1L-N). In summary, MPCi-treated cells display a consistent memory phenotype upon in vivo transfer in an infection model.

Adoptive Cell Transfer Therapy with MPCi-Treated CD8 T Cells is Better Able to Control Tumor Growth in a Mouse Melanoma Model

[0235] To assess the functional capacity of the generated memory T cells, the MPCi-treated cells were further tested in a mouse model of melanoma. Briefly, 10.sup.5 ovalbumin-expressing B16 melanoma cells were injected subcutaneously in 6-week-old mice. At day 6 post-engraftment, when a palpable tumor was present, mice were irradiated with 5Gy. The next day 10.sup.5 MPCi- or DMSO-treated OT1 cells were intravenously injected, followed by a subcutaneous vaccination of 50 μg CpG and 10 μg SIINFEKL (FIG. 2A). It has been shown before that memory CD8+ T cells are more potent in controlling tumor growth, and indeed, B16 tumor growth was strongly reduced in mice that received OT1 CD8+ T cells pre-treated with MPCi as compared to DMSO (FIG. 2B). However, upon dissection, the percentage of transferred cells out of total CD8+ T cells was not strikingly different in the spleen and tumor in MPCi mice (data not shown). Confirming the Listeria data, the proportion of central memory CD8+ T cells in the spleen was increased upon MPCi treatment (FIG. 2C). Interestingly, MPCi-treated T cells infiltrating the tumor had a significantly decreased expression of the immunosuppressive molecule PD-1 (FIG. 2D). When restimulating the single cell population of the tumor in vitro with the OVA peptide SIINFEKL, OT1 T cells treated with MPCi responded much better, with a higher proportion of cells being double positive for IFNγ and TNF (FIG. 2E).

An Epigenetic Mechanism Might be Responsible for the Durable In Vivo Memory Responses Upon In Vitro Metabolic Intervention

[0236] Mechanistically, it was hypothesized by the inventors that epigenetic processes might underlie the observed long-term memory phenotype and anti-tumor activity upon metabolic intervention with MPCi. Several metabolites were differentially abundant, but most strikingly was a strong increase in acetyl-CoA upon MPCi treatment (FIG. 3A). When trying to determine the carbon source of this increased acetyl-CoA, it was observed that 13C-labeled glucose is as expected incorporating less upon MPCi treatment. Instead 13C-glutamine incorporated dramatically more into acetyl-CoA after mitochondrial pyruvate carrier inhibition (FIG. 3B). Since acetyl-CoA is the acetyl donor for the acetylation of proteins, amongst which also histones, histone post-translational modifications were examined that are known to make chromatin accessible to transcription factors, a characteristic of memory T cells. Interestingly, an increase in acetylation of lysine 27 on histone 3 was observed, but also in trimethylation of lysine 4, upon UK5099 treatment (FIG. 3C). These are marks known to open chromatin and make it accessible for transcription factor docking. Indeed, ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) revealed that 981 genes were associated with more accessible chromatin upon MPCi treatment (FIG. 3D). Amongst those were Sell, Tcf7 and Ccr7, thereby correlating accessible chromatin with increased mRNA copies (FIG. 3E and see above).

[0237] Together, these results show that short-term metabolic intervention can lead to enhanced memory characteristics and improved anti-tumor control, likely by an epigenetic mechanism.

An MPC Inhibitor as Vaccine Adjuvant Induces a Better Memory CD8 T Cell Development and Protects Against Subsequent Tumor Challenge

