Use of an MPC inhibitor during in-vitro cell culture

Abstract

The present invention discloses compositions and methods for use in the treatment and / or prevention of cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease and / or an autoimmune disease. The invention also provides an in-vitro culture method of cells, such as e.g. immune cells.

Description

[0001] MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0002] Use of an MPC inhibitor during in-vitro cell culture

[0003] FIELD OF THE INVENTION

[0004] The present invention discloses compositions and methods for use in the treatment and / or prevention of cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease and / or an autoimmune disease. The invention also provides an in- vitro culture method of cells, such as e.g. immune cells.

[0005] BACKGROUND OF THE INVENTION

[0006] Adoptive T cell transfer with either ex vivo expanded tumor-infiltrating lymphocytes (TILs) or T cells genetically modified to express a T cell receptor (TCR T) or chimeric antigen receptor (CAR T), has shown remarkable therapeutic efficacy in relapsed or refractory melanoma, neuroblastoma and certain types of blood malignancies1. However, a large fraction of patients fails to achieve durable complete remission and eventually relapse.

[0007] An important factor that strongly correlates with this failure is the terminal differentiation and exhaustion of adoptively transferred cells2. Instead, it is well established that enhanced proportions of less differentiated central memory (TCM) or stem cell memory / naive T cells either at leukapheresis or in the drug product correlate to durable response with increased in vivo expansion and long-term persistence of therapeutic T cells3,4.

[0008] T cell activation and differentiation is closely intertwined with cellular metabolism. Activated effector T cells critically depend on glycolysis, but induce as well mitochondrial metabolic pathways such as oxidative and reductive metabolism of glutamine5. Instead, memory T cells depend more on mitochondrial metabolism and oxidative phosphorylation, and interference with those metabolic processes directly influences T cell differentiation. It has been previously found that genetic and pharmacological inhibition of the mitochondrial pyruvate carrier (MPC) favoured memory T cell differentiation6. Consequently, murine CAR T cells produced in the presence of the small molecule MPC inhibitor UK-5099 strongly suppressed melanoma tumor MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 growth. Furthermore, preliminary data with 1 human donor suggested that MPC inhibition with UK-5099 could as well enhance the anti-leukemic efficacy of anti-CD19 CAR T cells.

[0009] The MPC is a heterodimer consisting of an MPC1 and MPC2 protein, and is located in the inner mitochondrial membrane. Although its molecular identity was only uncovered about a decade ago7, Halestrap et al. described in 1975 the ability of UK-5099 to potently inhibit pyruvate-dependent oxygen consumption by rat heart mitochondria8. UK-5099 likely inhibits the MPC by covalently binding a cysteine residue on MPC29. Only recently, novel UK-5099 analogs were developed as MPC inhibitors to topically treat hair loss10. However, some of those novel MPC inhibitors possess a Michael acceptor unit, which have since long been controversial in drug design, due to fear of off-target effects and potential toxicity.

[0010] Therefore, there is an urgent need to develop new strategies to inhibit MPC, and thereby inducing a stem-cell memory T cell phenotype with a good safety profile that would lead to new therapeutic uses.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention provides an in-vitro cell culture method comprising a step of contacting an immune cell with an MPC inhibitor, preferably MITO-66, a tautomer, a geometrical isomer, an optically active form, a pharmaceutically acceptable salt or a pharmaceutically active derivative thereof, wherein the immune cell is selected from the group comprising a T cell, a gamma delta T cell (y8 T cell), a tumor infdtrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR-expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line derived from one of these cells or a pluripotent stem cell giving rise to one of these cells.

[0013] The present invention provides an in-vitro cell culture method comprising a step of contacting a cell, or a population of cells, with a mitochondrial pyruvate carrier (MPC) inhibitor, and MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 with a compound selected form a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate -depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HDAC inhibitor, or a combination thereof, wherein the cell is i) an immune cell, or a population of immune cells, selected from the group comprising a T cell, a gamma delta T cell (y3 T cell), a tumor infdtrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR-expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line derived from one of these cells or a pluripotent stem cell giving rise to one of these cells, or ii) a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, selected from the group comprising a mesenchymal stem cell, a multipotential stromal cell, a mesenchymal stromal cell, a mesenchymal progenitor cell and a multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR).

[0014] The present invention also provides an immune cell, or a population of immune cells, selected from the group comprising a T cell, a gamma delta T cell (y8 T cell), a tumor infiltrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR- expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line or a pluripotent stem cell derived from one of these cells, directly obtained by an in-vitro cell culture method of the invention.

[0015] Also provided is an immune cell selected from the group comprising a T cell, a gamma delta T cell (y8 T cell), a tumor infdtrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR-expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line or a pluripotent stem cell derived from one of these cells directly obtained by an in-vitro cell culture method according to the invention.

[0016] The present invention also provides a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, selected from the group comprising a MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 mesenchymal stem cell, a multipotential stromal cell, a mesenchymal stromal cell, and a mesenchymal progenitor cell, directly obtained by an in-vitro cell culture method of the invention.

[0017] The invention also provides a composition comprising an immune cell, or a population of immune cells, of the invention or a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, of the invention.

[0018] Also provided is a composition comprising an immune cell of the invention.

[0019] The present invention also provides a pharmaceutical composition comprising an immune cell, or population of immune cells, of the invention or a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, of the invention, and pharmaceutically acceptable carrier, diluent and / or excipient.

[0020] Also provided is a pharmaceutical composition comprising an immune cell of the invention, and pharmaceutically acceptable carrier, diluent and / or excipient.

[0021] The present invention also provides an in-vitro cell culture method comprising a step of contacting a cell, or population of cells, with a compound selected from a compound that inhibits alpha-ketoglutarate-dependent enzymes, thereby stabilizing HIF1 and / or HIF2, or a compound that inhibits histone and / or DNA demethylases (e.g. daprodustat, desidustat, enarodustat, molidustat, roxadustat (FG-4592), vadadustat, dimethyloxallyl glycine, FG-2216, IOX4, JNJ-42041935, MK-8617, S-2-hydroxyglutarate, fumarate, succinate, malate, deferasirox, deferoxamine, N-oxaloylglycine, N-oxalyl-2S-alanine, Alahopcin, dihydroxybenzoic acid, diazen-l-ium-l,2-diolate, diethylamine NONOate, Tilorone, a Factor inhibiting HIF (FIH) inhibitors, Von Hippel-Lindau (VHL) inhibitors or HIF allosteric agonists).

[0022] The invention also provides a cell culture medium comprising an MPC inhibitor and a compound selected from a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate -depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor and an HDAC inhibitor, or a combination thereof.

[0023] Also provided is an immune cell of the invention, or the pharmaceutical composition of the invention, for use in the treatment and / or prevention of a disease selected from the group MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 comprising a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease and / or an autoimmune disease.

[0024] The present invention also provides a method of treating and / or preventing a disease, comprising (i) providing a cell, or population of cells, (ii) expanding and culturing ex vivo or in vitro into a larger population of cells according to the in-vitro cell culture method of any one of the invention, and (iii) introducing (i.e. administering) said cell, or population of cells, into the patient or subject in need thereof.

[0025] The present invention also provides a method of treating and / or preventing a disease in a patient or subject in need thereof, comprising (i) providing cells isolated from the patient or subject in need thereof, (ii) expanding and culturing ex vivo or in vitro into a larger population of cells according to the in-vitro cell culture method of any one of the invention and (iii) reintroducing (i.e. administering) said immune cells into the patient or subject in need thereof.

[0026] Further provided are methods of treating and / or preventing a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease comprising (i) providing genetically engineered immune cells with one recombinant construct encoding a chimeric antigen receptor (CAR), (ii) expanding and culturing ex vivo or in vitro into a larger population of engineered immune cells according to the invention and (iii) reintroducing (i.e. administering) said engineered immune cells into the patient or subject in need thereof.

[0027] The present invention also provides a kit for treating and / or preventing a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease, the kit comprising a pharmaceutical composition of the invention.

[0028] The present invention also provides a kit for in-vitro cell culture, the kit comprising a cell culture medium of the invention.

[0029] MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0030] DESCRIPTION OF THE FIGURES

[0031] Figure 1. MITO-66 induces a stem cell memory phenotype in CAR T cells

[0032] (A) Experimental scheme depicting CD19-CART cell generation in the presence ofMITO-66.

[0033] (B) T cell expansion measured by machine-assisted trypan-blue-based cell counting on days 5, 7 and 9 post-activation. (5 donors, pooled data from 3 independent experiments). (C-D) CD62L-positive, CD45RO-negative stem cell memory T cells out of CD4 (C) or CD8 (D) CAR T cells at day 9 post-activation. (5 donors, pooled data from 3 independent experiments). (E- F) CAR-positive T cells in the CD4 (E) and CD8 (F) populations at day 9 post-activation. (4 donors, pooled data from 2 independent experiments). (G-H) CD62L-positive, CD45RO- negative stem cell memory T cells in the CD4 (G) or CD8 (H) CAR T populations at day 9 post-activation. (4 donors, pooled data from 2 independent experiments). (I- J) Mitochondrial membrane potential, measured by red / green signal ratio derived from IC-1 staining, expressed as fold change versus DMSO in CD4 (I) or CD8 (J) T cells at day 9 post-activation. (6 donors, pooled data from 4 independent experiments). (K-L) CD45RO-negative, CD62L / CD45RA / CCR7 / CD127-positive stem cell memory T cells in the CD4 (K) or CD8 (L) CAR T populations at day 9 post-activation. (6 donors, pooled data from 4 independent experiments).

[0034] Data is represented as mean ± standard deviation (SD). Statistics are based on one-way ANOVA (B, C, D) or on paired, two-tailed Students t-test (E-L).

[0035] Figure 2. MITO-66 conditioning during CAR T manufacturing enhances anti-tumor efficacy

[0036] (A) Experimental scheme depicting CD19-CART cell treatment of NALM6 leukemia-bearing mice. CAR T cells were prepared from 5 different donors during 3 independent manufacturing experiments and frozen at the end of the culture. At the time of ACT, the CAR T cells from all donors were thawed and administered to 1 or 2 recipient mice. (B) Survival of mice receiving no treatment, non-transduced T cells (NTD) or CAR T cells. (C) Number (No.) of CD 19- positive NALM6 cells in the blood of mice analysed by flow cytometry at the indicated timepoints. (D) Mouse weight in grams (g). (B-D: untreated, NTD DMSO and NTD MITO-66: n = 3 mice, CAR CTRL: n = 9 mice, CAR MITO-66: n = 7 mice.) (E-F) Number of CD4 (E) or CD8 (F) T cells in the blood at 11 days post-ACT. (G-J) Percentage of CD62L-positive cells out of CD4 or CD8 T cells at day 11 post-ACT (G, H) or day 33 post-ACT (I, I). (E-H: 4 MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 donors, pooled data from 2 independent experiments). (E-J: data from mice receiving CAR T cells from the same donor were pooled and average was calculated, represented here is the paired analysis of 5 donors (E-H) or 3 donors in surviving mice (I- J)).

[0037] Data is represented as mean ± standard error of mean (SEM). Statistics are based on two-way ANOVA (C) or on unpaired two-tailed Students t-test (D) or on paired, two-tailed Students t- test (E-J).

[0038] Figure 3. MITO-66 conditioned CAR T cells protect against cancer recurrence

[0039] (A) Experimental scheme depicting CD19-CART cell treatment of NALM6 leukemia-bearing mice following in vivo rechallenge with 2x106 NALM6 cells at days 33, 44 and 59 post-initial NALM6 engraftment and 5x106 NALM6 at day 70 post-initial NALM6 injection. CAR T cells were prepared from 2 different donors. CAR T cells from each donor were transferred in 5 different NALM6-bearing mice. Throughout the experiment, some mice were lost because of graft-versus-host disease. (B) NALM6 tumor burden measured by bioluminescence at the indicated days post-initial NALM6 engraftment. (C-D) Number of transferred CD3 T cells derived from donor 1 (C) or donor 2 (D) in the blood of mice analysed by flow cytometry at the indicated timepoints. (E-F) Percentage of CD62L-positive cells in the CD4 (E) or CD8 (F) T cell population in the blood at day 10 post-ACT.

[0040] Data is represented as mean ± SD. Statistics are based on two-way ANOVA (C-F).

[0041] Figure 4. Benchmarking MITO-66 with other small molecules influencing memory differentiation

[0042] (A) Experimental scheme depicting CD19-CAR T cell generation in the presence of different small molecule inhibitors. (B) T cell expansion measured by machine-assisted trypan-bluebased cell counting at day 5, 7 and 9 post-activation. (C-F) CD62L / CD45RO double positive cells out of CD4 (C) or CD8 (E) CAR T cells or CD62L-positive, CD45RO-negative stem cell memory T cells out of CD4 (D) or CD8 (F) CAR T cells at day 9 post-activation. (B-F: 6 donors, pooled data from 4 independent experiments). (G) Survival of mice receiving no treatment or inhibitor-conditioned CAR T cells. Surviving mice out of total treated mice are indicated between brackets. (H) Number (No.) of transferred CD3 T cells in the blood of mice analysed by flow cytometry at day 11 post-ACT. (I-K) Percentage of CD62L / CD45RO double positive cells (I), CD62L-positive, CD45RO-negative cells (J) or PD1 / TIM3 double positive cells (K) out of transferred CD3 T cells at day 11 post-ACT. (G-K: 3 human donors into 9-10 total mice, MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 pooled data from 2 independent experiments, only flow cytometry data with >20 events were used for phenotypic analysis).

[0043] Data is represented as mean ± standard error of mean (SEM). Statistics are based on one-way ANOVA.

[0044] Figure 5. MITO-66 induces a stem cell-like memory phenotype in CAR T cells from patients with B-cell malignancies

[0045] (A) T cell yield at day 10 post-activation, expressed as percentage of DMSO-conditioned CAR T cells. (B) Percentage of viable T cells at day 10 post-activation. (A-B: 8 patients, pooled data from 3 independent experiments). (C-D) Percentage of CD4 (C) and CD8 (D) T cells out of live cells at day 10 post-activation. (E) Percentage of CAR-positive T cells out of CD4 T cells at day 10 post-activation. (F-I) Percentage of CD 127-positive (F), CD62L-positive (G), CD62L / CD45RO-positive (H) and CD45RO-negative CD62L-positive (I) T cells out of CD4 CAR T cells at day 10 post-activation. (J) Percentage of CAR-positive T cells out of CD8 T cells at day 10 post-activation. (K-N) Percentage of CD 127-positive (K), CD62L-positive (L), CD62L / CD45RO-positive (M) and CD45RO-negative CD62L-positive (N) T cells out of CD8 CAR T cells at day 10 post-activation. (C-N: 11 patients, pooled data from 4 independent experiments).

[0046] Data is represented as mean ± SEM. Statistics are based on paired, two-tailed Students t-test.

[0047] Figure 6. MITO-66 is a novel small molecule inhibitor of the mitochondrial pyruvate carrier.

[0048] (A) BRET assay in HEK293T cells with MITO-1 or PBS as control. (B-C) Oxygen consumption rate (OCR) in HeLa cells measured by Seahorse using MPC inhibitor MITO-1

[0049] (B) or MITO-66 (C). IC50 was calculated based on drop in maximal oxygen consumption rate. (D) BRET assay in HEK293T cells with MITO-66 or PBS as control. (E) NanoDSF thermoshift analysis shows MPC1 / MPC2 heterocomplex stability induced by MITO-66 binding. Fluorescence-based melting curves at different concentrations of MITO-66. (F) Isothermal analysis for Kd estimation at 44.3°C (6.1°C above the protein Tm).

[0050] Data is represented as mean ± SEM.< MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0051] Figure 7. MITO-66 conditioning during CAR T cell manufacturing enhances anti-tumor efficacy

[0052] (A-B) CD4-positive (A) or CD8-positive (B) T cells in the live cell population at day 9 postactivation. (4 donors, pooled data from 2 independent experiments).

[0053] (C) Number of transferred CD4 or CD8 T cells in the blood of mice, derived from donors that induced survival following either DMSO- and MITO-66 conditioning.

[0054] Data is represented as mean ± standard error of mean (SEM). Statistics are based on paired, two-tailed Students t-test (A-B).

[0055] Figure 8. Benchmarking MITO-66 with other small molecules influencing memory differentiation.

[0056] (A) Number of transferred CD3 T cells in the blood of mice analysed by flow cytometry at day 11 post-ACT. Similar data as main Figure 5H, graph without rapamycin- and IDH2i-CAR T cells for clarity. (B-C) Number of transferred CD4 (B) or CD8 (C) T cells in the blood of mice analysed by flow cytometry at day 11 post-ACT. Similar data as main Figure 5H, graph without rapamycin- and IDH2i-CAR T cells for clarity. (D) Number of transferred CD 8 T cells in the blood of mice analysed by flow cytometry at day 11 post-ACT. Similar data as Figure 8C, graph without rapamycin-CAR T cells for clarity. (E-G) Percentage of CD62L / CD45RO double positive cells (E), CD62L-positive, CD45RO-negative cells (F) or PD1 / TIM3 double positive cells (G) out of transferred CD4 T cells at day 11 post-ACT. (H-J) Percentage of CD62L / CD45RO double positive cells (H), CD62L-positive, CD45RO-negative cells (I) or PD1 / TIM3 double positive cells (J) out of transferred CD8 T cells at day 11 post-ACT. (A-J: 3 human donors into 9-10 total mice, pooled data from 2 independent experiments. Only flow cytometry data with >20 events were used for phenotypic analysis).

[0057] Data is represented as mean ± standard error of mean (SEM). Statistics are based on one-way ANOVA.

[0058] Figure 9. Flow cytometry gating strategy.

[0059] Strategy used for selecting live, single cells for further downstream division in memory T cell subsets.

[0060] Figure 10. Inhibition of alpha-ketoglutarate-depending enzymes enhances TSCM differentiation and synergizes with MITO-66. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0061] (A-B) CD45RO-negative, CD62L / CD45RA / CCR7-positive stem cell memory T cells in the CD4 (A) or CD8 (B) CAR T populations at day 9 post-activation. (4 donors, pooled data from 2 independent experiments). Booster = Vadadustat; MITO Booster= MITO-66 + Vadadustat.

[0062] Figure 11. Effect of mitochondrial pyruvate carrier (MPC) inhibition and prolyl hydroxylase (PHD) inhibition on T cell viability, expansion, and stem cell memory T cell (TSCM) differentiation.

[0063] Cryopreserved T cells from a healthy donor were activated and expanded in a clinical-like manufacturing setting and treated with MPC inhibitors (MITO-66, UK-5099, 7ACC2, and MSDC-0160) and PHD inhibitors (FG-2216, Roxadustat, DMOG, and MK8617), alone or in combination. Cells were analyzed at day 9 post-activation. (A) The percentage of viable cells. (B) Total viable T cell counts. (C) The percentage of 2-marker TSCM cells (CCR7+CD45RA+) within the total T cell pool. (D) The percentage of 4-marker TSCM cells (CCR7+CD62L+CD45RA+CD45RO_) within the total T cell pool. (E) The calculated absolute number of 4-marker TSCM (% TSCM X viable T cell number).