[0238] One of the main goals of preventive and therapeutic vaccination in the context of pathogen infection and cancer is the establishment of a potent memory CD8 T cell pool. Since it was observed that MPC inhibition during in vitro priming of antigen-specific CD8 T cells increases their memory formation, it was investigated whether the MPCi could be used as a vaccine adjuvant. 10.sup.5 naïve OT1 CD8 T cells were transformed in healthy mice and the mice were subsequently vaccinated by a subcutaneous injection of 50 μg CpG and 10 μg SIINFEKL containing additionally 0.5 mg UK5099 or and equal volume of DMSO as control. 48 hours later, the mice received another subcutaneous injection of 0.5 mg UK5099 or and equal volume of DMSO (FIG. 4A). 14 days after vaccination, an increased proportion of the transferred OT1 CD8 T cells was observed that formed memory precursor cells (FIG. 4B). It was investigated whether this would render them better prepared for a subsequent antigen challenge, and injected 10.sup.5 ovalbumine-expressing B16 melanoma cells s.c. in the flank of the mice at 40 days post-vaccination (FIG. 4A). Strikingly, the mice that received the vaccine formulated with the MPCi were much better in controlling the tumor growth (FIGS. 4C and 4D). This preliminary experiment indicates that MPC inhibition during the priming phase of in vivo vaccination might induce a stronger memory pool formation and protect against future disease.

Mice and Tumor Lines

[0239] Mice were maintained in the animal facility of the University of Lausanne. OT1 mice were bred on site and C57BL/6 (B6) mice were obtained from ENVIGO. MPC1flox/flox mice were generated by Dr. Jared Rutter and intercrossed in our facility with CD4.CRE mice and OT1 mice. B16-Ova melanoma tumor cell line was generated previously in the laboratory. All experiments were performed in accordance with Swiss federal regulations and procedures approved by veterinary authority of the Canton de Vaud.

Mouse Splenocyte Culture

[0240] OT1 splenocytes were cultured for 3 days at a concentration of 106 cells per mL in RPMI medium (Gibco 61870-01) supplemented with 10% FBS, (Gibco 10270-106), 1% Penicillin/Streptomycin (Gibco 15070-063), 50 μM β-mercaptoethanol, 1% HEPES (Gibco 15630-080), 1× Non-essential amino acids (Gibco 11140-035), 1% L-glutamine (Gibco 25030-081), 1 mM Sodium Pyruvate (Gibco 11360-039). Cells were additionally supplemented with hIL-2 100 U/ml (Glaxo-IMB), ovalbumin N4 peptide (SIINFEKL) 1 μg/ml and either with 75 μM UK5099 (Sigma Aldrich) or with their solvent DMSO as a control. At day 3, splenocytes were collected, washed and split, and cells were cultured for 4 additional days with 100 U/ml hIL-2 and hIL-7 (Peprotech 200-07) supplemented either with 75 μM UK5099 (Sigma Aldrich) or DMSO. At day 7, flow cytometry analyses were performed for surface marker expression.

[0241] Naïve CD4 T cells were isolated by negative selection (Stem Cell Technologies) from the spleen of OT2 mice. Dendritic cells were isolated from spleens of C57BL/6 mice by CD11c positive selection (Stem Cell Technologies). 2×10.sup.5 CD4 T cells were co-cultured with 1×10.sup.6 dendritic cells in RPMI medium supplemented with 10% FBS, 1% Penicillin/Streptomycin, 50 μM β-mercaptoethanol, 1% HEPES, 1× Non-essential amino acids (Gibco 11140-035), 2 mM L-glutamine and 1 mM Sodium Pyruvate. T cells were activated by adding 1 μg/ml Ovalbumine peptide (323-339, ISQAVHAAHAEINEAGR) and 100 IU/ml rhIL-2, in the presence of 75 μM UK5099 or DMSO. Cells were split on day 4 and re-cultured in fresh medium containing 100 IU/ml rhIL-2, in the presence of 75 μM UK5099 or DMSO.

Human Cord Blood PBMC Culture

[0242] Peripheral blood mononuclear cells were isolated from fresh umbilical vein cord blood on a Percoll gradient. PBMCs were then cultured in RPMI supplemented with 10% human serum. 10.sup.4 PBMC's were seeded per well in a round bottom 96-well plate and activated with anti-CD3/CD28 beads at a 1:2 cell:bead ratio and 300 U/ml rhIL-2, in the presence of 25 μM UK5099 or DMSO. Cells were regularly split and CD62L expression was determined by flow cytometry on day 11.

Adoptive Cell Transfer

[0243] Activated CD45.1+OT-1 splenocytes were culture in vitro for 7 days as described above, collected and purified on a Ficoll gradient, allowing to separate dead and live splenocytes. Live splenocytes were counted with Trypan blue stain 0.4%. 100′000 live splenocytes were transferred into CD45.2+ host mice by tail vein injection.

In Vivo Listeria-Ova Infection Model

[0244] Recombinant bacteria Listeria monocytogenes deficient for actA and expressing the ovalbumin (Ova) peptide SIINFEKL were expanded and tittered. Optical density measured with a spectrophotometer was used to determine bacterial concentration and 2000 CFU were administered in each mouse by tail vein injection, 4 hours after adoptive cell transfer. Blood samples were collected and processed for surface markers and cytokine production analyses.

Melanoma Tumor Model

[0245] B16-OVA cells were cultured in DMEM (GIBCO) with 10% FBS and 1% P/S before their subcutaneous injection into the mouse flank. Each mouse received 100′000 cells in a volume of 200 μl of PBS. 6 days after B16-OVA cells injection, tumors were measured, mice were randomized and lymphodepleted by irradiation (5 Gray). 7 days after B16-Ova injection, mice were adoptively transferred with the ACT protocol described previously. Following the ACT, mice received a vaccination of CpG (50 μg/mouse) and N4 Ova peptide (10 μg/mouse) diluted in PBS to obtain a total volume of 100 μl/mouse, injected subcutaneously at the tail base. Tumors were measured every 2 days and the tumor volume was calculated according to the formula: V=π×[d2×D]/6, where d is the minor tumor axis and D is the major tumor axis. At day 26 days post-tumor engraftment, tumors and spleens were dissected were then stained for flow cytometry analyses.

Vaccination Model

[0246] Naïve CD8 T cells were isolated from OT1 spleens (negative selection, Stem Cell Technologies). 1×10.sup.5 T cells were i.v. transferred in WT C57BL/6 mice, followed by a subcutaneous injection of the vaccine, consisting of 10 μg SIINFEKL peptide, 50 μg CpG and 0.5 mg UK5099 or DMSO diluted in a total of 100 μl PBS per mouse. 2 days post-vaccination, mice received a second dose of s.c. 0.5 mg UK5099 or DMSO. After 2 weeks, blood was collected from the tail vein and analyzed by flow cytometry. 40 days post-vaccination, mice were challenged by an s.c. injection of 1×105 SIINFEKL-expressing B16 melanoma cells. 17 days later, mice were sacrificed and the tumor was dissected.

Flow Cytometry

[0247] Cells were incubated in a live/dead stain (Fixable aqua dead cell stain kit, Invitrogen) followed by an incubation with different antibody panels.

[0248] Antibodies used for in vitro surface analyses were: CD8-PE-texas-Red, CD62L-FITC, CD127-PE, CD27-PerCP-Cy5.5, CD71-PE-Cy7, CD44-APC-Cy7, CD98-APC, CD25-Pacific blue (BD Pharmingen and eBioscience, San Diego, CA, USA).

[0249] Antibody panels for blood analyses contained: CD8-PE-texas-Red, CD45.1-Pacific blue, CD45.2-FITC, CD127-PE, KLRG1-PE-Cy7, CD62L-APC, CD44-APC-Cy7, and CD27-PerCP-Cy5.5.

[0250] Antibody panel for intra-tumoral T cells and splenocytic T cells was: CD8-PE-texas-Red, CD45.1-Pacific blue, CD45.2-FITC, CD62L-PE-Cy7, CD44-APC-Cy7, PD1-APC, Lag3-PE, and CD127-FITC.

[0251] Antibody panel for cytokine detection contained: CD8-PE-texas-Red, CD45.1-FITC, CD45.2-APC, TNFa-Pacific blue, IFNg-PerCP-Cy5.5, IL-2-PE.

[0252] Cells were acquired on LSR-II flow cytometers from the flow cytometry facility of UNIL and data were analyzed with FlowJoTM10 software. Antibodies were purchased from BD Phamingen (San Diego, CA, USA), eBioscience (San Diego, CA, USA, and Biolegend (San Diego, CA, USA).