[0064] Figure 12. Immunophenotypic characterization and anti-inflammatory potential of mesenchymal stem / stromal cells (MSC) cultured with MITO-66 and FG-2216,

[0065] (A) Colony-forming unit (CFU) analysis of primary adipose-derived MSC from multiple donors cultured for 10 days under control conditions or with different concentrations of MITO- 66 and FG-2216 (10 pM MITO-66 + 30 pM FG-2216; 20 pM MITO-66 + 30 pM FG-2216; 20 pM MITO-66 + 40 pM FG-2216). CFU were quantified using 0.01% crystal violet staining.

[0066] (B) Flow cytometric analysis on the percent positive cells based on the expression of canonical MSC-defming surface markers (CD73, CD90, and CD105) in control and different concentrations of MITO-66+FG-2216 treated conditions. (B) Impact of MITO-66+FG-2216 treatment on MSC based on the expression of markers (CD 146, and CD 10) associated with anti-inflammatory and analgesic properties of MSC. (C) Determination of macrophage polarization by MSC treated with different concentrations of MITO-66+FG-2216. Macrophage polarization status was measured by qRT-PCR-directed mRNA expression of relevant genes and categorized as Ml, M2-like, or M2 phenotypes. Data are presented from three independent MSC donors. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0067] DESCRIPTION OF THE INVENTION

[0068] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0069] In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

[0070] The term "comprise / comprising" is generally used in the sense of "include / including", that is to say permitting the presence of one or more features or components. This term also encompasses the more restricted term "consist / consisting".

[0071] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0072] The term "amino acid" includes all of the naturally occurring amino acids as well as modified amino acids.

[0073] As used herein, "at least one" means "one or more", "two or more", "three or more", etc.

[0074] The term “about” particularly in reference to a given quantity, is meant to encompass deviations of plus or minus ten (10) percent (+ / - 10%).

[0075] “Homology” refers to the percent identity between two polynucleotide or two polypeptide moieties. Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% or at least MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence. Alternatively, homology can be determined by readily available computer programs or by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single stranded specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.

[0076] In general, “identity” refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. In some aspects, a nucleotide or amino acid sequence of the invention, or a portion thereof, is at least 80%, namely, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a corresponding nucleotide or amino acid sequence (SEQ ID NO identifier).

[0077] As used herein, the terms "peptide", "protein", "polypeptide", "polypeptide chain", "polypeptidic" and "peptidic" are used interchangeably to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.

[0078] The compounds disclosed herein, or their pharmaceutically acceptable salts may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (5)-. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (.S')-, isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included. Where compounds are represented in their chiral form, it is understood that the aspect encompasses, but is not limited to, the specific diastereomerically or enantiomerically enriched form. Where chirality is not specified but is present, it is understood that the aspect is directed to either the specific diastereomerically or enantiomerically enriched form; or a racemic or scalemic mixture of such compound(s). As used herein, “scalemic mixture” is a mixture of stereoisomers at a ratio other than 1: 1.

[0079] “Stereoisomer” as used herein refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof and includes “enantiomers”, which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0080] “Tautomer” as used herein refers to a proton shift from one atom of a molecule to another atom of the same molecule. In some aspects, the present disclosure includes tautomers of said compounds.

[0081] It was surprisingly found by the inventors that the use of an MPC inhibitor, preferably the novel MPC inhibitor MITO-66, during in-vitro cell culture of CAR T cells enlarged the proportion of stem cell memory CAR T cells resulting in a strongly enhanced in vivo antitumor efficacy and durability of response.

[0082] In particular, the obtained T-cells (CD4+ and CD8+ CAR T cells) are enriched for T- cells with a memory phenotype (TSCM) (as shown in Figure 1C and ID).

[0083] In addition, it was surprisingly found by the inventors that CAR T cells conditioned with MITO-66 efficiently cured leukemic mice, when compared to other small molecules influencing memory differentiation (as shown in Figure 4G).

[0084] It was also surprisingly found that following adoptive cell transfer, MITO-66- conditioned CAR T cells maintained an increased memory phenotype and protected cured mice against cancer cell rechallenge (as shown in Figure 3B). MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0085] The invention provides an in-vitro cell culture method comprising a step of contacting a cell with a mitochondrial pyruvate carrier (MPC) inhibitor, a pharmaceutically acceptable salt or a pharmaceutically active derivative thereof.

[0086] The present invention provides an in-vitro cell culture method comprising a step of contacting a cell, or a population of cells, with a mitochondrial pyruvate carrier (MPC) inhibitor, and with a compound selected from a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate -depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HDAC inhibitor, or a combination thereof, wherein the cell is i) an immune cell, or a population of immune cells, selected from the group comprising a T cell, a gamma delta T cell (y8 T cell), a tumor infdtrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR-expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line derived from one of these cells or a pluripotent stem cell giving rise to one of these cells, or ii) a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, selected from the group comprising a mesenchymal stem cell, a multipotential stromal cell, a mesenchymal stromal cell, and a mesenchymal progenitor cell.

[0087] In one aspect, the invention provides an in-vitro cell culture method comprising a step of contacting a cell, or a population of cells, with a mitochondrial pyruvate carrier (MPC) inhibitor, and with a compound selected from a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate -depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HDAC inhibitor, or a combination thereof, wherein the cell is i) an immune cell, or a population of immune cells, selected from the group comprising a T cell, a gamma delta T cell (y8 T cell), a tumor infdtrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR-expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line derived from one of these cells or a pluripotent stem cell giving rise to one of these cells, or ii) a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, selected from the group comprising a mesenchymal stem cell, a multipotential stromal cell, a mesenchymal stromal cell, a mesenchymal progenitor cell, and a multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR).

[0088] In one aspect, the multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR) is a mesenchymal stem cell expressing a chimeric antigen receptor (CAR).

[0089] In one aspect, the multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR) is a multipotential stromal cell expressing a chimeric antigen receptor (CAR).

[0090] In one aspect, the multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR) is a mesenchymal stromal cell expressing a chimeric antigen receptor (CAR).

[0091] In one aspect, the multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR) is a mesenchymal progenitor cell expressing a chimeric antigen receptor (CAR).

[0092] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is a T cell expressing a chimeric antigen receptor (CAR).

[0093] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is a gamma delta T cell (y8 T cell) expressing a chimeric antigen receptor (CAR).

[0094] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is a tumor infiltrating lymphocyte (TIL) expressing a chimeric antigen receptor (CAR).

[0095] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is an NK cell expressing a chimeric antigen receptor (CAR).

[0096] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is a regulatory T cell (Treg cell) expressing a chimeric antigen receptor (CAR).

[0097] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is a macrophage expressing a chimeric antigen receptor (CAR).

[0098] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is a TCR- expressing cell and expressing a chimeric antigen receptor (CAR). MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0099] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is an eosinophil expressing a chimeric antigen receptor (CAR).

[0100] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is a basophil expressing a chimeric antigen receptor (CAR).

[0101] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is a neutrophil expressing a chimeric antigen receptor (CAR).

[0102] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is a myeloid cell expressing a chimeric antigen receptor (CAR).

[0103] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is a B cell expressing a chimeric antigen receptor (CAR).

[0104] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is a plasma cell expressing a chimeric antigen receptor (CAR).

[0105] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is a regulatory B cell (Breg) expressing a chimeric antigen receptor (CAR).

[0106] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is an innate lymphoid cell 1 (ICL1) expressing a chimeric antigen receptor (CAR).

[0107] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is an innate lymphoid cell 2 (ICL2) expressing a chimeric antigen receptor (CAR).

[0108] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is an innate lymphoid cell 3 (ICL3) expressing a chimeric antigen receptor (CAR).

[0109] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is a dendritic cell expressing a chimeric antigen receptor (CAR).

[0110] In one aspect, the immune cell expressing a chimeric antigen receptor (CAR) is an NK-T cell expressing a chimeric antigen receptor (CAR).

[0111] As used herein, the term “first class of compounds” refers to compounds selected from the list of mitochondrial pyruvate carrier (MPC) inhibitors provided in the present invention.

[0112] The term “second class of compounds” refers to compounds that are distinct from those of the first class, and are selected from the list provided in the present invention, such as a prolyl hydroxylase (PHD) inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0113] (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate -depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HDAC inhibitor.

[0114] According to one aspect, the invention provides an in-vitro cell culture method comprising a step of contacting a cell with an MPC inhibitor, e.g. MITO-66, a tautomer, a geometrical isomer, an optically active form, a pharmaceutically acceptable salt or a pharmaceutically active derivative thereof.

[0115] As used herein, the term "stabilizes" in relation to a protein refers to any process, treatment, or modification that enhances or maintains the structural integrity, functional activity, or biological availability of the protein over time or under specific conditions. Stabilization may prevent or reduce degradation, denaturation, aggregation, oxidation, hydrolysis, or other forms of chemical or physical instability that could impair the protein’s intended function.

[0116] According to one aspect, the terms "a cell, or a population of cells" refer to a cell or a cell population of multipotential stem / progenitor cells commonly referred to as mesenchymal stem cells, multipotential stromal cells, mesenchymal stromal cells, mesenchymal progenitor cells, and a multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR) .

[0117] According to one aspect, the terms "a cell, or a population of cells" refer to an immune cell or a population of immune cells, preferably an autologous or an allogeneic immune cell. In one aspect, the immune cell is selected from the non-limiting group comprising a T cell, a gamma delta T cell (y§ T cell), a tumor infiltrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR-expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line derived from one of these cells or a pluripotent stem cell giving rise to one of these cells.

[0118] In one aspect, the invention provides an in-vitro cell culture method comprising a step of contacting an immune cell with MITO-66, a tautomer, a geometrical isomer, an optically active form, a pharmaceutically acceptable salt or a pharmaceutically active derivative thereof, wherein the immune cell is selected from the group comprising a T cell, a gamma delta T cell (y§ T cell), a tumor infiltrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR-expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0119] ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line derived from one of these cells or a pluripotent stem cell giving rise to one of these cells.

[0120] As used herein, the term "contacting" refers to the deliberate in vitro exposure or application of a compound, composition, or agent to a cell, cell population, or cell culture under conditions suitable to allow the in vitro physical interaction between the compound and the cells. In the context of the present invention, "contacting" includes, but is not limited to, adding the compound directly to the cell culture medium in which the cells are maintained, thereby permitting diffusion or transport of the compound to the cellular environment. The term encompasses both transient and sustained exposure and includes any method of delivery that results in the compound being available to interact in vitro with the cells in a manner that promotes cell growth, proliferation, viability, or survival.

[0121] As used herein, the terms "a pluripotent stem cell giving rise to one of these cells", in relation to the list of immune cells as defined in the invention, refer to the differentiation of a pluripotent stem cell into an immune cell as defined in the invention. As used herein, the differentiation process refers to the process by which a pluripotent stem cell is induced, under defined in vitro or ex vivo conditions, to acquire the phenotypic and functional characteristics of an immune cell. This process involves directed lineage specification through exposure to specific culture conditions, signaling factors, or genetic modifications that guide the stem cell through intermediate stages toward a terminal immune cell fate.

[0122] In one aspect, the invention provides an in-vitro cell culture method, wherein the MPC inhibitor is selected from the group comprising, or consisting of a cinnamic acid, a-cyano- 4- hydroxycinnamate (CHC), diones, thiazolidines and thiazolidinediones (TZDs), the cGMP- specific phosphodiesterase (PDE) inhibitor zaprinast, the anti-cancer agent lonidamine, coumarin derivatives, and quinolone antibiotics, or a combination thereof.

[0123] As used herein, an "MPC inhibitor" or "mitochondrial pyruvate carrier (MPC) inhibitor" refers to a compound inhibiting MPC, including cinnamic acids (such as e.g. UK5099, an alpha- cyano-cinnamate derivative), a-cyano- 4 -hydroxycinnamate (CHC), several diones, thiazolidines and thiazolidinediones (TZDs), the cGMP-specific phosphodiesterase (PDE) inhibitor zaprinast, the anti-cancer agent lonidamine, coumarin derivatives, and quinolone antibiotics. For a review of MPC inhibitors see, e.g. Tavoulari S, et al. Mol Metab. 2022 Jun;60: 101469. doi: 10.1016 / j.mohnet.2022.101469; Du J, et al. J Biol Chem. 2013 Dec MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0124] 13;288(50):36129-40; Divakaruni AS, et al. Proc Natl Acad Sci U S A. 2013 Apr 2;110(14):5422-7; Nancolas B, et al. Biochem J. 2016 Apr l;473(7):929-36; Corbet C, et al. Nat Commun. 2018 Mar 23;9(1): 1208; Hodges WT, et al. J Biol Chem. 2022 Feb;298(2): 101554).

[0125] In one aspect, the invention provides an in-vitro cell culture method, wherein the cinnamic acid is a cyano-cinnamate selected from the group comprising, or consisting of CHC, UK5099, BE1976, BE1978, BE1980, BE1984, BE2617, BE2623, JXL020, JXL069, JXL050, JXL051, JXL069, 2-cyano-3-(5-phenyl-2-fiiryl)acrylic acid, and 2-cyano-3-[5-(2-nitrophenyl)-2-fiiryl] acrylic acid, or a derivative, a tautomer, a geometrical isomer and an optically active form of any one of these compounds, or a combination thereof.

[0126] Non-limiting examples of cyano-cinnamates and derivatives are selected from the group comprising CHC, UK5099, BE1976, BE1978, BE1980, BE1984, BE2617, BE2623, JXL020, JXL069, JXL050, JXL051, JXL069, 2-cyano-3-(5-phenyl-2-fiiryl)acrylic acid, and 2-cyano- 3-[5-(2-nitrophenyl)-2-fiiryl] acrylic acid, or derivatives thereof (see e.g. Table 1 of Tavoulari S27, et al. 2023 Aug;238(4):el4016), or a tautomer, a geometrical isomer, an optically active form of any one of these compounds.

[0127] As used herein, the term “geometrical isomer” refers to a type of stereoisomer in which compounds with the same molecular formula and atom-to-atom connectivity differ in the spatial arrangement of substituents or groups relative to a rigid structural element, such as a carbon-carbon double bond, a cyclic structure, or other configurationally restricted systems. Such isomerism typically arises due to the lack of free rotation about the double bond or within the ring, resulting in distinct configurations — commonly referred to as cis (or Z) and trans (or E) — depending on whether the substituents are positioned on the same side or opposite sides of the rigid framework. Geometrical isomers often exhibit different physicochemical or biological properties despite having the same molecular connectivity.

[0128] As used herein, the term “optically active form” refers to a stereoisomer of a compound that has the ability to rotate the plane of plane-polarized light, a property known as optical activity. This phenomenon arises due to molecular asymmetry, typically the presence of one or more chiral centers, and is quantitatively expressed by the specific rotation of the compound under defined conditions (e.g., concentration, temperature, wavelength, and path length). Optically active forms exist as enantiomers, which are non-superimposable mirror MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 images that rotate polarized light in equal magnitude but opposite directions, either dextrorotatory (clockwise, denoted as (+)) or levorotatory (counterclockwise, denoted as (-)).

[0129] In one aspect, the invention provides an in-vitro cell culture method, wherein the dione, thiazolidine, and thiazolidinedione MPC inhibitor is selected from the group comprising, or consisting ofGW604714X, GW450863X, Pioglitazone, Rosiglitazone, MSDC-0160, Carsalam, Nitrofurantoin, (E)-5-(4-Hydroxybenzylidene) thiazolidine-2, 4-dione, and (E)-5- (3 -Hydroxy-4- methoxybenzylidene) thiazolidine-2, 4-dione or a derivative, a tautomer, a geometrical isomer and an optically active form of any one of these compounds or a combination thereof.

[0130] Non-limiting examples of diones, thiazolidine s, and thiazolidinediones MPC inhibitors are selected from the group comprising GW604714X, GW450863X, Pioglitazone, Rosiglitazone, MSDC-0160, Carsalam, Nitrofurantoin, (E) -5 -(4 -Hydroxybenzylidene) thiazolidine-2, 4- dione, and (E)-5-(3-Hydroxy-4- methoxybenzylidene) thiazolidine-2, 4-dione (see e.g. Table 2 of Tavoulari S, et al. 2023 Aug;238(4):el4016), or atautomer, a geometrical isomer, an optically active form of any one of these compounds .

[0131] In one aspect, the invention provides an in-vitro cell culture method, wherein the MPC inhibitor is selected from the group comprising, or consisting of, Zaprinast, Lonidamine, 7ACC2, 7ACC1, Entacapone, Quinolone antibiotics (e.g. Pefloxacine), and Clinafloxacin, or a tautomer, a geometrical isomer and an optically active form of any one of these molecules, or a combination thereof.

[0132] Non-limiting examples of other MPC inhibitors are selected from the group comprising (but not limited to) Zaprinast, Lonidamine, 7ACC2, 7ACC1, Entacapone, Quinolone antibiotics (e.g. Pefloxacine), and Clinafloxacin (see e.g. Table 3 of Tavoulari S, et al. 2023 Aug;238(4):el4016), compounds (and their respective synthesis) described in PCT / EP2022 / 072681 Formula I (WO2023 / 017154, MPC therapeutics and Universite de Geneve) or a tautomer, a geometrical isomer, an optically active form of any one of these compounds and characterized by Formula I: MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 wherein Rl is a moiety R3-R4; R2 is selected from H, optionally substituted Ci-Ce alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryl Ci-Ce alkyl, optionally substituted heteroaryl Ci-Cg alkyl, optionally substituted Ci-Cg alkoxy such as optionally substituted Ci-Cg alkoxy substituted Ci-Cg alkyl, optionally substituted C’s-C’s heterocycloalkyl and optionally substituted Cs-Cs cycloalkyl such as optionally substituted cyclopropyl; R3 is selected from a bond, S, and NR5; R4 is a group -(CR7R8)n-R6 wherein n is an integer between 0 and 1 ; R5 is H or optionally substituted Ci-Cg alkyl; R6 is selected from optionally substituted heterocycle, optionally substituted aryl; R7 and R8 are independently selected from is H and optionally substituted Ci-Cg alkyl; as well as tautomers, geometrical isomers, optically active forms and pharmaceutically acceptable salts and pharmaceutically active derivative thereof.