Metabolomics

[0253] OT1 splenocytes were activated and cultured as described above. After 66 hours, the cells were collected and the medium was replaced with glucose- and glutamine-free RPMI, supplemented with 10% dialyzed FBS, 1% P/S, 10 mM HEPES, 50 μM β-mercaptoethanol and either 11 mM normal glucose or 11 mM U-.sup.13C6-glucose (Cambridge Isotopes) with respectively 4 mM .sup.13C5-glutamine (Cambridge Isotopes or 4 mM normal L-glutamine. After 6 hours labelling at 37° C., cells were collected and lysed in Methanol. Next Chloroform was added, and after centrifugation, the fraction containing the polar metabolites was collected and dried on a SpeedVac. Samples were then resuspended and loaded on a mass spectrometer. The spectra of AcetylCoA were analyzed with the M+0 representing the unlabeled fraction, and the M+2 representing the Acetyl group derived from the .sup.13C-labelled substrate (glucose or glutamine).

RNA Sequencing and Analysis

[0254] mRNA was extracted from OT1 T cells on day 3 of culture (Qiagen RNeasy kit) and sequenced on the Illumina HiSeq platform. Purity-filtered reads were adapters and quality trimmed with Cutadapt (v. 1.8, Martin 2011). Reads matching to ribosomal RNA sequences were removed with fastq_screen (v. 0.9.3). Remaining reads were further filtered for low complexity with reaper (v. 15-065, Davis et al. 2013). Reads were aligned against Mus musculus.GRCm38.86 genome using STAR (v. 2.5.2b, Dobin et al. 2013). The number of read counts per gene locus was summarized with htseq-count (v. 0.6.1, Anders et al. 2014) using Mus musculus.GRCm38.86gene annotation. Quality of the RNA-seq data alignment was assessed using RSeQC (v. 2.3.7, Wang et al. 2012). Reads were also aligned to the Mus musculus.GRCm38.86 transcriptome using STAR (v. 2.5.2b, Dobin et al. 2013) and the estimation of the isoforms abundance was computed using RSEM (v. 1.2.31, Li and Dewey 2011). Statistical analysis was performed for genes in R (R version 3.4.0). An analysis with all genes, including mitochondria, was done. Genes with less than one count per million in all samples were filtered out. Library sizes were scaled using TMM normalization (EdgeR package version 3.14.0; Robinson et al. 2010) and log-transformed with EdgeR cpm function.

[0255] Differential expression was computed with limma (Ritchie et al. 2015) by fitting the samples into a linear model using all conditions as factors and correcting for batch effect by introducing factors for the replicates (=paired analysis).

ATAC Sequencing

[0256] After 3 days of OT1 CD8 T cell culture, 5×10.sup.4 cells were collected and transposed as described before (Buenrostro et al, Transposition of native chromatin for multimodal regulatory analysis and personal epigenomics. Nat Methods. 2013 December; 10(12): 1213-1218.). Amplified transposed fragments were sequenced on an Illumina HiSeq platform.

[0257] Computations of the analysis were performed at the Vital-IT Center for high-performance computing of the SIB Swiss Institute of Bioinformatics. Sequencing reads contained in fastq files were aligned to the Mouse mm10 reference genome using Bowtie2 v.2.3.4.1, and alignment files were manipulated with samtools v.1.8. Peak calling was performed with Macs2 v.2.1.1. Differential peak analysis was performed in R v.3.5.1 with package DiffBind v.2.10.0. Genomic feature annotation was performed using CHIPpeakAnno v.3.16.0 and rGREAT v.1.14.0.

Western Blot

[0258] Cells were lysed in RIPA lysis buffer (50 mM TrisHCl pH8, 150 mM NaCl, 1% Triton X 100, 0.5% Sodium deoxycholate, 0.1% SDS) and Halt protease/phosphatase cocktail inhibitors (Roche) and denatured by heat. Proteins were quantified by BCA protein assay kit (Thermo Scientific). Proteins were separated on 12.5% polyacrylamide gradient gels and transferred onto nitrocellulose membranes 0.2 μm (Biorad). Non-specific binding sites were blocked in milk 5% and membranes were incubated with primary antibodies (Cell Signaling). Membranes were then incubated with HRP-labeled secondary antibodies anti-rabbit (1:1000) and anti-mouse (1:10′000) (Santa Cruz Biotechnology), and blots were visualized by chemiluminescence with ECL and femto reagents (Super Signal West, Thermo Scientific).

Statistical Analyses

[0259] Statistical analyses were performed in GraphPad Prism 7 software using different statistical tests indicated for each experiment. Results are shown in mean±SEM and P<0.05 was considered statistically significant.

Mouse and Human CD4 Data

[0260] An experiment has been performed on mouse T cells in order to evaluate if MPC inhibition also promotes memory marker expression in CD4 T cells. As indicated in the experimental scheme (FIG. 5A), CD4 T cells from OT2 mice were activated by co-culture with dendritic cells. OT2 mice are transgenic for an D DTCR recognizing specifically a chicken ovalbumine peptide 323-339 associated with the mouse MHC class II molecule I-Ab. We activated OT2 CD4 T cells by co-culture with dendritic cells loaded with 1p g/ml of the ovalbumine peptide in the presence of 100 IU/ml recombinant human IL2 in the presence of 75 μM UK5099 or DMSO control. After 4 days, cells were collected, washed and split, and brought back in culture with 100 IU/ml rhIL2 and 75 μM UK5099 or DMSO control. At day 7, CD62L expression was determined by flow cytometry and showed a strong increase in the CD62L-positive population upon IDH2i treatment (FIGS. 5B and 5C).

[0261] CD62L expression in human CD4 T cells was also analyzed. Human cord blood PBMCs were cultured and activated as described before, in the presence of 25p M UK5099 or DMSO control. About 98% of the cells in culture on day 11 are CD3-positive. Since CD3 is exclusively expressed on CD4 and CD8 T cells, we can deduct that all CD3-positive, CD8-negative must be CD4 T cells. When analyzing the CD3-pos/CD8-neg population we observed that CD62L expression is increased upon MPCi treatment (FIG. 5D).

Measurement of MPC Inhibitory Activity

[0262] Potential drug interactions with the MPC were assessed using a bioluminescence energy transfer (BRET)-based MPC activity system called reporter sensitive to pyruvate (RESPYR). Briefly, described MPC2-RLuc8 and MPC1-Venus fusion proteins were stably expressed in HEK293 cells using lentiviral transduction. Cells were plated in white 96-well plates 48 hr before recording. Cells were washed with PBS-CM (PBS supplemented with 1 mM CaCl.sub.2) and 0.5 mM MgCl2), and readings were performed 5 min after addition of 5 mM coelenterazine h substrate (Invitrogen). Signals were detected with two filter settings (R-Luc8 filter, 460±40 nm; and Venus filter, 528±20 nm) at 37 C using the Synergy 2 plate reader (Biotek). The BRET value was defined as the difference between the emission at 528 nm/460 nm of co-transfected R-Luc8 and Venus fusion proteins (MPC2-R+MPC1-V) and the emission at 528 nm/460 nm of the R-Luc8 fusion protein alone (MPC2-R). Results were expressed in milliBRET units (mBU). MPC inhibitors are added after baseline readings in the absence of pyruvate.

MPC Inhibition During the Production of Murine CAR T Cells Improves their Memory Phenotype and Antitumor Function Upon Adoptive Cell Transfer Therapy.

[0263] The data obtained in mice made use of CD8 T cells isolated from transgenic OT1 mice, which were designed to express one unique T cell receptor recognizing a peptide sequence of the chicken ovalbumin protein (SIINFEKL) when presented on MHC class I molecules. The inventors were able to show that OT1 T cells, activated and cultured in vitro in the presence of an MPC inhibitor, displayed improved anti-tumor activity when transferred in mice bearing melanoma B16 tumor that were genetically engineered to express the SIINFEKL peptide.

[0264] It was intended to extend this data by evaluating if this method can also be applied during the production of mouse CAR T cells. Therefore, a CAR construct recognizing human HER2 was used, an oncogene frequently involved in human breast cancer, containing a 4-1BB costimulatory domain.