[0133] In one aspect, the invention also provides an in-vitro cell culture method, wherein the MPC inhibitor is of formula I: wherein Rl is a moiety R3-R4;

[0134] R2 is selected from H, optionally substituted Ci-Cg alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryl Ci-Ce alkyl, optionally substituted heteroaryl Ci-Ce alkyl, optionally substituted Ci-Ce alkoxy such as optionally substituted Ci-Ce alkoxy MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 substituted Ci-Ce alkyl, optionally substituted Cs-Cs heterocycloalkyl and optionally substituted Cs-Cs cycloalkyl such as optionally substituted cyclopropyl;

[0135] R3 is selected from a bond, S, and NR5;

[0136] R4 is a group -(CR7R8)n-R6 wherein n is an integer between 0 and 1 ;

[0137] R5 is H or optionally substituted Ci-Cg alkyl;

[0138] R6 is selected from optionally substituted heterocycle, optionally substituted aryl;

[0139] R7 and R8 are independently selected from H and optionally substituted Ci-Ce alkyl; or a tautomer, a geometrical isomer, an optically active form, a pharmaceutically acceptable salt or a pharmaceutically active derivative thereof.

[0140] In one aspect, the invention provides an MPC inhibitor of Formula (I) wherein R1 is a moiety S-R6.

[0141] In one aspect, the invention provides an MPC inhibitor of Formula (I) wherein R1 is a moiety NR9-R6.

[0142] In one aspect, the invention provides an MPC inhibitor of Formula (I) wherein R1 is a moiety NH-R6.

[0143] In one aspect, the invention provides an MPC inhibitor of Formula (I) wherein R1 is a moiety R6.

[0144] In one aspect, the invention provides an MPC inhibitor of Formula (I) wherein R2 is optionally substituted C1-C6 alkyl.

[0145] In one aspect, the invention provides an MPC inhibitor of Formula (I) wherein R2 is optionally substituted propyl.

[0146] In one aspect, the MPC inhibitor is MITO-66, a tautomer, a geometrical isomer, an optically active form, a pharmaceutically acceptable salt or a pharmaceutically active derivative thereof.

[0147] The chemical structure of MITO-66 is as shown below and has been described in PCT / EP2022 / 072681. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0148] 3-(benzylsulfanyl)-5-propyl[l,2,4]triazolo[4,3-a]pyrimidin-7(8H)-one

[0149] MITQ 6

[0150] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell or the population of cells is contacted with the MPC inhibitor of formula

[0151] 3-(benzylsulfanyl)-5-propyl[l,2,4]triazolo[4,3-a]pyrimidin-7(8H)-one

[0152] MITO-66 or a tautomer, a geometrical isomer, an optically active form, a pharmaceutically acceptable salt or a pharmaceutically active derivative thereof.

[0153] As used herein, the terms “pharmaceutically active derivative” refer to any compound that, upon in vitro administration on cells in the present in vitro cell culture method, directly or indirectly provides a pharmacological effect equivalent to that of the reference compound. The terms “pharmaceutically active derivative” include, but is not limited to, prodrugs, metabolites, salts, esters, solvates, or other chemical forms that are converted in the in vitro cell culture method of the invention into the active compound or exhibit comparable therapeutic activity.

[0154] As used herein, the term "MITO-66" is interchangeable with the term "3-(benzylsulfanyl)-5- propyl[l,2,4]triazolo[4,3-a]pyrimidin-7(8H)-one", and refers to an MPC inhibitor as described in the invention. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0155] The in vitro cell culture may comprise a culture vessel (e.g. a dish, a well plate, a bioreactor, a flask, or a bottle), a culture medium comprising MITO-66 as provided herein, and immune cells. The culture may be static or dynamic (e.g. agitated by means of rotation or stirring).

[0156] In one aspect, the invention provides an in-vitro cell culture method, wherein the compound selected from a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha- ketoglutarate-depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HDAC inhibitor, or a combination thereof is selected from daprodustat, desidustat, enarodustat, molidustat, Roxadustat (FG-4592), vadadustat, dimethyloxallyl glycine (DMOG), FG-2216, IOX4, JNJ-42041935, MK-8617, S-2-hydroxyglutarate, fumarate, succinate, malate, deferasirox, deferoxamine, N-oxaloylglycine, N-oxalyl-2S-alanine, Alahopcin, dihydroxybenzoic acid, diazen-l-ium-l,2-diolate, diethylamine NONOate, Tilorone, a Factor inhibiting HIF (FIH) inhibitors, Von Hippel-Lindau (VHL) inhibitors or HIF allosteric agonists, or a combination thereof.

[0157] In one aspect, the invention provides an in-vitro cell culture method, wherein the compound is selected from daprodustat, desidustat, enarodustat, molidustat, Roxadustat (FG-4592), vadadustat, dimethyloxallyl glycine (DMOG), FG-2216, IOX4, JNJ-42041935, MK-8617, S- 2 -hydroxyglutarate, fumarate, succinate, malate, deferasirox, deferoxamine, N-oxaloylglycine, N-oxalyl-2S-alanine, Alahopcin, dihydroxybenzoic acid, diazen-l-ium-l,2-diolate, diethylamine NONOate, Tilorone, a Factor inhibiting HIF (FIH) inhibitors, Von Hippel-Lindau (VHL) inhibitors or HIF allosteric agonists, or a combination thereof.

[0158] In one aspect, the invention provides an in-vitro cell culture method, wherein the compound selected form a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha- ketoglutarate-depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HDAC inhibitor, or a combination thereof is selected from Roxadustat, Vadadustat, , FG-2216, and MK-8617.

[0159] As used herein, the term " FG-2216" is interchangeable with the term "N-[( 1 -chloro-4-hydroxy- 3-isoquinolinyl)carbonyl]-glycine", and refer to an inhibitor of hypoxia-inducible factor prolyl hydroxylase 2 (HIF-PH2) as described in the invention. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0160] In one aspect, the in-vitro cell culture method of the invention further comprises contacting the cell, e.g. the immune cell with an isocitrate dehydrogenase 2 (IDH2) inhibitor, a mammalian target of rapamycin (mTOR) inhibitor, an AKT inhibitor, a phosphatidylinositol 3 -kinase (PI- 3K) inhibitor, a MEK inhibitor, a tyrosine kinase inhibitor (such as e.g. Dasatinib), a Bruton tyrosine kinase (BT inhibitor, a bromodomain and extra-terminal (BET) inhibitor, a peroxisome proliferator-activated receptor (PPAR) inhibitor, metformin, a disubstituted pyrrolopyrimidine inhibitor of glycogen synthase kinase 3 beta (GSK30) (such e.g. TWS119) and / or a Histone deacetylase HD AC inhibitor, a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite that inhibits alpha-ketoglutarate -depending enzymes such as prolyl hydroxylases, Jumanji-domain containing demethylases etc., or a combination thereof.

[0161] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell or the population of cells is contacted with the MPC inhibitor of formula: 3 -(benzylsulfanyl)-5 -propyl [ 1 ,2,4]triazolo [4,3 -a]pyrimidin-7(8H)-one

[0162] MITO-6® or a tautomer, a geometrical isomer, an optically active form, a pharmaceutically acceptable salt or a pharmaceutically active derivative thereof, and wherein the compound is selected from Roxadustat or FG-2216.

[0163] In one aspect, the invention provides an in vitro cell culture method, wherein the cell or the population of cells is contacted simultaneously or in a staggered manner in time with the MPC inhibitor and with the compound selected from a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate-depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HD AC inhibitor, or a combination thereof

[0164] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted simultaneously with both MITO-66 (3-(benzylsulfanyl)-5- MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 propyl[l,2,4]triazolo[4,3-a]pyrimidin-7(8H)-one) and one compound selected from Roxadustat, Vadadustat, FG-2216, and MK-8617 (both compounds are present in the same cell culture medium).

[0165] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted first with MITO-66 (3-(benzylsulfanyl)-5- propyl[l,2,4]triazolo[4,3-a]pyrimidin-7(8H)-one) and then, in a second time (e.g. 1-10 minutes, 1 to 10 hours, 2 to 5 days, . . .) with one compound selected from Roxadustat, Vadadustat, FG- 2216, and MK-8617.

[0166] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted first with a compound selected from Roxadustat, Vadadustat, FG-2216, and MK-8617 and then, in a second time (e.g. 1-10 minutes, 1 to 10 hours, 2 to 5 days, ...) with MITO-66 (3-(benzylsulfanyl)-5-propyl[l,2,4]triazolo[4,3-a]pyrimidin-7(8H)- one).

[0167] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both UK-5099 and Roxadustat.

[0168] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both UK-5099 and DMOG.

[0169] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both UK-5099 and FG-2216.

[0170] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both UK-5099 and MK-8617.

[0171] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both UK-5099 and Vadadustat.

[0172] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both MITO-66 (3-(benzylsulfanyl)-5- propyl[l,2,4]triazolo[4,3-a]pyrimidin-7(8H)-one) and Roxadustat.

[0173] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both MITO-66 (3-(benzylsulfanyl)-5- propyl[l,2,4]triazolo[4,3-a]pyrimidin-7(8H)-one) and DMOG. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0174] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both MITO-66 (3-(benzylsulfanyl)-5- propyl[l,2,4]triazolo[4,3-a]pyrimidin-7(8H)-one) and FG-2216.

[0175] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both MITO-66 (3-(benzylsulfanyl)-5- propyl[l,2,4]triazolo[4,3-a]pyrimidin-7(8H)-one) and MK-8617.

[0176] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both MITO-66 (3-(benzylsulfanyl)-5- propyl[l,2,4]triazolo[4,3-a]pyrimidin-7(8H)-one) and Vadadustat.

[0177] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both 7ACC2 and Roxadustat.

[0178] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both 7ACC2 and DMOG.

[0179] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both 7ACC2 and FG-2216.

[0180] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both 7ACC2 and Vadadustat.

[0181] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both 7ACC2 and MK-8617.

[0182] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both MSDC-0160 and Roxadustat.

[0183] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both MSDC-0160 and Vadadustat.

[0184] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both MSDC-0160 and DMOG.

[0185] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both MSDC-0160 and FG-2216.

[0186] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell, or the population of cells are contacted with both MSDC-0160 and MK-8617.

[0187] T1 MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0188] In one aspect, the invention provides an in-vitro cell culture method, further comprising adding nutrients and / or other supplements selected from the group comprising, or consisting of pyruvate, galactose, nicotinamide adenine dinucleotide (NAD), lactate, any amino acid, nucleic acids, inosine, fatty acids, acetate, ketone bodies, a respiratory chain uncoupler agent (e.g. FCCP), a mitochondrial ATPase inhibitor (e.g. Oligomycin A), a PP AR alpha ligand (e.g. Bezafibrate) and urolithin A, or a combination thereof.

[0189] As used herein, the expression “adding nutrients and / or other supplements” refers to the step of introducing one or more exogenous substances into the cell culture medium of the invention in order to support, enhance, or maintain the viability, proliferation, metabolism, differentiation, or productivity of cells cultured therein.

[0190] The step of adding nutrients and / or other supplements as defined in the invention may occur prior to, at the time of, or during the culture process (e.g., as part of a feeding strategy), and may be performed as a single addition, multiple additions, or in a continuous or semi- continuous manner, depending on the cell type, culture system, and desired outcome.

[0191] In one aspect, the step of addition of nutrients and / or supplements to the cell culture medium can be performed before the step(s) of contacting the cell, or the population of cells with the first class and second class of compounds.

[0192] In one aspect, the step of addition of nutrients and / or supplements to the cell culture medium can be performed between the step of contacting the cell, or the population of cells with the first class of compounds and the step of contacting the cell, or the population of cells with the second class of compounds.

[0193] In one aspect, the step of addition of nutrients and / or supplements to the cell culture medium can be performed after the step(s) of contacting the cell, or the population of cells with the first class and second class of compounds.

[0194] In one aspect, the step of addition of nutrients and / or supplements to the cell culture medium can be performed between the step of contacting the cell, or the population of cells with the second class of compounds and the step of contacting the cell, or the population of cells with the first class of compounds.

[0195] Unless otherwise specified, the expression “adding nutrients and / or other supplements” also encompasses the addition of such components in defined, partially defined, or complex formulations, and includes both manual and automated modes of administration. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0196] In one aspect, the cell, e.g. the immune cell is contacted first with the MPC inhibitor and then, in a second time (e.g. 1-10 minutes, 1 to 10 ours, 2 to 5 days, ...) with a compound selected from an isocitrate dehydrogenase 2 (IDH2) inhibitor, a mammalian target of rapamycin (mTOR) inhibitor, an AKT inhibitor, a phosphatidylinositol 3-kinase (PI-3K) inhibitor, a MEK inhibitor, a tyrosine kinase inhibitor (such as e.g. Dasatinib), a Bruton tyrosine kinase (BT inhibitor, a bromodomain and extra-terminal (BET) inhibitor, a peroxisome proliferator-activated receptor (PPAR) inhibitor, metformin, a disubstituted pyrrolopyrimidine inhibitor of glycogen synthase kinase 3 beta (GSK3P) (such e.g. TWS119) and / or a Histone deacetylase HDAC inhibitor, a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite that inhibits alpha-ketoglutarate- depending enzymes such as prolyl hydroxylases, Jumanji -domain containing demethylases etc., or a combination thereof.

[0197] In one aspect, the cell, e.g. the immune cell is contacted first with a compound selected from an isocitrate dehydrogenase 2 (IDH2) inhibitor, a mammalian target of rapamycin (mTOR) inhibitor, an AKT inhibitor, a phosphatidylinositol 3-kinase (PI-3K) inhibitor, a MEK inhibitor, a tyrosine kinase inhibitor (such as e.g. Dasatinib), a Bruton tyrosine kinase (BT inhibitor, a bromodomain and extra-terminal (BET) inhibitor, a peroxisome proliferator-activated receptor (PPAR) inhibitor, metformin, a disubstituted pyrrolopyrimidine inhibitor of glycogen synthase kinase 3 beta (GSK3P) (such e.g. TWS119) and / or a Histone deacetylase HDAC inhibitor, a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite that inhibits alpha-ketoglutarate- depending enzymes such as prolyl hydroxylases, Jumanji -domain containing demethylases etc., or a combination thereof and then, in a second time (e.g. 1-10 minutes, 1 to 10 hours, 2 to 5 days, or more) with the MPC inhibitor.

[0198] In one aspect, the cell, e.g. the immune cell is contacted with the MPC inhibitor and a compound selected from an isocitrate dehydrogenase 2 (IDH2) inhibitor, a mammalian target of rapamycin (mTOR) inhibitor, an AKT inhibitor, a phosphatidylinositol 3-kinase (PI-3K) inhibitor, a MEK inhibitor, a tyrosine kinase inhibitor (such as e.g. Dasatinib), a Bruton tyrosine kinase (BT inhibitor, a bromodomain and extra-terminal (BET) inhibitor, a peroxisome proliferator-activated receptor (PPAR) inhibitor, metformin, a disubstituted pyrrolopyrimidine inhibitor of glycogen synthase kinase 3 beta (GSK3p) (such e.g. TWS119) and / or a Histone deacetylase HDAC inhibitor, a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 metabolite that inhibits alpha-ketoglutarate -depending enzymes such as prolyl hydroxylases, Jumanji-domain containing demethylases etc., or a combination thereof at the same time, i.e. both compounds are present in the same culture medium.

[0199] The cell culture method of MITO-66 in combination with another small molecule inhibitor can be further supplemented with nutrients or other supplements, such as pyruvate, galactose, nicotinamide adenine dinucleotide (NAD), lactate, any amino acid, nucleic acids, inosine, fatty acids, acetate, ketone bodies, a respiratory chain uncoupler agent (e.g. FCCP), a mitochondrial ATPase inhibitor (e.g. Oligomycin A), a PPAR alpha ligand (e.g. Bezafibrate) and urolithin A, or a combination thereof.

[0200] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell is an autologous, an allogeneic cell or a cell line.

[0201] As used herein, the term “autologous cell” refers to a cell that is derived from the same individual (donor and recipient are the same person). In the context of the present invention, autologous cells are isolated from a subject, optionally modified or expanded ex vivo, and then reintroduced into the same subject.

[0202] As used herein, the term “allogeneic cell” refers to a cell that is derived from a different individual of the same species (donor and recipient are distinct individuals). In the context of the present invention, allogeneic cells are typically obtained from a healthy donor and administered to a recipient for whom the cells are not genetically identical.

[0203] In a preferred aspect, the method of the invention further comprises a step of obtaining immune cells with a memory phenotype from the culture. Thus, the inventive methods provided herein may comprise a further step of obtaining the immune cells, in particular an immune cell selected from the group comprising a T cell, a gamma delta T cell (y§ T cell), a tumor infiltrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR- expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line derived from one of these cells or a pluripotent stem cell giving rise to one of these cells, from the culture, thereby producing a cell population comprising immune cells with a memory phenotype, as described herein.

[0204] Preferably herein, the immune cells are human cells. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0205] Where the immune cells are e.g. T-cells, these T-cells are autologous or allogeneic CD 8+ or CD4+ T cells obtained from human umbilical cord blood (CB) cells and / or peripheral blood mononuclear cells (PBMC). Thus, the invention further relates to a cell or population of cells comprising T-cells, preferably human T-cells, with a memory phenotype obtained by the method of the invention.

[0206] Where the immune cells are e.g. NK-cells, these NK-cells are autologous or allogeneic NK- cells obtained from blood, including human umbilical cord blood (CB) cells and / or peripheral blood mononuclear cells (PBMC). Thus, the invention further relates to a cell or population of cells comprising NK-cells, preferably human NK-cells, with a memory phenotype obtained by the method of the invention.

[0207] In one aspect, the invention provides an in-vitro cell culture method, wherein the immune cell or the population of immune cells is an NK cell line selected from the group comprising NK- 92, YTS and KHYG1 cell lines.

[0208] As used herein, the term “YTS cells” refers to a human natural killer (NK) cell line originally derived from a patient with NK-like large granular lymphocyte leukemia.

[0209] As used herein, the term “NK-92 cells” refers to an interleukin-2 (IL-2)-dependent human natural killer (NK) cell line derived from the peripheral blood of a patient with non -Hodgkin’s lymphoma.

[0210] As used herein, the term “KHYG-1 cells” refers to a human NK / T-cell lymphoma-derived cell line established from the peripheral blood of a patient with aggressive NK-cell leukemia.

[0211] In one aspect, the NK cell is an NK cell line selected from the group comprising NK-92, YTS or KHYG1 cell lines. Thus, the invention further relates to a cell or population of cells comprising NK-cell line(s), preferably human NK-cell line(s), with a memory phenotype obtained by the method of the invention.