[0265] For retrovirus preparation, a modified protocol from Tschumi et al, J Immunother Cancer. 2018 Jul. 13; 6(1):71 was used.

[0266] For each retroviral preparation, 8×10.sup.6 Phoenix ECO cells (ATCC, CRL-3214) were plated in a T150 tissue culture flask in RPMI medium supplemented with 10% FCS, 10 mM HEPES and 50 U/ml Penicillin-Streptomycin. On the next day, cells were transfected with 21 μg of the retroviral construct with Turbofect transfection reagent (Thermo Fischer Scientific), according to the manufacturer protocol. The medium was changed daily and collected at 48 h and 72 h post transfection. 48 h and 72 h virus supernatants were pooled and sedimented at 22000rcf for 2 h at 4° C. Finally, retrovirus pellets were resuspended in 2 ml of full RPMI medium and divided in 8 aliquots of 250 μl each, which were snap-frozen on dry ice and stored at −80° C.

[0267] For T cell transduction, a modified protocol from Tschumi et al, J Immunother Cancer. 2018 Jul. 13; 6(1):71 was used.

[0268] Spleens from wild type CD45.1.2 mice were smashed on a 70 m cell strainer. CD8 T cells were purified using the EasySep™ Mouse CD8+ T Cell Isolation Kit (StemCell) according to the manufacturer protocol. 0.5×10.sup.6 CD8 T cells were plated in 48 well plates in 0.5 ml of complete RPMI 1640 medium supplemented with 10% FCS, antibiotics and 50 IU/ml of recombinant human IL-2, and exposed to either DMSO or 20 μM UK5099. Mouse T-cells were activated with Activator CD3/CD28 Dynabeads (Gibco) at a ratio of 2 beads per cell. Retroviral infection was conducted at 37° C. for 24 h. Untreated 48-well plates were coated for 24 h with g/ml of recombinant human fibronectin (Takara Clontech) at 4° C., followed by PBS 2% BSA for 30 min at RT and finally washed with PBS. One aliquot of concentrated retroviruses was plated in each fibronectin-coated 48-well plates and centrifuged for 90 min at 2000rcf and 32° C. Then, 0.5×10.sup.6 of 24 h-activated CD8 T cells were added on top of the viruses and spun for 10 min at 400rcf and 32° C. On day 3, the medium was replaced with 10 IU/ml recombinant human IL-2, 10 ng/ml recombinant human IL-7 and 10 ng/ml recombinant human IL-15, containing either DMSO or 20 μM UK5099. Cells were then split every second day.

[0269] For adoptive cell transfer a modified protocol from Tschumi et al, J Immunother Cancer. 2018 Jul. 13; 6(1):71 was used.

[0270] CD45.2 C57BL/6 mice were engrafted subcutaneously with 4×10.sup.5 B16F10 tumors modified to express HER2. Six days later, mice were lymphodepleted with 100 mg/kg cyclophosphamide (Sigma Aldrich, C7397) injected i.p., and homogeneous groups were constituted with regard to tumor volume. T cells (5×10.sup.6) were adoptively transferred i.v. on the next day. Tumor volumes were measured three times a week with a caliper and calculated using the formula: V=π×[d2×D]/6, where d is the minor tumor axis and D is the major tumor axis. Tumors were collected and separated from skin. Single cell suspensions were obtained with the Mouse Tumor Dissociation Kit (Miltenyi, 130-096-730) according to the manufacturer protocol. Spleen and draining lymph node were smashed on a 70 m cell strainer. Single cell suspensions were stained with antibodies before flow cytometry analysis.