[0212] In one aspect, the invention provides an in-vitro cell culture method, wherein the immune cell is an immune cell expressing a CAR selected from the group comprising, or consisting of a CAR-T cell, a TRUCK T cell, an armored CAR-T cell, a CAR-NK cell, and a CAR macrophage. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0213] Where the immune cells are e.g. CAR cells (e.g. CD8+ or CD4+ CAR T-cells, TRUCK T cells, CAR NK-cells, CAR macrophages or CAR-Treg cells), these CAR cells are autologous or allogeneic immune cells (e.g. T- or NK cells, macrophages or Treg cells) obtained from blood, human umbilical cord blood (CB) cells and / or peripheral blood mononuclear cells (PBMC) and engineered using techniques and methods known in the art. These cells are then contacted with MPC inhibitors (including, but not limited to UK-5099, MITO-66, 7ACC2, MSDC-0160) and / or with a compound selected form a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate -depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HDAC inhibitor, or a combination thereof, according to in-vitro cell culture method of the invention. Thus, the invention further relates to a cell or population of cells comprising CAR cells, preferably human CAR cells, with a memory phenotype obtained by the method of the invention.

[0214] In addition to T cells, the carrier cells for chimeric antigen receptor (CAR) expression may comprise alternative immune or non-immune cell types. Suitable alternative carrier cells include, but are not limited to, natural killer (NK) cells, including primary NK cells and NK cell lines such as NK-92, YTS, and KHYG-1 ; y8 T cells; natural killer T (NKT) cells, including invariant NKT (iNKT) cells; and mucosal-associated invariant T (MAIT) cells. Further suitable cell types comprise macrophages, monocytes, dendritic cells, and neutrophils. In certain aspects, stem cells, including hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), may be genetically modified to express a CAR, either prior to or following differentiation into immune effector cells. In some aspects, the cell is an induced pluripotent stem cell (iPSC)-derived immune cell, including, for example, iPSC-derived NK cells, T cells, or hybrid cell types. In further aspects, other cell types such as basophils, eosinophils, mast cells, or genetically engineered stromal cells, fibroblasts, or endothelial cells may be employed, for example, for modifying the tumor microenvironment or delivering immunomodulatory agents. The choice of cell type may depend on factors such as the therapeutic indication, the desired effector function, the route of administration, and whether the product is intended for autologous or allogeneic use.

[0215] In one aspect, the invention provides an in-vitro cell culture method, wherein the cell or the population of cells contacted with an MPC inhibitor and with a compound of the invention is selected from a multipotential stem / progenitor cell, or a population of multipotential MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 stem / progenitor cells, selected from the group comprising a mesenchymal stem cell, a multipotential stromal cell, a mesenchymal stromal cell, a mesenchymal progenitor cell, and a multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR). A person skilled in the art would know how to prepare such multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR) (as disclosed in Vladislav Volarevic et al.25and in Yan L. et al.26).

[0216] As used herein, the term “carrier cells” refers to cells that are genetically modified to express a chimeric antigen receptor (CAR) and serve as vehicles for delivering the therapeutic function of the CAR construct. Carrier cells may include, but are not limited to, immune cells such as T cells, natural killer (NK) cells, y8 T cells, NKT cells, macrophages, or other suitable cell types capable of mediating an immune response or therapeutic effect upon administration.

[0217] As used herein, the term “TRUCK T cells” (T cells Redirected for Universal Cytokine- mediated Killing) refers to T cells that are genetically modified to express a chimeric antigen receptor (CAR) and one or more additional transgenes under the control of a CAR-inducible promoter. Upon antigen recognition, TRUCK T cells not only exert CAR-mediated cytotoxic activity but also locally release therapeutic proteins to enhance anti-tumor efficacy. The transgenes may encode, for example, cytokines (e.g., IL-12, IL-15, IL-18), chemokines (e.g., CCL19, CCL21), immune checkpoint modulators (e.g., PD-1 blockers), or co-stimulatory ligands (e.g., CD40L). TRUCK T cells are particularly suited for use in the treatment of solid tumors, where modulation of the tumor microenvironment is required, but may also be applied in hematologic malignancies, infectious diseases, or autoimmune disorders.

[0218] In one aspect, the invention provides an in-vitro cell culture method, wherein the immune cell or the population of immune cells is a macrophage cell line, wherein the macrophage cell line is a macrophage differentiated from the monocytic THP-1 cell line.

[0219] As used herein, the term “THP-1” refers to macrophage-like cells derived from the human monocytic leukemia cell line THP-1, typically obtained by treatment with phorbol esters such as phorbol 12-myristate 13 -acetate (PMA). The THP-1 cell line exhibits morphological and functional characteristics of human macrophages and are commonly used as an in vitro model in Research and Development.

[0220] In one aspect, the macrophage is a macrophage cell line wherein the macrophage differentiated from the monocytic THP-1 cell line. Thus, the invention further relates to a cell or population MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 of cells comprising macrophage cell line(s), preferably human macrophage cell line(s), with a memory phenotype obtained by the method of the invention.

[0221] In one aspect, the invention provides an in-vitro cell culture method, wherein the pluripotent stem cell is a hematopoietic stem and progenitor cell (hPSC) or an induced pluripotent stem cells (iPSC).

[0222] In one aspect, a pluripotent stem cell (hPSC) giving rise to one of the immune cells described herein is selected from an hematopoietic stem cell, a progenitor cell and / or an induced pluripotent stem cell (iPSC). Thus, the invention further relates to a cell or population of cells comprising an hPSC with a memory phenotype obtained by the method of the invention.

[0223] The term “memory phenotype”, as used herein, is defined as a cell state which resembles a memory T-cell at least in some aspects. The term “memory-like T-cell” is used herein interchangeably with the term “memory phenotype”. An important feature associated with a memory phenotype is the longevity of the cell. Longevity means that the cell or a progenitor survives long enough, e.g. without dividing or slowly dividing, in a subject to be able to elicit a therapeutic effect. In particular, a cell with a memory phenotype has stem cell -like properties. The longevity is preferably due to self-renewal which comprises proliferation. Self-renewal, as used herein, is not meant in a strict sense, but also includes the capacity to maintain a largely similar, although not necessarily identical, phenotype for a therapeutically relevant period of time. The self-renewal can be maintained for the entire life-time or even beyond, but it is sufficient, as used herein, if it is maintained long enough for the therapeutic purpose. A therapeutically relevant period of time means that the transferred cells or their progeny persist long enough in a subject to have a therapeutic effect.

[0224] Also encompassed in the present invention is a cell directly obtained by an in-vitro cell culture method of the invention. Non-limiting examples of a cell comprise a cell and a cell population of multipotential stem / progenitor cells such as mesenchymal stem cells, multipotential stromal cells, mesenchymal stromal cells, mesenchymal progenitor cells, and a multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR).

[0225] Also encompassed in the present invention is an immune cell selected from the group comprising a T cell, a gamma delta T cell (y8 T cell), a tumor infdtrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR-expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line or a pluripotent stem cell derived from one of these cells directly obtained by an in-vitro cell culture method of the invention.

[0226] The invention further provides an immune cell, or a population of immune cells, selected from the group comprising a T cell, a gamma delta T cell (y8 T cell), a tumor infiltrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR- expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line or a pluripotent stem cell derived from one of these cells, directly obtained by an in-vitro cell culture method of the invention.

[0227] In one aspect, the invention provides an immune cell, or a population of immune cells, of the invention, wherein the immune cell expressing a CAR is selected from the group comprising a CAR-T cell, a TRUCK T cell, an armored CAR-T cell, a CAR-NK cell, and a CAR macrophage.

[0228] In one aspect, the invention provides a carrier cell or the immune cell for chimeric antigen receptor (CAR) expression, which may include natural killer (NK) cells, including primary NK cells and NK cell lines such as NK-92, YTS, and KHYG-1; y8 T cells; natural killer T (NKT) cells, including invariant NKT (iNKT) cells; mucosal-associated invariant T (MAIT) cells; macrophages; monocytes; dendritic cells; neutrophils; hematopoietic stem cells (HSCs); mesenchymal stem cells (MSCs); induced pluripotent stem cell (iPSC)-derived immune cells, including iPSC-derived T cells, iPSC-derived NK cells, and hybrid immune cell types; basophils; eosinophils; mast cells; stromal cells; fibroblasts; and endothelial cells.

[0229] In one aspect, the immune cell obtained by an in-vitro cell culture method of the invention expresses a surface marker selected from the group comprising CD45RA+, CCR7+, CD27+, CD62L, CD127+, CD69, NKG2D, DNAM-1 and NKp46, or a combination of two or more thereof. These markers are not expressed, or to a lesser extent, on the cell surface of an immune cell not obtained by an in-vitro cell culture method of the invention. The determination and the analysis of the levels of expression of cell surface markers is part of the expertise of a person skilled in the art.

[0230] In one aspect, the invention provides an immune cell, or a population of immune cells, of the invention, wherein the immune cell expresses i) a surface marker selected from the group MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 comprising, or consisting of CD45RA+, CCR7+, CD27+, CD62L+, CD127+, CD69+, NKG2D+, DNAM-1+ and NKp46+, or a combination of two or more thereof, and / or ii) a nuclear expression of FOXO1 and TCF1, or a combination thereof.

[0231] As used herein, the notation of a “+” sign following the name of a cell surface marker, for example, “CD45RA+”, indicates that the marker is positively expressed on the surface of a cell. Specifically, a cell described as “CD45RA+” expresses detectable levels of the CD45RA protein on its plasma membrane, as determined by methods known in the art, such as flow cytometry using marker-specific antibodies. Conversely, a sign following a marker name indicates absence or lack of detectable expression of that marker on the cell surface.

[0232] As used herein, the terms " nuclear expression of " FOXO1 and TCF1 refer to the detectable presence, localization, and / or activity of one or more of these transcription factors within the nuclear compartment of a cell, wherein said nuclear expression is indicative of a particular cellular phenotype, functional state, lineage, differentiation status, pathological condition, or other biologically relevant characteristic.

[0233] For the purposes of the present invention, nuclear expression of a transcription factor may be determined by any suitable method known in the art, including but not limited to immunocytochemistry, immunofluorescence, subcellular fractionation, or reporter assays, and is considered a phenotypic marker insofar as it correlates with or defines a discernible and reproducible attribute of a cell population

[0234] As used herein, the term “CD45RA” refers to a cell surface isoform of the protein tyrosine phosphatase receptor type C (PTPRC), characterized by the inclusion of the A exon in the extracellular domain. CD45RA corresponds to isoform(s) encoded by the gene PTPRC.

[0235] The term “CCR7” refers to the C-C chemokine receptor type 7, a G-protein coupled receptor expressed as a cell surface molecule, involved in chemotactic migration toward lymphoid tissues.

[0236] The term “CD27” denotes a member of the tumor necrosis factor receptor superfamily, expressed as a cell surface molecule, playing a role in co-stimulatory signaling and cell survival.

[0237] As used herein, “CD62L” refers to L-selectin, a cell adhesion molecule involved in lymphocyte homing to peripheral lymph nodes, expressed as a cell surface molecule. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0238] As used herein, “CD 127” refers to the alpha subunit of the interleukin-7 receptor (IL7Ra), a cytokine receptor expressed as a cell surface molecule, critical for T cell development and homeostasis.

[0239] As used herein, “CD69” is an early activation marker expressed as a cell surface molecule, functioning as a C-type lectin receptor involved in immune regulation.

[0240] As used herein, “NKG2D” refers to a type II transmembrane C-type lectin-like activating receptor expressed as a cell surface molecule, encoded by the KLRK1 gene, which recognizes stress-induced ligands on target cells and mediates immune activation.

[0241] As used herein, “DNAM-1 ” (DNAX accessory molecule-1) refers to a transmembrane glycoprotein of the immunoglobulin superfamily expressed as a cell surface molecule, involved in adhesion and activation through interaction with ligands CD112 and CD155.

[0242] As used herein, “NKp46” (natural cytotoxicity triggering receptor 1, NCR1) refers to a type I transmembrane activating receptor expressed as a cell surface molecule, which mediates recognition and killing of target cell.

[0243] As used herein, "FOXO 1 " (Forkhead box protein 01) refers to a transcription factor belonging to the FOXO subfamily, which is involved in the regulation of gene expression related to cell cycle control, apoptosis, metabolism, and oxidative stress response.

[0244] As used herein, "TCF1" (T cell factor 1) refers to a transcription factor encoded by the TCF7 gene, which functions in the Wnt / -catenin signaling pathway and is involved in T cell development and the regulation of immune responses.

[0245] In one aspect, the immune cell obtained by an in-vitro cell culture method of the invention expresses a nuclear expression of FOXO 1 and TCF1, or a combination thereof.

[0246] In one aspect, the invention provides an immune cell, or a population of immune cells, of the invention, wherein the immune cell shows an increased secretion of inflammatory mediators selected from the group comprising, or consisting of granzymes, perforins, interferons, TNF, and IL-2, or a combination thereof, upon stimulation or repeated stimulation by target cells.

[0247] As used herein, the terms "increased secretion" refer to an increase in the secretion levels of inflammatory mediators in the cells produced / treated by the in vitro cell culture method of the invention, relative to a reference or control with the same cells but not produced / treated by the in vitro cell culture method of the invention. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0248] As used herein, the term "target cells" refers to cells interacting and thereby stimulating the cell, or a population of cells, of the invention. Target cells can refer to the primary stimulators of CAR-T cells, which are tumor or target cells that express the specific antigen recognized by the CAR, leading to T cell activation and cytotoxic response. Additional interactions occur with antigen-presenting cells such as dendritic cells and macrophages, which, although not required for antigen presentation, can enhance CAR-T function through co -stimulatory signals and cytokine secretion. Other cell types, including endothelial cells, fibroblasts, and various immune cells, can influence CAR-T cell trafficking, activation, or suppression within the tumor microenvironment through direct contact or by modulating local immune conditions. In one aspect, the immune cell obtained by an in-vitro cell culture method of the invention shows an increased secretion of inflammatory mediators selected from the group comprising granzymes, perforins, interferons, TNF, and IL-2, or a combination of two or mor thereof, upon stimulation or repeated stimulation by target cells, when compared to the secretion level of these inflammatory mediators in an immune cell not obtained by an in-vitro cell culture method of the invention.

[0249] In one aspect, the invention further provides a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, selected from the group comprising, or consisting of a mesenchymal stem cell, a multipotential stromal cell, a mesenchymal stromal cell, a mesenchymal progenitor cell, and a multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR), directly obtained by an in-vitro cell culture method of the invention.

[0250] As used herein, the terms " Multipotential stem cells " or "multipotent stem cells" refer to undifferentiated cells capable of self-renewal and differentiation into multiple, but lineage- restricted, cell types within a specific germ layer or tissue system. Subcategories of multipotential stem cells include hematopoietic stem cells, which give rise to all blood lineages; mesenchymal stem cells, capable of differentiating into osteoblasts, chondrocytes, and adipocytes; neural stem cells, which generate neurons and glial cells; and epithelial stem cells, such as those residing in the skin, cornea, and intestinal crypts.

[0251] As used herein, the terms "multipotential progenitor cell" include, but is not limited to common myeloid progenitors and common lymphoid progenitors, which give rise to hematopoietic lineages; mesenchymal progenitor cells committed to osteogenic, chondrogenic, or adipogenic fates; neural progenitor cells differentiating into neurons, astrocytes, or oligodendrocytes; as MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 well as tissue-specific progenitors such as hepatic progenitor cells, pancreatic progenitor cells, and endothelial progenitor cells. These multipotential progenitor cell exhibit reduced selfrenewal compared to stem cells but retain multilineage differentiation potential within their respective lineages.

[0252] In one aspect, the invention provides a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, of the invention, wherein the multipotential stem / progenitor cell expresses i) a surface marker selected from the group comprising, or consisting of CD73+, CD90+, and CD105+, or a combination of two or more thereof, and / or ii) a nuclear expression of a transcription factor selected from the group comprising, or consisting of RUNX2, SOX9, and PPARy, or a combination of two or more thereof, and wherein the multipotential stem / progenitor cell does not express a surface marker selected from the group comprising, or consisting of CD34-, CD45-, and HLA-DR-, or a combination of two or more thereof.

[0253] In one aspect, the multipotential stem / progenitor cell of the invention comprises a specific phenotypic profile of the mesenchymal lineage potential characterized by the expression of: i) a surface marker selected from the group comprising, or consisting of CD73+, CD90+, and CD105+, or a combination of two or more thereof, ii) a nuclear expression of a transcription factor selected from the group comprising, or consisting of RUNX2, SOX9, and PPARy, or a combination of two or more thereof, and wherein the multipotential stem / progenitor cell does not express a surface marker selected from the group comprising, or consisting of CD34-, CD45-, and HLA-DR-, or a combination of two or more thereof.

[0254] In one aspect, the invention provides a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, of the invention, wherein the multipotential stem / progenitor cell expresses a surface marker selected from the group comprising, or consisting of CD 10+ and CD146+, or a combination thereof.

[0255] As used herein, CD10, also known as Neprilysin, is atype II integral membrane zinc -dependent metallopeptidase that functions in the degradation of a variety of physiologically active peptides through its endopeptidase activity, thereby regulating processes such as blood pressure, immunomodulation, inflammation (anti-inflammatory), and tissue remodeling.

[0256] As used herein, CD 146, also known as Melanoma Cell Adhesion Molecule (MCAM), is a cell surface glycoprotein belonging to the immunoglobulin superfamily that mediates calcium- MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 independent cell-cell adhesion and plays a key role in processes such as angiogenesis, immunomodulation, inflammation (anti-inflammatory) and tumor progression.

[0257] In one aspect, the invention provides cell surface markers and / or nuclear expression markers used for phenotypic characterization (or phenotypic profiling) of cells, which may refer to the identification and analysis of observable cellular traits, including morphology, protein expression, surface markers, and / or functional behaviors. It is used to classify cells based on their state, type, or response to specific conditions or treatments.

[0258] As used herein, the term " CD73" refers to ecto-5 '-nucleotidase (NT5E) and is a human cell surface enzyme that hydrolyzes extracellular AMP to adenosine, thereby modulating purinergic signaling and immune responses.

[0259] As used herein, the term " CD90" refers to Thy-1 membrane glycoprotein and is a human cell surface protein involved in cell -cell and cell-matrix interactions, particularly in the regulation of adhesion, migration, and signal transduction in various cell types including stem cells and neurons.

[0260] As used herein, the term "CD 105" refers to endoglin and is a human transmembrane glycoprotein that functions as part of the TGF-P receptor complex and plays a critical role in angiogenesis, vascular development, and endothelial cell function.

[0261] As used herein, the term "RUNX2" refers to Runt-related transcription factor 2 and is a human transcription factor essential for osteoblast differentiation and skeletal development, acting as a key regulator of bone gene expression.

[0262] As used herein, the term "SOX9" refers to transcription factor SOX-9 and is a human transcription factor that plays a crucial role in chondrogenesis, sex determination, and regulation of embryonic development by controlling the expression of genes involved in cell differentiation and lineage commitment.

[0263] As used herein, the term "PPARy" refers to peroxisome proliferator-activated receptor gamma and is a human nuclear receptor that regulates adipocyte differentiation, lipid metabolism, and insulin sensitivity by modulating the expression of target genes in response to ligand binding.