[0271] Polyclonal CD8 T cells from wild type mice were activated in the presence of DMSO or 20 μM UK5099 and then retrovirally transduced with a control blue fluorescent protein-expressing retroviral construct (BFP) or with HER2-CAR (FIG. 6A). When adoptively transferring (ACT) the T cells in mice bearing B16 melanoma tumors expressing HER2, we could observe that only UK5099-treated HER2CAR T cell ACT was able to significantly suppress tumor growth (FIG. 6B). More UK5099-HER2CAR T cells were engrafted in the tumor-draining lymph nodes, while CAR T cell numbers in the spleen were similar (FIGS. 6C and 6D). However, a higher percentage of splenic UK5099-HER2CAR T cells was expressing the memory-specific transcription factor TCF1 (FIG. 6E). Tumors of UK5099-HER2CAR-treated mice contained significantly more CAR T cells (FIG. 6F). As in the spleen, more tumor-infiltrating UK5099-treated CAR T cells expressed TCF1 (FIG. 6G). Interestingly, when looking at the co-expression of TCF1 with PD1, UK5099-treated CAR T cells formed more progenitor exhausted T cells (TCF1-positive) and less terminally differentiated exhausted T cells (TCF1-negative) (FIGS. 6H and 6I), indicative of an increased stem cell-like phenotype which might benefit from combination therapy with checkpoint blockade immunotherapy. Finally, fewer UK5099-treated CAR T cells were expressing the inhibitory molecules PD1 and TIM3, indicating a less exhausted phenotype (FIG. 6J).

MPC Inhibition During the Activation and Culture of Adult Human T Cells Induces a Memory Phenotype.

[0272] MPC inhibition during activation of umbilical cord blood T cells induces a memory phenotype. This data demonstrates that these largely naive and stem-cell-like T cells can benefit from MPC inhibition, as those cord blood cells can have important applications as source cells for adoptive cell transfer therapies. However, the majority of the CAR T cell products currently approved or in clinical trials derive from patient (adult) T cells. It was thus intended to include new data showing the induction of memory phenotypes when adult T cells are activated and cultured with an MPC inhibitor (FIG. 7). As can be seen in that figure, not only the MPC inhibitor (UK5099) was used, but also the IDH2 inhibitor AG221 and a competitive molecule inhibiting PI3Kdelta (CAL101).

Isolation and Culture of Peripheral Blood Mononuclear Cells (PBMCs)

[0273] Heparinised blood was diluted with PBS and pipetted on top of Ficoll-paque (LymphoPrep). After centrifugation at 1800 rpm for 20 minutes at room temperature, the layer of cells at the intersection, containing the PBMCs was removed, washed and resuspended at 5×10.sup.5 cells/ml of RPMI medium containing 8% human serum (AB serum), antibiotics, 2 mM L-Glutamine, 1% non-essential amino acids, 1 mM sodium pyruvate and 50 μM β-mercaptoethanol (all Gibco). Cells were activated with Human T-activator CD3/CD28 Dynabeads at a 1 bead/cell ratio with 150 IU/ml human recombinant IL2 and DMSO or 25 μM UK5099. Throughout the culture, cells were regularly split when necessary and beads were removed at day 5. At day 9, a fraction of the cells was stained with antibodies for flow cytometry analysis or for mitochondrial analysis.

Mitochondrial Staining

[0274] 2×10.sup.5 cells were stained with 25 nM TMRM and 50 nM MitoGreen (Molecular Probes, Invitrogen) for 30 minutes in RPMI with 5% FCS at 37° C. Cells were then washed, stained with a Live/Dead dye and antibodies before acquisition by flow cytometry.

[0275] The activation of PBMCs in vitro (FIG. 7A) resulted in a CD8 T cell population that was largely positive for the memory marker CD62L. Nevertheless, both UK5099- and AG221-treatment were able to further induce the population of CD8 T cells positive for CD62L (FIG. 2B). More strikingly, the mean fluorescent intensity of the CD62L signal in the CD62L-positive cells was increased upon UK5099 and AG221 treatment (FIG. 7C). This means that the CD62L-positive cells express more CD62L molecules on a per cell basis. When looking at several markers known in previous literature to identify stem cell-like memory T cells, it was found that this population of cells increased upon treatment with UK5099 (FIG. 7D). Intact mitochondrial membrane function is a known characteristic of long-lasting memory T cells. UK5099 treatment did not impact the total mitochondrial mass (FIG. 7E), but significantly increased mitochondrial membrane potential (FIG. 7F), resulting in more polarised mitochondria (FIG. 7G), which could be indicative of an improved mitochondrial respiratory capacity.