[0264] As used herein, the term "CD34" refers the hematopoietic progenitor cell antigen CD34 and is a human cell surface glycoprotein that serves as a marker of hematopoietic stem and progenitor MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 cells and is involved in cell adhesion, migration, and signal transduction within the vascular and immune systems.

[0265] As used herein, the term "CD45" refers to the protein tyrosine phosphatase receptor type C (PTPRC) and is a human transmembrane protein tyrosine phosphatase expressed on all nucleated hematopoietic cells that functions as a key regulator of antigen receptor signaling and immune cell activation.

[0266] As used herein, the term "HLA-DR" refers to a human major histocompatibility complex (MHC) class II cell surface receptor involved in the presentation of antigenic peptides to helper T cells, thereby playing a central role in the adaptive immune response.

[0267] The invention further provides a composition comprising, or consisting of an immune cell, or a population of immune cells, of the invention or a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, of the invention.

[0268] The invention further provides a pharmaceutical composition comprising, or consisting of an immune cell, or population of immune cells, of the invention or a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, of the invention, and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0269] In one aspect, the invention provides an immune cell, or population of immune cells, of the invention, for use in the treatment and / or prevention of a disease selected from the group comprising a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease and / or an autoimmune disease.

[0270] In one aspect, the invention provides a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, of the invention, for use in the treatment and / or prevention of a disease selected from the group comprising a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease and / or an autoimmune disease.

[0271] In one aspect, the invention provides a pharmaceutical composition of the invention, for use in the treatment and / or prevention of a disease selected from the group comprising a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease and / or an autoimmune disease.

[0272] The invention also provides an in-vitro cell culture method comprising a step of contacting a cell, or population of cells, with a compound selected from a compound that MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 inhibits alpha-ketoglutarate -dependent enzymes, that could lead to the inhibition of prolyl hydroxylases, thereby stabilizing HIF1 and / or HIF2, or a compound that inhibits histone and / or DNA demethylases (e.g. daprodustat, desidustat, enarodustat, molidustat, roxadustat (FG- 4592), vadadustat, dimethyloxallyl glycine (DMOG), FG-2216, IOX4, JNJ-42041935, MK- 8617, S-2-hydroxyglutarate, fumarate, succinate, malate, deferasirox, deferoxamine, N- oxaloylglycine, N-oxalyl-2S-alanine, Alahopcin, dihydroxybenzoic acid, diazen-l-ium-1,2- diolate, diethylamine NONOate, Tilorone, a Factor inhibiting HIF (FIH) inhibitors, Von Hippel-Lindau (VHL) inhibitors or HIF allosteric agonists).

[0273] In one aspect, the invention provides an in-vitro cell culture method comprising a step of contacting a cell, or population of cells, with a compound selected from FG-2216, roxadustat (FG-4592), vadadustat, dimethyloxallyl glycine (DMOG) and MK-8617, or a combination thereof.

[0274] In one aspect, the invention provides an in-vitro cell culture method comprising a step of contacting a cell, or population of cells, with a compound selected from FG-2216 and roxadustat (FG-4592), or a combination thereof.

[0275] In one aspect, the invention provides an in-vitro cell culture method comprising a step of contacting a cell, or population of cells, with FG-2216.

[0276] In one aspect, the invention provides an in-vitro cell culture method comprising a step of contacting a cell, or population of cells, with roxadustat (FG-4592).

[0277] In one aspect, the invention provides an in-vitro cell culture method comprising a step of contacting a cell, or population of cells, with vadadustat.

[0278] In one aspect, the invention provides an in-vitro cell culture method comprising a step of contacting a cell, or population of cells, with dimethyloxallyl glycine (DMOG).

[0279] In one aspect, the invention provides an in-vitro cell culture method comprising a step of contacting a cell, or population of cells, with MK-8617.

[0280] The invention also provides an in-vitro cell culture method comprising, or consisting of a step of contacting a cell, or population of cells, with a compound selected from a compound that inhibits alpha-ketoglutarate -dependent enzymes, that could lead to the inhibition of prolyl hydroxylases, thereby stabilizing HIF1 and / or HIF2, and / or MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 a compound that inhibits histone and / or DNA demethylases (e.g. daprodustat, desidustat, enarodustat, molidustat, roxadustat, vadadustat, dimethyloxallyl glycine, FG-2216, FG-4592, IOX4, JNJ-42041935, MK-8617, S-2-hydroxyglutarate, fumarate, succinate, malate, deferasirox, deferoxamine, N-oxaloylglycine, N-oxalyl-2S-alanine, Alahopcin, dihydroxybenzoic acid, diazen-l-ium-l,2-diolate, diethylamine NONOate, Tilorone, a Factor inhibiting HIF (FIH) inhibitors, Von Hippel-Lindau (VHL) inhibitors and HIF allosteric agonists), or a combination thereof.

[0281] The invention also provides a cell culture medium comprising, or consisting of an MPC inhibitor and a compound selected from a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate -depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor and an HDAC inhibitor, or a combination thereof.

[0282] In one aspect, the invention provides a cell culture medium comprising, or consisting of an MPC inhibitor and a prolyl hydroxylase inhibitor.

[0283] In one aspect, the invention provides a cell culture medium comprising, or consisting of an MPC inhibitor and a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein.

[0284] In one aspect, the invention provides a cell culture medium comprising, or consisting of an MPC inhibitor and a small molecule or metabolite inhibiting alpha-ketoglutarate -depending enzymes.

[0285] In one aspect, the invention provides a cell culture medium comprising, or consisting of an MPC inhibitor and an isocitrate dehydrogenase 2 (IDH2) inhibitor.

[0286] In one aspect, the invention provides a cell culture medium comprising, or consisting of an MPC inhibitor and an HDAC inhibitor.

[0287] In one aspect, the invention provides a cell culture medium comprising an MPC inhibitor selected from UK-5099, MITO-66 (3-(benzylsulfanyl)-5-propyl[l,2,4]triazolo[4,3- a]pyrimidin-7(8H)-one), 7ACC2 and MSDC-0160, or a combination thereof and a compound selected from Roxadustat, DMOG, MK-8617, FG-2216 and Vadadustat, or a combination thereof. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0288] In one aspect, the invention provides a cell culture medium comprising UK-5099 and Roxadustat.

[0289] In one aspect, the invention provides a cell culture medium comprising UK-5099 and DMOG.

[0290] In one aspect, the invention provides a cell culture medium comprising UK-5099 and FG-2216.

[0291] In one aspect, the invention provides a cell culture medium comprising UK-5099 and MK- 8617.

[0292] In one aspect, the invention provides a cell culture medium comprising UK-5099 and Vadadustat.

[0293] In one aspect, the invention provides a cell culture medium comprising MITO-66 (3- (benzylsulfanyl)-5-propyl[l,2,4]triazolo[4,3-a]pyrimidin-7(8H)-one) and Roxadustat.

[0294] In one aspect, the invention provides a cell culture medium comprising MITO-66 (3- (benzylsulfanyl)-5 -propyl [ 1 ,2,4]triazolo [4,3 -a]pyrimidin-7(8H)-one) and DMOG.

[0295] In one aspect, the invention provides a cell culture medium comprising MITO-66 (3- (benzylsulfanyl)-5 -propyl [ 1 ,2,4]triazolo [4,3 -a]pyrimidin-7(8H)-one) and FG-2216.

[0296] In one aspect, the invention provides a cell culture medium comprising MITO-66 (3- (benzylsulfanyl)-5 -propyl [ 1 ,2,4]triazolo [4,3 -a]pyrimidin-7(8H)-one) and MK-8617.

[0297] In one aspect, the invention provides a cell culture medium comprising MITO-66 (3- (benzylsulfanyl)-5 -propyl [ 1 ,2,4]triazolo [4,3 -a]pyrimidin-7(8H)-one) and Vadadustat.

[0298] In one aspect, the invention provides a cell culture medium comprising 7ACC2 and Roxadustat.

[0299] In one aspect, the invention provides a cell culture medium comprising 7ACC2 and DMOG.

[0300] In one aspect, the invention provides a cell culture medium comprising 7ACC2 and FG-2216.

[0301] In one aspect, the invention provides a cell culture medium comprising 7ACC2 and Vadadustat.

[0302] In one aspect, the invention provides a cell culture medium comprising 7ACC2 and MK-8617.

[0303] In one aspect, the invention provides a cell culture medium comprising MSDC-0160 and Roxadustat.

[0304] In one aspect, the invention provides a cell culture medium comprising MSDC-0160 and Vadadustat. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0305] In one aspect, the invention provides a cell culture medium comprising MSDC-0160 and DMOG.

[0306] In one aspect, the invention provides a cell culture medium comprising MSDC-0160 and FG- 2216.

[0307] In one aspect, the invention provides a cell culture medium comprising MSDC-0160 and MK- 8617.

[0308] As used herein, the term "cell culture medium" refers to a nutrient-rich solution designed to support the growth, survival, and function of cells in vitro. It typically contains a balanced mixture of amino acids, vitamins, salts, glucose, and other essential nutrients, and may be supplemented with the molecules of the invention, growth factors, hormones, or serum depending on the cell type. In one aspect, the invention provides a cell culture medium comprising, or consisting of an MPC inhibitor and a compound selected from a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate -depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor and an HDAC inhibitor, or a combination thereof, for use in the in-vitro cell culture method of the invention.

[0309] Also encompassed in the present invention is an in-vitro cell culture method comprising a step of contacting an immune cell with a compound that stabilizes HIF1 and / or HIF2, such as prolyl hydroxylase inhibitors (daprodustat, desidustat, enarodustat, molidustat, roxadustat, vadadustat, dimethyloxallyl glycine), Factor inhibiting HIF (FIH) inhibitors, Von Hippel-Lindau (VHL) inhibitors or HIF allosteric agonists.

[0310] Also encompassed in the present invention is an in-vitro cell culture method comprising a step of contacting an immune cell with a compound that inhibits enzymes that depend on the cofactor alpha-ketoglutarate for their enzymatic function, such as prolyl hydroxylases, histone and DNA methylases etc. In one aspect, the in-vitro cell culture method comprises a step of contacting an immune cell with a compound that inhibits alpha-ketoglutarate-dependent enzymes, that could lead to the inhibition of prolyl hydroxylases, thereby stabilizing HIF1 and / or HIF2, or inhibition of histone and / or DNA demethylases (daprodustat, desidustat, enarodustat, molidustat, Roxadustat (FG-4592) (CAS#808118-40-3), vadadustat

[0311] (CAS# 1000025 -07-9), dimethyloxallyl glycine (DMOG) (CAS# 89464-63-1), FG-2216 (CAS#223387-75-5), IOX4 (CAS#1154097-71-8), JNJ-42041935 (CAS#1193383-09-3), MK- 8617 (CAS# 1187990-87-9), S-2-hydroxyglutarate, fumarate, succinate, malate, deferasirox, MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 deferoxamine, N-oxaloylglycine, N-oxalyl-2S-alanine, Alahopcin, dihydroxybenzoic acid, diazen-l-ium-l,2-diolate, diethylamine NONOate, Tilorone), Factor inhibiting HIF (FIH) inhibitors, Von Hippel-Lindau (VHL) inhibitors or HIF allosteric agonists. S-2- hydroxyglutarate, fumarate, succinate, malate, deferasirox, deferoxamine, N-oxaloylglycine, N-oxalyl-2S-alanine, Alahopcin, dihydroxybenzoic acid, diazen-l-ium-l,2-diolate, diethylamine NONOate, Tilorone), Factor inhibiting HIF (FIH) inhibitors, Von Hippel-Lindau (VHL) inhibitors or HIF allosteric agonists.

[0312] The above targets can be inhibited by either a small molecule, RNA interference, antibody- mediated blockade or genetic deletion.

[0313] As used herein, the term "RNA interference (RNAi)" includes but is not limited to: small interfering RNA (siRNA) molecules, including chemically synthesized, in vitro transcribed, or vector-expressed double -stranded RNA capable of inducing sequence-specific degradation of target mRNA; short hairpin RNA (shRNA) molecules, comprising RNA sequences that form a stem-loop structure and are processed intracellularly into active siRNA species; microRNA (miRNA) molecules, whether endogenous or synthetic, that modulate gene expression through partial base-pairing with target mRNAs; Dicer-substrate RNA (dsRNA) molecules, typically 25-30 nucleotides in length, which are substrates for Dicer-mediated processing; antisense oligonucleotides that inhibit gene expression via recruitment of the RNA-induced silencing complex (RISC) or through RNase H-mediated degradation; chemically modified or artificial RNAi molecules, including but not limited to 2’-O-methyl, locked nucleic acid (LNA), or phosphorothioate-modified oligonucleotides; and RNAi-expressing vectors, such as plasmids or viral vectors, comprising nucleotide sequences encoding shRNA or miRNA operably linked to regulatory elements suitable for expression in a target cell.

[0314] As used herein, the terms " antibody-mediated blockade", refer to full-length immunoglobulin molecules as well as antigen-binding fragments and engineered derivatives capable of specifically binding to and inhibiting a target molecule. The terms "antibody-mediated blockade" include monoclonal, polyclonal, chimeric, humanized, and fully human antibodies, as well as functional fragments such as Fab, F(ab')2, Fv, single-chain variable fragments (scFv), and single-domain antibodies (sdAb or nanobodies). The terms "antibody-mediated blockade" as used herein, also include antibody-drug conjugates (ADCs), comprising antibodies linked to cytotoxic or functional agents. For intracellular targets, the use of such antibodies (for antibody-mediated blockade) is feasible provided that they are capable of entering cells under MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 in vitro culture conditions. Cellular entry may be achieved through direct conjugation to cellpenetrating peptides, encapsulation in liposomes or nanoparticles, use of electroporation, or other suitable delivery systems known in the art.

[0315] Alternatively, wildtype or degradation-resistent HIF can be overexpressed in T cells.

[0316] In one aspect, the above in vitro method as defined in the invention can be combined in coculture with an "MPC inhibitor" (including, but not limited to UK-5099, MITO-66, 7ACC2, MSDC-0160) and / or with a compound selected from a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate-depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HDAC inhibitor, or a combination thereof.

[0317] Also encompassed in the present invention is a composition comprising a cell, preferably an immune cell described herein.

[0318] Also provided herein are pharmaceutical compositions.

[0319] In one aspect, the pharmaceutical composition comprises, or consists of, a cell, preferably an immune cell of the invention, and pharmaceutically acceptable carrier, diluent and / or excipient.

[0320] In one aspect, the pharmaceutical composition is for use in the treatment and / or prevention of disease. Preferably, the disease is selected from a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease.

[0321] Where the disease to be treated is an inflammatory or inflammation-induced disease, it is selected from the non-limiting group comprising fibrosis (e.g. lung, renal, or cardiac fibrosis), chronic obstructive pulmonary disease, cardiovascular diseases, diabetes, asthma, fatty liver disease, gout, and scleroderma, or a combination thereof.

[0322] Where the disease to be treated is a chronic disease, it is selected from the non-limiting group comprising fibrosis, Alzheimer disease, lupus erythematosus, and chronic kidney disease or a combination thereof.

[0323] Where the disease to be treated is an infectious disease, it is selected from the non-limiting group comprising HIV, Hepatitis C and Human Cytomegalovirus or a combination thereof.

[0324] Where the disease to be treated is cancer, it is selected from a solid cancer or a liquid cancer. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0325] Where the cancer to be treated is a solid cancer, it is selected from the non-limiting group comprising lung cancer, breast cancer, ovarian cancer, cervical cancer, uterus cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal carcinoma, kidney cancer, prostate cancer, gastric cancer, bronchus cancer, pancreatic cancer, urinary bladder cancer, hepatic cancer and brain cancer and skin cancer, in particular melanoma, or a combination thereof.

[0326] Where the cancer to be treated is a liquid cancer, it refers to cancer cells that are present in body fluids, such as blood, lymph and bone marrow. Liquid cancer is selected from the nonlimiting group comprising leukemia, myeloma, myelodysplastic syndrome (MDS), and liquid lymphomas. For example, liquid cancer can be acute myeloid leukemia (AML). Liquid lymphomas include lymphomas that contain cysts or liquid areas, such as e.g. Diffuse large B cell lymphoma (DLBCL), or Follicular lymphoma.

[0327] Where the disease to be treated is an autoimmune disease, it is selected from the non-limiting group comprising rheumatoid arthritis (RA), multiple sclerosis (MS), endometriosis, inflammatory bowel disease (IBD), psoriasis, and psoriatic arthritis, or a combination thereof.

[0328] As used herein the terms "subject" / "subject in need thereof, or "patient" / "patient in need thereof " are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some cases, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other aspects, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. Preferably, the subject is a human, most preferably a human that suffer or might be at risk of suffering from a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease.

[0329] The term "therapeutically effective amount" as used herein means an amount of an immune cell high enough to significantly positively modify the symptoms and / or condition to be treated, but low enough to avoid serious side effects (at a reasonable risk / benefit ratio), within the scope of sound medical judgment.

[0330] In certain aspects, the in-vitro cell culture method of the invention comprises the use of the following concentrations of compounds of the invention ranging from 0.1 pM to 100 pM. or MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 even higher. For example, in-vitro cell culture method of the invention comprises the use of the following concentrations of compounds of the invention selected from:

[0331] From 0.1 pM to 1.0 pM, including specifically 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM, and 1.0 pM;

[0332] From 2 pM to 10 pM, including specifically 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, and 10 pM;

[0333] From 10 pM to 100 pM, including specifically 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, and 100 pM.

[0334] In some aspects, concentrations exceeding 100 pM may be used, for example, from 100 pM to 500 pM, or from 100 pM to 1000 pM, depending on the therapeutic context and drug tolerability.

[0335] All numerical ranges provided herein are intended to include the endpoints as well as all intermediate values to the level of precision indicated.

[0336] The therapeutically effective amount of a host cell, e.g. an immune cell, as described herein is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient or subject; the severity of the condition or disease (e.g. cancer, infection or autoimmune disease ) to be treated; the route of administration; the renal and hepatic function of the patient or subject. A physician of ordinary skill in the art can readily determine and prescribe the effective amount of the host cell required to prevent, counter or arrest the progress of the disease, such as e.g. a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease.

[0337] “Pharmaceutically acceptable carrier or diluent” means a carrier or diluent that is useful in preparing pharmaceutical compositions that is generally safe, non-toxic, and desirable, and includes carriers or diluents that are acceptable for human pharmaceutical use.

[0338] Such pharmaceutical compositions, comprising the cells of the invention, may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0339] Any of the compositions, comprising the cells of the invention, provided herein can be provided in any appropriate pharmaceutical composition and be administered by any suitable route of administration. Suitable routes of administration include, but are not limited to, inhalation, intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary, and subcutaneous routes. Pharmaceutical compositions of the present invention are preferably formulated for intravenous administration.

[0340] The pharmaceutical compositions (solutions, suspensions or the like), comprising the cells of the invention, may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.

[0341] The pharmaceutical compositions, comprising the cells of the invention, can further comprise at least one additional therapeutic agent or therapy. A variety of other additional therapeutic agents may be used in conjunction with the compositions described herein.

[0342] In one aspect, said at least one additional therapeutic agent or therapy is an anticancer agent or anticancer therapy, useful to treat a cancer. Preferably, the one or more anti-cancer therapy will be selected from the group comprising radiotherapy, chemotherapy, immune checkpoint inhibitor, immunotherapy and hormone therapy, or a combination of one of more thereof.

[0343] Preferably, the immune checkpoint inhibitor is selected from the non-limiting group a CTLA- 4 inhibitor, a TIM3 inhibitor, a TIGIT inhibitor, a PD-1 inhibitor, and a PD-L1 inhibitor or a combination of one or more thereof, e.g. PD-1 / PD-L1 inhibitor or TIM3 / PD-1 / PD-L1 inhibitor.

[0344] For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0345] For example, potentially useful additional therapeutic agents include PD-L1 inhibitors such as atezolizumab, avelumab, AMP-224, MEDI-0680, RG-7446, GX-P2, durvalumab, KY-1003, KD-033, MSB-0010718C, TSR-042, ALN-PDL, STI-A1014, CX-072, and BMS-936559.

[0346] Non-limiting examples of CTLA-4 inhibitors include ipilimumab (Yervoy) (also known as BMS-734016, MDX-010, MDX-101) and tremelimumab (formerly ticilimumab, CP-675,206).

[0347] A chemotherapy of the present invention can concern agents that damage DNA and / or prevent cells from multiplying, such as genotoxins.

[0348] Genotoxins can be selected from the group comprising alkylating agents, antimetabolites, DNA cutters, DNA binders, topoisomerase poisons and spindle poisons. Examples of alkylating agents are lomustine, carmustine, streptozocin, mechlorethamine, melphalan, uracil nitrogen mustard, chlorambucil, cyclosphamide, iphosphamide, cisplatin, carboplatin, mitomycin, thiotepa, dacarbazin, procarbazine, hexamefhyl melamine, triethylene melamine, busulfan, pipobroman, mitotane and other platine derivatives.

[0349] An example of DNA cutters is bleomycin.

[0350] Topoisomerases poisons can be selected from the group comprising topotecan, irinotecan, camptothecin sodium salt, daorubicin, doxorubicin, idarubicin, mitoxantrone teniposide, adriamycin and etoposide.

[0351] Examples of DNA binders are dactinomycin and mithramycin whereas spindle poisons can be selected among the group comprising vinblastin, vincristin, navelbin, paclitaxel and docetaxel.

[0352] A chemotherapy of the present invention can concern antimetabolites selected among the following coumpounds: methotrexate, trimetrexate, pentostatin, cytarabin, ara-CMP, fludarabine phosphate, hydroxyurea, fluorouracyl, fioxuridine, chlorodeoxyadenosine, gemcitabine, thioguanine and 6-mercaptopurine.

[0353] Radiotherapy refers to the use of high-energy radiation to shrink tumors and kill cancer cells. Examples of radiation therapy include, without limitation, external radiation therapy and internal radiation therapy (also called brachytherapy).

[0354] External radiation therapy is most common and typically involves directing a beam of direct or indirect ionizing radiation to a tumor or cancer site. While the beams of radiation, the photons, the Cobalt or the particule therapy are focused to the tumor or cancer site, it is nearly impossible MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 to avoid exposure of normal, healthy tissue. Energy source for external radiation therapy is selected from the group comprising direct or indirect ionizing radiation (for example: x-rays, gamma rays and particle beams or combination thereof).

[0355] Internal radiation therapy involves implanting a radiation-emitting source, such as beads, wires, pellets, capsules, etc., inside the body, at, or near to the tumor site. Energy source for internal radiation therapy is selected from the group of radioactive isotopes comprising: iodine (iodine 125 or iodine 131), strontium89, radioisotopes of phosphorous, palladium, cesium, indium, phosphate, or cobalt, and combination thereof. Such implants can be removed following treatment, or left in the body inactive. Types of internal radiation therapy include, but are not limited to, interstitial, and intracavity brachytherapy (high dose rate, low dose rate, pulsed dose rate).

[0356] A currently less common form of internal radiation therapy involves biological carriers of radioisotopes, such as with radio -immunotherapy wherein tumor-specific antibodies bound to radioactive material are administered to a patient or subject. The antibodies bind tumor antigens, thereby effectively administering a dose of radiation to the relevant tissue.

[0357] Methods of administering radiation therapy are well known to those of skill in the art.

[0358] A variety of other additional therapeutic agents may be used in conjunction with the compositions described herein. These additional therapeutic agents may be administered before, concomitantly and / or after administration of the immune cells obtained according to a method of the invention.

[0359] Additional therapeutic agents suitable for use in combination with the invention include, but are not limited to, ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (KADCYLA®), imatinib (Gleevec®), cetuximab (Erbitux®), panitumumab (Vectibix®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, mTOR inhibitors such as Everolimus and MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0360] Temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, CDK inhibitors such as CDK inhibitor (palbociclib).

[0361] In additional aspects, the additional therapeutic agent can be an anti-inflammatory agent. Antiinflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and my cophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox- 2 inhibitors, and sialylates. Exemplary analgesics include acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists, (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitors and adhesion molecule inhibitors. The biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofm) and intramuscular) and minocycline.

[0362] The present invention further contemplates methods of treating and / or preventing a disease, preferably the disease is selected from a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease.

[0363] The invention further provides a method of treating and / or preventing a disease, comprising (i) providing a cell, or population of cells, (ii) expanding and culturing ex vivo or in vitro into a larger population of cells according to the in-vitro cell culture method of the invention, and (iii) introducing (i.e. administering) said cell, or population of cells, into the patient or subject in need thereof. providing a cell, or population of cells:

[0364] As used herein, the terms "providing a cell, or population of cells" refer to providing a biological specimen comprising one or more cells, which may be isolated, cultured, or present within a mixed population. The cell, or population of cells of the invention may originate from MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 a human or non -human animal subject, including but not limited to a patient or a healthy individual, and may be obtained from a tissue biopsy, bodily fluid (e.g., blood, urine, cerebrospinal fluid, or saliva), swab, lavage, or other clinical specimen.

[0365] The cell, or population of cells of the invention may alternatively comprise cells derived from an established or primary cell line, including immortalized cell lines, genetically modified cells, stem cells (e.g., pluripotent, multipotent, or induced pluripotent stem cells), progenitor cells, or differentiated cells obtained through in vitro differentiation protocols.

[0366] In one aspect, the cell, or population of cells of the invention is selected from: i) an immune cell, or a population of immune cells, selected from the group comprising a T cell, a gamma delta T cell (y3 T cell), a tumor infiltrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR-expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line derived from one of these cells or a pluripotent stem cell giving rise to one of these cells, or ii) a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, selected from the group comprising a mesenchymal stem cell, a multipotential stromal cell, a mesenchymal stromal cell, a mesenchymal progenitor cell, and a multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR).

[0367] In one aspect, the cell, or population of cells of the invention can originate from the same patient / subject or from one or more patients / subjects different from the one patient / subject that will receive (that is to be administered) the cell, or population of cells of the invention. expanding and culturing ex vivo or in vitro into a larger population of cells according to the in-vitro cell culture method of the invention:

[0368] As used herein, the terms " expanding and culturing ex vivo or in vitro " refer to a population of cells maintained or propagated outside of a living organism under controlled environmental conditions. Such cultures involve the growth, maintenance, or manipulation of cells in an artificial medium (or the cell culture medium of the invention) that supports cellular viability and function. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0369] As used herein, the term "ex vivo" specifically denotes cells or tissues that have been isolated from an organism and are maintained in a viable state outside the organism for experimental, therapeutic, diagnostic, or research purposes. As used herein, the term "in vitro" refers more generally to any culture of cells performed outside of their natural biological environment, typically in a laboratory setting.

[0370] The cell culture of the invention may be conducted under static or dynamic conditions and may involve two-dimensional (2D) or three-dimensional (3D) systems or organoid systems. The culture environment may be adapted to support proliferation, differentiation, activation, preservation, or analysis of the cells, and may optionally include co-culture systems, perfusion systems, or microphysiological platforms.

[0371] In one aspect, step (ii) of the method of treating and / or preventing a disease comprises a step of contacting a cell, or a population of cells, with a mitochondrial pyruvate carrier (MPC) inhibitor, and with a compound selected form a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate -depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HDAC inhibitor, or a combination thereof. introducing (i.e. administering) said cell, or population of cells, into the patient or subject in need thereof:

[0372] As used herein, the terms "introducing" or "administration" refer to suitable routes of administration of the cell, or population of cells of the invention including, but are not limited to, inhalation, intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary, and subcutaneous routes.

[0373] In one aspect, the cell, or population of cells of the invention, to be introduced (administered) into the patient, can originate from the same patient / subject or from one or more patients / subjects different from the one patient / subject that receives (that is to be administered) the cell, or population of cells of the invention.

[0374] In one aspect, the invention provides a method of treating and / or preventing a disease, wherein the cell is a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, selected from the group comprising, or consisting of a mesenchymal MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 stem cell, a multipotential stromal cell, a mesenchymal stromal cell, a mesenchymal progenitor cell, and a multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR).

[0375] In one aspect, the invention provides a method of treating and / or preventing a disease, wherein the cell is an immune cell, or a population of immune cells, selected from the group comprising, or consisting of a T cell, a gamma delta T cell (y8 T cell), a tumor infiltrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR-expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line derived from one of these cells or a pluripotent stem cell giving rise to one of these cells.

[0376] In one aspect, the method of treating and / or preventing a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease comprises (i) providing genetically engineered immune cells with one recombinant construct encoding a chimeric antigen receptor (CAR), (ii) expanding and culturing ex vivo or in vitro into a larger population of engineered immune cells according to the invention and (iii) reintroducing (i.e. administering) said engineered immune cells into the patient or subject in need thereof.

[0377] In one aspect, the method of treating and / or preventing a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease comprises (i) providing genetically engineered immune cells with one recombinant construct encoding a chimeric antigen receptor (CAR), (ii) expanding and culturing ex vivo or in vitro into a larger population of engineered immune cells according to the invention and (iii) introducing or reintroducing (i.e. administering) said engineered immune cells into the patient or subject in need thereof.

[0378] In one aspect, the invention provides a method of treating and / or preventing a disease, wherein the immune cell, or the population of immune cells is genetically engineered with one recombinant construct encoding a chimeric antigen receptor (CAR).

[0379] As used herein, the terms "genetically engineered" refer to cells whose genetic material has been intentionally modified by the introduction, deletion, substitution, or rearrangement of nucleic acid sequences using molecular biology techniques. The genetic modification may be achieved through the use of recombinant DNA technology, genome editing tools (e.g., CRISPR / Cas systems, zinc finger nucleases, TALENs), transposon systems, or viral or non- MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 viral vectors. The genetically engineered cells of the invention may comprise altered gene expression, the expression of heterologous and / or synthetic sequences, and / or the knockdown, knockout, and / or correction of endogenous genes. The genetic modification(s) introduced in the genetically engineered cells of the invention may be stable or transient and may be carried out ex vivo or in vitro prior to use in research, diagnostic, or therapeutic applications.

[0380] As used herein, the term “recombinant construct” refers to an artificially assembled nucleic acid molecule comprising at least two nucleic acid sequences that are not naturally contiguous. The recombinant construct of the invention may include regulatory elements (such as promoters, enhancers, polyadenylation signals, or selection markers) operably linked to a coding or non-coding sequence of interest. The recombinant construct of the invention may be in the form of a plasmid, viral vector, linear DNA, RNA molecule, or any other suitable format and may be used for the purpose of introducing genetic material into a host cell of the invention, directing gene expression, or mediating genome modification. Unless otherwise specified, the term recombinant construct includes expression constructs, gene editing constructs, and any other engineered nucleic acid molecule designed for functional use in vitro or ex vivo. The recombinant construct of the invention may be prepared according to established protocols (as mentioned in Green, M.R. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, Fourth Edition (2012)).

[0381] The term “nucleic acid” refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3' position of one nucleotide to the 5' end of another nucleotide. The nucleic acid is not limited by length, and thus the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The term nucleic acid also comprises DNA molecules including genomic DNA, comprising both coding and non-coding regions, wherein coding regions encode proteins and non-coding DNA includes introns, regulatory elements, and structural sequences such as telomeric, centromeric, and satellite DNA. Other forms of DNA include complementary DNA (cDNA) synthesized from RNA templates, mitochondrial DNA (mtDNA) and chloroplast DNA (cpDNA) of organellar origin, as well as extrachromosomal DNA (ecDNA) such as plasmids and circular DNAs, which may play roles in gene regulation, replication, or amplification. The term nucleic acid also comprises examples of RNAs or RNA molecules (included within total RNA) such as but not limited to: mRNA, amplicons, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, ncRNA (e.g. IncRNA), truncated RNA, ribozyme, riboswitch and viral RNA (e.g., retroviral RNA). MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0382] As used herein, the terms "encoding a chimeric antigen receptor (CAR)" refer to the presence of a nucleotide sequence (e.g., DNA or RNA) that, through the processes of transcription and translation, directs the biosynthesis of a specific / corresponding CAR polypeptide or protein. The sequence encodes a series of codons that correspond to the amino acid sequence of the protein according to the universal genetic code. The terms "encoding a chimeric antigen receptor (CAR)" encompass both full-length coding sequences and fragments thereof, and includes natural, synthetic, or modified sequences, provided they are capable of being expressed to produce a detectable or functional protein product under suitable conditions. Unless otherwise indicated, the terms "encoding a chimeric antigen receptor (CAR)" include sequences that encode fusion proteins, tagged proteins, or variants containing conservative substitutions, deletions, or insertions that do not abolish the protein’s intended function.

[0383] In one aspect, the chimeric antigen receptor (CAR) of the invention is selected from the group comprising, or consisting of CD19, CD20, CD22, CD33, CD70, CD123, BCMA, GD2, IL13 receptor, HER2, GPC3, PSMA, EGFR, MSLN, CLDN18.2 , or a combination thereof.

[0384] In one aspect, the invention provides a method of treating and / or preventing a disease, wherein the disease is selected from a cancer, an infectious disease, an inflammatory or inflammation- induced disease, a chronic disease or an autoimmune disease.

[0385] In one aspect, the method of treating and / or preventing a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease comprises (i) providing isolated cells, i.e. immune cells , (ii) expanding and culturing ex vivo or in vitro into a larger population of immune cells according to the method described herein and (iii) reintroducing (i.e. administering) said immune cells into the patient or subject in need thereof.

[0386] The present invention also provides a method of treating and / or preventing a disease in a patient or subject in need thereof, comprising (i) providing cells isolated from the patient or subject in need thereof, (ii) expanding and culturing ex vivo or in vitro into a larger population of cells according to the in-vitro cell culture method of the invention and (iii) reintroducing (i.e. administering) said immune cells into the patient or subject in need thereof. providing cells isolated from the patient or subject in need thereof:

[0387] As used herein, the terms "providing cells isolated from the patient or subject" refer to cells that have been obtained from a human subject, typically through a clinical or diagnostic MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 procedure such as a biopsy, blood draw, lavage, or other sampling method, and subsequently separated from their native tissue, fluid, or cellular environment. The isolation process may involve mechanical, enzymatic, or chemical steps, and may be followed by enrichment, purification, or selection procedures. Such isolated cells may be used immediately or subjected to further manipulation, such as culture, expansion, or genetic modification, and may retain or be induced to exhibit specific biological characteristics relevant to diagnostic, therapeutic, or research applications. The isolated cells of the invention may originate from a human or nonhuman animal subject, including but not limited to a patient or a healthy individual or a diseased patient, and may be obtained from atissue biopsy, bodily fluid (e.g., blood, urine, cerebrospinal fluid, or saliva), swab, lavage, or other clinical specimen.

[0388] In one aspect, the isolated cells of the invention are selected from: i) an immune cell, or a population of immune cells, selected from the group comprising a T cell, a gamma delta T cell (y8 T cell), a tumor infiltrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR-expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line derived from one of these cells or a pluripotent stem cell giving rise to one of these cells, or ii) a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, selected from the group comprising a mesenchymal stem cell, a multipotential stromal cell, a mesenchymal stromal cell, a mesenchymal progenitor cell, and a multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR).

[0389] In one aspect, the isolated cells of the invention originate from the same patient / subject from the one patient / subject that will receive (that is to be administered) the isolated cell, or population of cells of the invention. expanding and culturing ex vivo or in vitro into a larger population of cells according to the in-vitro cell culture method of the invention:

[0390] As used herein, the terms " expanding and culturing ex vivo or in vitro " refer to a population of cells maintained or propagated outside of a living organism under controlled environmental conditions. Such cultures involve the growth, maintenance, or manipulation of cells in an MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 artificial medium (or the cell culture medium of the invention) that supports cellular viability and function.

[0391] As used herein, the term "ex vivo" specifically denotes cells or tissues that have been isolated from an organism and are maintained in a viable state outside the organism for experimental, therapeutic, diagnostic, or research purposes. As used herein, the term "in vitro" refers more generally to any culture of cells performed outside of their natural biological environment, typically in a laboratory setting.

[0392] The cell culture of the invention may be conducted under static or dynamic conditions and may involve two-dimensional (2D) or three-dimensional (3D) systems or organoid systems. The culture environment may be adapted to support proliferation, differentiation, activation, preservation, or analysis of the cells, and may optionally include co-culture systems, perfusion systems, or microphysiological platforms.

[0393] In one aspect, step (ii) of the method of treating and / or preventing a disease comprises a step of contacting a cell, or a population of cells, with a mitochondrial pyruvate carrier (MPC) inhibitor, and with a compound selected form a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate -depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HDAC inhibitor, or a combination thereof. iii): reintroducing (i.e. administering) said immune cells into the patient or subject in need thereof:

[0394] As used herein, the terms "reintroducing" or "administration" refer to suitable routes of administration of the isolated cells of the invention including, but are not limited to, inhalation, intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary, and subcutaneous routes.

[0395] In one aspect, the isolated cells of the invention, to be reintroduced (administered) into the patient, originate from the same patient / subject from the one patient / subjectthat receives (that is to be administered) the isolated cells of the invention.

[0396] In one aspect, the invention provides a method of treating and / or preventing a disease in a patient or subject in need thereof, wherein the cell is a multipotential stem / progenitor cell, or a MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 population of multipotential stem / progenitor cells, selected from the group comprising a mesenchymal stem cell, a multipotential stromal cell, a mesenchymal stromal cell, a mesenchymal progenitor cell, and a multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR).

[0397] In one aspect, the invention provides a method of treating and / or preventing a disease in a patient or subject in need thereof, wherein the cell is an immune cell, or a population of immune cells, selected from the group comprising a T cell, a gamma delta T cell (y§ T cell), a tumor infiltrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR- expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line derived from one of these cells or a pluripotent stem cell giving rise to one of these cells.

[0398] In one aspect, the invention provides a method of treating and / or preventing a disease in a patient or subject in need thereof, wherein the immune cell, or the population of immune cells is genetically engineered with one recombinant construct encoding a chimeric antigen receptor (CAR).

[0399] In one aspect, the invention provides a method of treating and / or preventing a disease in a patient or subject in need thereof, wherein the disease is selected from a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease.

[0400] The invention also contemplates kits for treating and / or preventing a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease. In one aspect of the invention, the kit comprises a pharmaceutical composition of the invention.

[0401] The kits of the invention may also comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the disease of disorder of the invention and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Alternatively, or additionally, the kits may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer (such as bacteriostatic water for injection MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0402] (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution) and / or at least one additional therapeutic agent (such as e.g. a ligand of the invention). It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fdters, needles, and syringes.

[0403] The label or package insert may comprise instructions for use thereof. Instructions included may be affixed to packaging material or may be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure.

[0404] The present invention also provides a kit for treating and / or preventing a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease, the kit comprising a pharmaceutical composition of the invention.

[0405] As used herein, the kit of the present invention refers to a combination of components packaged together for use in the preparation, delivery, or administration of the pharmaceutical composition of the invention comprising living cells, wherein the cells are intended for use in the treatment, prevention, or management of a disease or medical condition in a subject. The kit of the invention may comprise one or more populations of viable cells, which may be autologous, allogeneic, or xenogeneic, and may be genetically modified, activated, expanded, or otherwise manipulated ex vivo or in vitro prior to use.

[0406] The kit of the invention may further comprise one or more of the following: a cell culture medium or supplement of the invention, a cryopreservation solution, a delivery device (e.g., syringe, catheter, or infusion system), reagents or buffers for cell washing or resuspension, instructions for preparation or administration, and optionally, agents or compositions for preconditioning the subject, tracking the cells post-administration, or enhancing the therapeutic effect. Unless otherwise specified, the components of the kit are provided in separate containers but are intended to be used in a coordinated manner for cell-based therapeutic purposes.

[0407] In one aspect, the invention provides a kit for in-vitro cell culture, the kit comprising a cell culture medium of the invention.

[0408] In one aspect, the invention provides a kit for in-vitro cell culture, the kit comprising one or more cell culture medium of the invention. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0409] In one aspect, the kit of the present invention comprises the cell culture medium of the invention, which comprises an MPC inhibitor and a compound selected from a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate- depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor and an HDAC inhibitor, or a combination thereof.

[0410] In one aspect, the invention provides a kit further comprising one or more vial(s), wherein one vial comprises the cell culture medium of the invention, wherein one vial comprises an MPC inhibitor and / or wherein one vial comprises a compound selected from a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate- depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HDAC inhibitor, or a combination thereof.

[0411] In one aspect, the invention provides a kit with three distinct vials, wherein one vial comprises the cell culture medium of the invention, and wherein one vial comprises an MPC inhibitor and wherein one vial comprises a compound selected from a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate -depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HDAC inhibitor, or a combination thereof.

[0412] In one aspect, the invention provides a kit with two distinct vials, wherein one vial comprises the cell culture medium of the invention, and wherein one vial comprises an MPC inhibitor and a compound selected from a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate-depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HDAC inhibitor, or a combination thereof.

[0413] In one aspect, the invention provides a kit with a vial, wherein the vial comprises the cell culture medium of the invention and an MPC inhibitor and a compound selected from a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate- depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HDAC inhibitor, or a combination thereof. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0414] In one aspect, the kit of the invention comprises one or more vial(s), wherein the one or more vial(s) can be mixed before and / or during the use in the in vitro cell culture method of the invention.

[0415] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991); Carey and Sundberg Advanced Organic Chemistry 3.sup.rd Ed. (Plenum Press) Vols A and B (1992).

[0416] The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

[0417] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein. Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety. The foregoing description will be more fully understood with reference to the following Examples. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0418] EXAMPLES

[0419] Material & Methods

[0420] Mice & cell lines

[0421] NOD-SCID-yc-7-(NSG) mice were purchased from the Jackson laboratory. Males and females between 6 to 10 weeks old were used for experiments. Sample size was chosen based on previous experience. Researchers were not blinded to the different treatment groups. Mice were kept in the animal facility of Agora in Lausanne in individually ventilated cages, between 19- 23 °C with 45-65% humidity and a 12-hour dark / light cycle. Experimentation was performed respecting the protocols approved by the veterinary authorities of the Canton de Vaud (VD3763b).

[0422] HEK293T, NALM6 and T2 cells were obtained from the American Type Culture Collection and were not further authenticated. All cell lines were cultured in RPMI, containing 10% fetal bovine serum (FBS), 1% Penicillin / Streptomycin (P / S). Polymerase chain reaction (PCR) testing was systematically done to confirm that cell lines were mycoplasma-free.

[0423] Peripheral blood from de -identified healthy human volunteers was obtained from the Center of Interregional Blood Transfusion SRK Bern. Peripheral blood mononuclear cells were obtained from patients with B cell malignancies (Table 1) at the Geneva University Hospital under a research protocol approved by the cantonal ethical commission for human research (CCER- 2023-02042), and after obtaining informed consent.

[0424] MITO-66 IC50 determination

[0425] 1) Bioluminescence resonance energy transfer (BRET)

[0426] BRET analyses were performed as previously described11, using HEK293T cells stably transfected with MPCl-Rluc8 and MPC2 -Venus. Cells were maintained in DMEM, 10% FBS, 1% P / S, 2mM glutamine. For the assay, cells were resuspended at 106cells / ml and 100 pl seeded in white 96 well plates (Greiner) and grown overnight. The cells were carefully washed in PBS, ImM CaCh and 0.5mM MgCh and the assay was performed in the same buffer supplemented with 5p.M Coelenterazine H, the luminescent substrate for Rluc8. The test compounds, typically a 1:3 dilution series starting at 30 .M were added, and compound- induced changes in luminescence intensity were measured overtime in a Synergy 2 plate reader (Biotek). BRET values were quantified as described previously11. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0427] 2) Seahorse determination of Oxygen Consumption Rate (OCR)

[0428] Mito stress test assays were performed in the Seahorse XFe24 Flux Analyzer (Seahorse Biosciences) using the protocols provided by the manufacturer. Briefly, Hela cells were resuspended at 2xl05 / ml and 200 pl aliquots added to each well. Cells were allowed to settle undisturbed to ensure uniform distribution, then incubated overnight at 37°C, 5% CO2. For the assay, cells were washed twice in the assay medium, PBS supplemented with ImM CaCh, 0.5mM MgCh, ImM pyruvate. After washing, 450 pl fresh assay buffer was added to each well and the plate was transferred to the XFe24 Flux Analyser, in which the injection ports had been primed with lOx reagents as follows: Port A: different MPC inhibitors; Port B: 20pM oligomycin A; Port C: lOpM FCCP; Port D: lOpM Rotenone + lOpM Antimycin. Compound potency was determined from the data following injection of the uncoupler FCCP, during which the maximal rate of oxygen consumption is measured.

[0429] Thermoshift analysis

[0430] Stock solution of purified MPC1 / MPC2 was incubated with a serial dilution of MITO-66 and resulting protein concentration was 1 1 pM. For the measurement, a nanoDSF Prometheus NT.48 device from NanoTemper Technologies GmbH (Mtinchen, Germany) with Prometheus High Sensitivity Capillaries was used. Excitation LED power was set to 100%, and fluorescence data was collected in the temperature range from 15°C to 80°C with temperature ramp of l°C / min.

[0431] Estimation of binding affinity is based on the analysis of melting temperature shift in the presence of MITO-66. Analysis was done with help of eSPC online data-analysis platform12, based on the FoldAffinity method13. Fluorescence ratio 350nm / 330nm was fit in the temperature range of 20°C - 70°C. Estimation of Kd was done by fitting a model with single binding site in the temperature range of 43°C - 47°C, with best fits being at 44.3°C. The confidence interval (marginal asymmetric confidence interval at a 95% confidence level) was estimated as suggested by Vaida Paketuryte et al14.

[0432] Human anti-CD19 CAR T cell preparation

[0433] The hCD19-28z CAR was constructed by ligating the hCD19 scFv (FMC63) into the CAR backbone sequences of the third-generation viral vector pTRPE-28z. Briefly, 95% confluent HEK293T cells were transfected with pTRPE, psPAX2 encoding gag-pol and pMD2.G encoding the VSV-G envelope, using Lipofectamine 2000 (Invitrogen). Viral supernatant was MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 collected 24 and 48h post-transfection, fdtered, concentrated by ultracentrifugation and frozen. Viral titer was determined by serial dilution transfection in T2 cells for 72 hours, followed by flow cytometry-based determination of transduction efficiency. For the generation of CAR T cells from healthy donors, T cells were enriched from peripheral blood by Rosette Sep (Stem Cell Technologies). For the generation of CAR T cells from patient samples, whole peripheral blood mononuclear cells were cultured at 105cells per lOO L in round-bottom 96 well plates. T cells were activated with human anti-CD3 / CD28 Dynabeads (Gibco) and cultured in RPMI supplemented with 10% FBS, ImM HEPES, 1% Penicillin / streptomycin and 1% non-essential amino acids (all Gibco). 24h later, T cells were transduced at an MOI of 3 with the lentiviral vector encoding anti -human CD19scFv fused to CAR backbones containing human CD28 and CD3^(CD247) signaling domains and expanded ex vivo for 10-12 days. Transduced T cells were maintained at a concentration of 0.75x106cells / ml throughout the culture period by cell enumeration every 2-3 days. T cells were exposed to the indicated concentrations of small molecule inhibitors or solvent only (DMSO) throughout the entire culture period.

[0434] B-ALL xenograft model

[0435] NSG mice were inoculated with 106NALM6 cells in the tail vein. 14 or 15 days after NALM6 infusion, human CAR T cells were thawed, washed and 2xl06CAR+cells were adoptively transferred in the tail vein. Body weight and health of the mice were regularly monitored. When the physical state and behavior of mice declined below the levels established by the Swiss cantonal authorities, or body weight decreased by more than 15%, mice were sacrificed. NALM6 cell numbers in the blood were measured by anti -human CD 19 flow cytometry analysis.

[0436] B-ALL xenograft in vivo serial rechallenge

[0437] Luciferase-expressing NALM6 (NALM6-Luc) cells were generated by lentiviral transduction of NALM6 cells with the lentiviral vector “Lenti-luciferase-P2A-Neo”, a gift from Christopher Vakoc15(Addgene #105621; http: / / n2t.net / addgene: 105621;RRID:Addgene_105621). 106NALM6-Luc were intravenously injected in NSG mice. 7 days later, 3xl06DMSO or MITO- 66-conditioned CD19-CAR T cells were transferred in the tail vein. Blood was sampled at the indicated time points for flow cytometric analysis. Tumor growth was monitored by bioluminescence imaging using the Xenogen in vivo imaging system (Caliper Life Sciences), following intraperitoneal injection of 150mg / kg D -Luciferin (Promega). MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0438] Flow Cytometry

[0439] Fluorochrome -conjugated antibodies were all from Biolegend or BD Biosciences. Staining for flow cytometry was done in PBS with 2% FBS and 2mM EDTA, at 4°C in the dark. The Live / Dead Fixable yellow Cell Stain Kit (Thermo Fisher Scientific) was used to mark dead cells. CD19-CAR T cells were stained with recombinant human AlexaFluor647-tagged CD19 protein (R&D Systems).

[0440] For the flow cytometry-based measurement of the mitochondrial membrane potential, 5xl05cells were incubated in 2 pM JC-1 and CD4 and CD8 antibodies in PBS for 25 minutes at 37°C. Cells were then washed with PBS and immediately measured on the flow cytometer.

[0441] Acquisition was done on Fortessa and Symphony flow cytometers with FACSDiva Software (BD Biosciences).

[0442] The flow cytometry gating strategy is illustrated in Figure 9.

[0443] Quantification and statistical analysis

[0444] FlowJo vlO was used to analyse flow cytometry data. Prism v9 software (GraphPad) was used for statistical analyses. Results are represented as mean ± standard deviation (SD). Information on each statistical test used is provided in figure legends, with sample size and number of independent repeats. Two group comparisons were tested by unpaired two-tailed Student’s t tests. Comparisons of more than two groups were done with one-way ANOVA. Grouped data comparisons were calculated using two-way ANOVA. Graft-versus-host disease or death led to the exclusion of some mice from analyses. Samples were excluded from flow cytometry analyses when the number of events recorded was lower than 20 in the population of interest, preventing accurate analyses.

[0445] Results

[0446] MITO-66 is a novel small molecule inhibitor of the Mitochondrial Pyruvate Carrier

[0447] In order to screen for novel MPC inhibitors, we used our previously developed bioluminescence resonance energy transfer (BRET)-based biosensor, RESPYR, in which MPC1 is fused to the donor group RLuc8 (a variant of Renilla luciferase) and MPC2 is fused to the acceptor group Venus (a variant of yellow fluorescent protein)11. In this system, conformational changes due to substrate or inhibitor binding alter the proximity between the termini and will result in a measurable change in energy transfer and an increase in MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 luminescence. We thus screened a library of -70’000 small molecules, and selected a top hit (MITO-1, figure 6A). The inhibitory effect of MITO- 1 was further confirmed by its capacity to inhibit oxygen consumption of HeLa cells, in a Seahorse assay in which the only carbon source available was pyruvate, with an IC50 of 690 nM (Figure 6B). Medicinal chemistry performed on this molecule resulted in the synthesis of a novel and more potent compound, which we called MITO-66, having a Seahorse IC50 of 119 nM (Figure 6C), and a RESPYR IC50 of 105 nM (figure 6D). To test whether MITO-66 was inhibiting pyruvate import through direct binding to the MPC carrier, we purified the MPC 1 / MPC2 heterodimer and performed a thermoshift assay (Figure 6E). The results showed a shift in the melting temperature of the unbound MPC1 / MPC2 heterodimer from 38.2°C to 56.1 °C in the presence of MITO-66, leading to a Kd estimation of320 nM (Figure 6F). Overall, these results support a direct binding of MITO-66 to the MPC. Importantly, DEREK16studies predicted no mutagenic activity of the molecule.

[0448] Thus, MITO-66 is a novel and potent MPC inhibitor.

[0449] MITO-66 induces a stem cell memory phenotype in CAR T cells

[0450] We next determined the effect of MITO-66 on axi-cel / brexu-cel-like CAR T cells, which are FDA-approved for the treatment of large B-cell lymphoma, acute lymphoblastic leukemia and mantle cell lymphoma. These CAR T cells express second-generation CAR constructs targeting CD 19, contain an intracellular CD28 costimulatory domain and are manufactured with IL-217,18. For our experiments, CD I 9-28t^ CAR T cells were generated from T cells enriched from the peripheral blood of healthy donors in the presence of different concentrations of MITO-66, or DMSO alone as a control (Figure 1A). MITO-66 slightly increased dose- dependently the total yield of the T cell product by the end of the culture, 9 days post-activation (Figure IB). Importantly, MITO-66 also dose-dependently increased the proportion of stem cell memory-like CD45RO-negative, CD62L-positive CD4 and CD8 CART cells (Figures 1C- D), most potently and significantly at 25 pM. We thus selected 25 pM MITO-66 for subsequent testing. We confirmed induction of a TSCM phenotype in 4 additional donors, while CAR transduction efficiency and the proportions of CD4 and CD 8 T cells were not affected (Figure 1E-H and Figure 7A-B). Of note, MITO-66 also induced TSCM cells when this population was identified more stringently by including cell surface markers CD45RA, CCR7 and CD 127 in addition to CD45RO and CD62L (Figure 1K-L). Interestingly, MITO-66 increased the mitochondrial membrane potential in both CD4 and CD8 T cells (Figure 1I-J), suggestive of MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 enhanced mitochondrial activity, which was indeed shown to compensate for the loss of MPC function6. Finally, after performing a minimal wash-out procedure, consisting of one centrifugation-resuspension step, only trace amounts of MITO-66 were detected in the cell pellet (9.14ng ± 4.14 per 108cells), suggesting negligible patient exposure.

[0451] Thus, we conclude that MPC inhibition by MITO-66 during CD19-CAR T cell generation induces a TSCM phenotype with no negative impact on yield and transduction efficiency.

[0452] MITO-66 conditioning during CAR T manufacturing enhances anti-tumor efficacy

[0453] Memory phenotype and mitochondrial fitness have been associated with superior anti-tumor function of CAR T cells4,19. We tested the anti -tumor potential of MITO-66-conditioned CD 19- CAR T cells following adoptive cell transfer in a NALM6-based mouse model of pre-B cell acute lymphoblastic leukemia. We opted for a stress-test model, in which 2.5xl06CAR T cells were adoptively transferred in NOD- scid-yc / _(NSG) mice 15 days following NALM6 engraftment, when the tumor burden is high (Figure 2A). Non-transduced T cells, either treated or untreated with MITO-66, had no effect on survival when compared to untreated mice, while DMSO-conditioned CAR T cell transfer prolonged survival and cured -44% of mice (Figure 2B). Remarkably, CD19-CAR T cells generated in the presence of MITO-66 cured 100% of mice (Figure 2B). NALM6 leukemic cells in the blood were efficiently suppressed by MITO- 66-conditioned CAR T cells 7 days post-ACT, while the disease continued to progress in most mice treated with DMSO-conditioned CAR T cells (Figure 2C). Accordingly, MITO-66- conditioned CAR T cell-treated mice experienced less severe weight loss (Figure 2D). 11 days post-ACT, almost no NALM6 leukemic cells were detected in the blood of cured mice (Figure 2C), while the number of CAR T cells generated with MITO-66 was, in 4 out of 5 donors, much higher in the blood compared to DMSO-conditioned CAR T cells (Figure 2E-F). When looking at individual in vivo CD4 and CD8 T cell expansion curves of the donor T cells that cured mice, we observed that MITO-66 conditioning resulted in either a higher peak expansion or anticipated expansion (Figure 7C). Phenotypically, CAR T cells generated with MITO-66 from 4 out of 5 donors displayed increased CD62L-positive memory T cell differentiation by day 11 post-ACT (2G and H), which was maintained in CD8 T cells at day 33 post-ACT (Figures 21 and J).

[0454] Thus, MITO-66 conditioning during preparation of CD 19 CAR T cells induces a memory phenotype, which results in strongly enhanced anti -tumor efficacy and in vivo memory CAR T cell establishment. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0455] MITO-66 conditioned CAR T cells protect against cancer recurrence

[0456] Despite very high overall response rates following CAR T cell treatment in B cell malignancies, a large proportion of patients relapse within a year, either due to CAR T cell exhaustion, lack of persistence, or due to antigen downregulation15. Since MITO-66-conditioned CD19-CAR T cells maintain an increased memory phenotype in vivo, we wondered whether this would ensure protection against antigen-positive cancer recurrence. We thus generated CD19-CAR T cells with or without MITO-66, which were adoptively transferred in NSG mice at day 7 post- NALM6 engraftment, to obtain a higher number of mice cured with control CD 19-CAR T cells (Figure 3A). Indeed, 15 days post-ACT, all mice receiving CAR T cells were cancer-free (Figure 3B). Interestingly, the first two in vivo re-administrations of NALM6 cells did not induce cancer relapse, while the third rechallenge resulted in a strong cancer recurrence in DMSO-CAR-treated mice, but was much better controlled in MITO-66-CAR T cell-treated mice, although differences were not statistically different. A fourth rechallenge with a 5 -fold higher NALM6 dose than used for the first tumor challenge eventually induced cancer recurrence in 6 out of 7 DMSO-CAR T cell-treated mice, while only 4 out of 7 MITO-66-CAR T cell-treated mice relapsed (Figure 3B). Interestingly, ACT at 7 days post-NALM6 resulted in a higher number of MITO-66-conditioned CAR T cells 22 days post-ACT (Figures 3C-D). More importantly, we confirmed the increased proportion of CD62L-positive memory cells amongst the transferred CD4 and CD8 T cells (Figures 3E-F).

[0457] Therefore, MITO-66 conditioning during CAR T manufacturing induces a memory phenotype that persists in vivo and strongly reduces cancer recurrence.

[0458] Benchmarking MITO-66 with other small molecules influencing memory differentiation

[0459] Previous studies on the induction of memory T cell differentiation have focused on small molecule interference with components in the signaling cascade downstream of T cell activation, such as AKT, PI-3K8 and mTOR20'22, using AKT-VIII (AKTi), idelalisib (PI3K5i) or rapamycin (mTORi), respectively. Direct metabolic targeting has also been proposed, such as interference with glycolysis using 2-deoxyglucose (2-DG)23, and we have recently investigated the possibility of targeting either the MPC using UK-50996or targeting isocitrate dehydrogenase 2 (IDH2) using enasidenib (IDH2i)5. We therefore decided to perform a side- by-side comparison of MITO-66 versus the above-mentioned small molecule inhibitors in CD 19-CAR T cells (Figure 4A). 2-DG significantly affected CAR T cell proliferation, while inhibition of the other targets had no significant impact (Figure 4B). While AKT, PI-3K8 and MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0460] IDH2 inhibition induced a CD45RO / CD62L double positive TCM phenotype, MPC inhibition with either MITO-66 or UK5099 induced a CD45RO-negative / CD62L-positive TSCM phenotype (Figure 4C-F) reflecting the distinction between more differentiated central memory T cells (TCM) and the less differentiated, stem-like memory T cells (TSCM) with greater selfrenewal and long-term persistence potential.. CAR T cells were frozen at day 9, then thawed and infused into NSG mice, 14 days post-NALM6 engraftment. Of note, the 2-DG-conditioned CAR T cells were not used for in vivo studies, because insufficient number of cells were generated. IDH2i-, mTORi- and MPCi-conditioned CAR T cells most efficiently mediated long-term survival of leukemic mice, with MITO-66 performing slightly better than UK-5099 (Figure 4G). Surprisingly, PI3K8i conditioning did not enhance antitumor efficacy, while AKTi conditioning partially enhanced mouse survival (Figure 4G). At day 11 post-ACT, mTORi- and IDH2i -conditioned CAR T cells were the most abundant in the blood, followed by MITO-66-conditioned CAR T cells (Figures 4H and Figures 8A-D). Interestingly, the enhanced central memory phenotype was maintained in IDH2i-conditioned CAR T cells at 11 days post-ACT, while MITO-66-conditioned CAR T cells maintained an increased TSCM phenotype (Figure 41 and J, and Figures 8E, F, H and I). mTORi and IDH2i- conditioned CAR T cells showed increased proportions of CD8 TSCM cells (Figure 81), while AKTi and PI3K6i did not induce major phenotypic changes when compared to DMSO -conditioned CAR T cells. Finally, AKTi- and IDH2i-conditioned CAR T cells displayed higher levels of PD1 / TIM3 double positive T cells, markers associated with T cell exhaustion, at day 11 post-ACT when compared to DMSO-conditioned CAR T cells, whereas this phenotype was not observed for MITO-66-conditioned CAR T cells (Figure 4K and Figure 8G and 8J).8G and 8J).

[0461] In conclusion, MPC inhibition using MITO-66 during preparation of the CAR T cells is among the best solutions to enhance CAR T cell anti-tumor efficacy, and has the distinct advantage that the increased TSCM phenotype following in vivo transfer is maintained.

[0462] MITO-66 induces a stem cell-like memory phenotype in CAR T cells from patients with B-cell malignancies

[0463] Healthy donor T cells significantly differ from leukemic patient T cells, which often show signs of exhaustion due to disease state or treatment history24. We therefore generated CD19-CAR T cells in the presence of 25 pM MITO-66 from peripheral blood of a heterogeneous cohort of patients, mainly aged Diffuse large B cell lymphoma (DLBCL) patients with various treatment histories (Table 1). Similar to healthy donors, presence of MITO-66 during CD19-CAR T cell MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 manufacturing from patient T cells did not affect cell yield, viability, CD4 / CD8 ratio or CAR transduction efficiency (Figures 5A-D, 5E and 5J). MITO-66 induced IL7-receptor alpha (CD 127) expression in both CD4 and CD8 CAR T cells of several, but not all, patients (Figures 5F and 5K). A TSCM phenotype was significantly induced by MITO-66 in both CD4 and CD8 patient CAR T cells, while CD8 CAR T cells also showed an increase in CD62L-positive cells, resulting in a significant increase in TCM cells as well (Figures 5G-I and 5L-N).

[0464] In summary, MITO-66 treatment during CD 19-CAR manufacturing induces a TSCM phenotype in normal as well as in patient T cells.

[0465] Table 1

[0466] Clinical parameters of patient samples used for experiments in Figure 5.

[0467] R-CHOP: rituximab, cyclophosphamide, doxorubicin hydrochloride (hydroxydaunorubicin), vincristine sulfate (Oncovin), and prednisone; R-ICE: rituximab, ifosfamide, carboplatin and etoposide; R-DHAP: rituximab, dexamethasone, cytarabine, cisplatin; MATRIx: methotrexate, cytarabine, thiotepa, and rituximab.

[0468] Inhibition of alpha-ketoglutarate-depending enzymes enhances TSCM differentiation and synergizes with MITO-66

[0469] We next manufactured CAR T cells in the presence of MITO-66 in combination with the small molecule Vadadustat, which inhibits prolyl hydroxylases to stabilize HIF, and hypothesized MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 that this could synergize with MPC inhibition to induce a TSCM phenotype. However, because of its mode of action, it is highly likely that Vadadustat also inhibits other enzymes that use alpha-ketoglutarate as a cofactor. We modified our manufacturing parameters to better match a particular clinical -grade manufacturing pipeline. Briefly, we activated T cells from healthy donors with TransAct (Miltenyi) in PrimeXV medium (FujiFilm / Irvine Scientific) in a 24 well G-Rex system (Wilson Wolf) in the presence of MITO-66, Vadadustat, a combination of MITO-66 and Vadadustat, or DMSO as solvent control. After 24 hours, T cells were transduced with an anti-CD19-CAR-encoding lentiviral vector. Fresh medium containing the small molecule inhibitors was used to replace old medium at days 5 and 7. When analyzing the TSCM phenotype at day 9 post-activation, we could not see any effect of MITO -66 as compared to DMSO (Figures 10A-B). We hypothesize that the different manufacturing parameters induced a metabolic CAR T cell state in which the inhibition of the MPC by MITO-66 was not able to increase TSCM differentiation. However, Vadadustat treatment enhanced TSCM differentiation, although not statistically significant. Surprisingly, combining MITO-66 with Vadadustat strongly and significantly enhanced TSCM differentiation (Figures 10A-B).

[0470] Stabilizing hypoxia inducible factor (HIF) through prolyl hydroxylase (PHD) inhibition enhances TSCM differentiation and synergizes with MPC inhibitors

[0471] We wanted to assess if the synergistic observations made with MITO-66 and Vadadustat were generalizable to MPC inhibition in combination with HIF stabilization. Therefore, CAR-T cells were manufactured in the presence of different MPC inhibitors (MITO-66, 10 pM; UK5099, 10 pM; 7ACC2, 10 pM; and MSDC-0160, 10 pM) and PHD inhibitors (FG-2216, 30 pM; Roxadustat, 10 pM; DMOG, 25 pM; and MK8617, 2.5 pM) alone and all possible dual permutations and combinations. All the above-mentioned PHD inhibitors stabilize HIF by competitively binding to the alpha-ketoglutarate binding pocket of PHD. However, it cannot be ruled out that the used PHD inhibitors also inhibit other enzymes that require alpha- ketoglutarate as a cofactor.

[0472] Manufacturing parameters were selected to align with “clinical-like” manufacturing pipelines. Briefly, on day 0, cryopreserved T-cells from healthy donors were activated with TransAct (Miltenyi) in PrimeXV medium (FujiFilm / Irvine Scientific) in a 24-well G-Rex system (Wilson Wolf) in the presence of the above-mentioned MPC and PHD inhibitors alone and in combinations. An equal volume of DMSO has been used as a vehicle control. T-cell activation was performed for the first 72 hours in the presence of IL-2 (30 lU / ml), IL-7 (10 ng / ml), and MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0473] IL- 15 (10 ng / ml). After the activation process, T-cells were cultured in the presence of small molecule inhibitors and the cytokines IL-7 (10 ng / ml) and IL- 15 (10 ng / ml) from day 3 onwards. A 75% medium replacement was done on the 6thday, and the T-cell expansion was terminated on the 9thday. On day 6, the small molecule inhibitors were added based on the volume of the fresh medium added. However, the cytokines were added based on the final volume. On 9, T- cells were harvested, and live cells were counted using a trypan blue exclusion assay to measure viability. Then, T-cells were analyzed by flow cytometry to determine the immunophenotype.

[0474] When comparing the combinations, T-cell viability is least impacted in all the MITO-66- and MSCD-0160-containing combinations (Fig. 11 A). Similarly, between the combination treatments, total viable T-cell numbers are least impacted in all the MITO -66- and MSDC- 0160-containing combinations (Fig 11B). Notably, in all combinations, total viable T-cell numbers are more negatively impacted compared to their corresponding single small molecule treatments (Fig. 11B).

[0475] When analyzing a stem cell-like memory T cell phenotype based on 2 surface markers (CCR7+CD45RA+) at day 9 post-activation, we found that, all PHD inhibitors alone increase TSCM differentiation, while this increase was further amplified in combination with MPC inhibitors (Fig. 11C). Surprisingly, this synergic effect was not observed for MPC inhibitors in combination with DMOG. A similar observation was made when looking at a more stringent definition of TSCM cells with 4 surface markers (CCR7+CD62L+CD45RA+CD45ROneg) (Fig. 1 ID). In the combination treatments, the combination with UK-5099 as MPC inhibitor resulted in the highest % of TSCM, followed by MITO-66. However, UK5099 also most strongly negatively impacted total T cell expansion (Fig. 11A, C and D). In order to take both manufacturing parameters into account, we mathematically calculated the total TSCM number (=%Tscm x Total viable T-cell number) in the final T-cell pool as the function for “balanced T-cell quality and quantity”. Out of this screen, it appears that MITO-66 + FG-2216, UK-5099 + FG-2216, MITO-66 + FG-4592 and UK-5099 + FG4592 are the combinations that most potently induce high percentages and high total yields of TSCM (Fig. 1 ID and 1 IE).

[0476] MITO-66 and FG-2216 treatment triggers anti-inflammatory potential of mesenchymal stem / stromal cells (MSC) while preserving characteristics of surface markers expression

[0477] To evaluate the effects of combined MPC and HIF-prolyl hydroxylase inhibition on MSC, we performed a series of experiments. MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0478] First, to evaluate the impact of combined inhibition of MPC and PHD on clonogenic potential of MSC, primary adipose -derived MSC from multiple human donors were treated with different concentrations of MITO-66 and FG-2216 (10 pM MITO-66 + 30 pM FG-2216 ("1 / 3 MITO-66 / FG-2216"); 20 pM MITO-66 + 30 pM FG-2216 ("2 / 3 MITO-66 / FG-2216"); 20 pM MITO-66 + 40 pM FG-2216 ("2 / 4 MITO-66 / FG-2216")) for 10 days. Subsequently, colonyforming units (CFU) were measured using cytochemical staining with 0.01% crystal violet. We found that MITO-66 and FG-2216 treatment is neutral on MSC-CFU. Interestingly, the treatment of MITO-66 and FG-2216 synchronizes MSC-CFU between the donors compared to the control (Figure 12A).

[0479] To evaluate the quality of MSC under such conditions, a flow cytometric detection was performed on canonical MSC-defming markers (CD73, CD90, CD 105) upon MITO-66 and FG-2216 treatment. MSC, cultured under these conditions, maintain expression of the above- mentioned CD markers indicating preservation of MSC identity. In addition, inhibitors-treated MSC display equivalent expression of functional markers such as CD10 and CD146, which are associated with immunomodulatory and anti-inflammatory properties (Figure 12B).

[0480] Figure 12C demonstrates the impact of MITO-66 and FG-2216 treatment on the immunomodulatory capacity of MSC in a co-culture assay with THP-l-derived macrophages. Different concentrations of MITO-66 and FG-2216-treated MSCs shifts macrophage polarization from the pro-inflammatory Ml phenotype and promoted differentiation toward to anti-inflammatory M2 -like and M2 phenotypes.

[0481] Together, these results indicate that MSC treated with MPC and a HIF-prolyl hydroxylase inhibitor not only preserve their sternness-defining phenotype but also gain enhanced immunomodulatory capacity, suggesting their therapeutic use in inflammatory and degenerative pathophysiology.

[0482] MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0483] References

[0484] 1 Park, J. H. et al. Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic

[0485] Leukemia. N Engl J Med 378, 449-459 (2018). https: / / doi.org / 10.1056 / NEJMoal709919

[0486] 2 Fraietta, J. A. et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med 24, 563-571 (2018). https: / / doi.org / 10.1038 / s41591-018-001Q-l

[0487] 3 Bai, Z. et al. Single-cell antigen-specific landscape of CAR T infusion product identifies determinants of CD19-positive relapse in patients with ALL. SciAdvB, eabj2820 (2022). https: / / doi.org / 10.1126 / sciadv.abi2820

[0488] 4 Chen, G. M. et al. Integrative Bulk and Single-Cell Profiling of Premanufacture T-cell Populations Reveals Factors Mediating Long-Term Persistence of CAR T-cell Therapy. Cancer Discov 11, 2186-2199 (2021). https: / / doi.org / 10.1158 / 2159-8290.CD-20-1677

[0489] 5 Jaccard, A. et al. Reductive carboxylation epigenetically instructs T cell differentiation. Nature 621, 849-856 (2023). https: / / doi.org / 10.1038 / s41586-023-06546-y

[0490] 6 Wenes, M. et al. The mitochondrial pyruvate carrier regulates memory T cell differentiation and antitumor function. Cell Metab 34, 731-746 e739 (2022). https: / / doi.Org / 10.1016 / j.cmet.2022.03.013

[0491] 7 Herzig, S. et al. Identification and functional expression of the mitochondrial pyruvate carrier. Science 337, 93-96 (2012). https: / / doi.org / 10.1126 / science.1218530

[0492] 8 Halestrap, A. P. The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors. Biochem J 148, 85-96 (1975). https: / / doi.org / 10.1042 / bjl480085

[0493] 9 Yamashita, Y., Vinogradova, E. V., Zhang, X., Suciu, R. M. & Cravatt, B. F. A Chemical Proteomic Probe for the Mitochondrial Pyruvate Carrier Complex. Angew Chem Int Ed Engl 59, 3896-3899 (2020). https: / / doi.org / 10.1002 / anie.201914391

[0494] 10 Liu, X. et al. Development of Novel Mitochondrial Pyruvate Carrier Inhibitors to Treat

[0495] Hair Loss. J Med Chem 64, 2046-2063 (2021). https: / / doi.org / 10.1021 / acs.jmedchem.0c01570

[0496] 11 Compan, V. et al. Monitoring Mitochondrial Pyruvate Carrier Activity in Real Time Using a BRET-Based Biosensor: Investigation of the Warburg Effect. Mol Cell 59, 491- 501 (2015). https: / / doi.Org / 10.1016 / j.molcel.2015.06.035

[0497] 12 Burastero, O. et al. eSPC: an online data-analysis platform for molecular biophysics.

[0498] Acta Crystallogr D Struct Biol 77, 1241-1250 (2021). https: / / doi.org / 10.1107 / S20597983210Q8998

[0499] 13 Niebling, S. et al. FoldAffinity: binding affinities from nDSF experiments. Sci Rep 11, 9572 (2021). https: / / doi.org / 10.1038 / s41598-Q21-88985-z

[0500] 14 Paketuryte, V. et al. Uncertainty in protein-ligand binding constants: asymmetric confidence intervals versus standard errors. Eur Biophys J 50, 661-670 (2021). https: / / doi.org / 10.1007 / sQ0249-021-01518-4

[0501] 15 Ruella, M. et al. Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell. Nat Med 24, 1499-1503 (2018). https: / / doi.org / 10.1038 / s41591-018-Q201-9 MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00

[0502] 16 Sanderson, D. M. & Earnshaw, C. G. Computer prediction of possible toxic action from chemical structure; the DEREK system. Hum Exp Toxicol 10, 261-273 (1991). https: / / doi.org / 10.1177 / 0960327191010004Q5

[0503] 17 Kite Pharma. YESCARTA (package insert). U.S. Food and Drug Administration, 2022. https: / / www.fda.gov / media / 108377 / download.

[0504] 18 Kite Pharma. TECARTUS (package insert). U.S. Food and Drug Administration, 2022. https: / / www.fda.go / media / 140409 / download.

[0505] 19 Dumauthioz, N. et al. Enforced PGC-lalpha expression promotes CD8 T cell fitness, memory formation and antitumor immunity. Cell Mol Immunol 18, 1761-1771 (2021). https: / / doi.org / 10.1038 / s41423-020-Q365-3

[0506] 20 Klebanoff, C. A. et al. Inhibition of AKT signaling uncouples T cell differentiation from expansion for receptor-engineered adoptive immunotherapy. JCI Insight 2 (2017). https: / / doi.org / 10.1172 / jci.insight.95103

[0507] 21 Araki, K. et al. mTOR regulates memory CD8 T-cell differentiation. Nature 460, 108- 112 (2009). https: / / doi.org / 10.1038 / nature08155

[0508] 22 Abu Eid, R. et al. Enhanced Therapeutic Efficacy and Memory of Tumor-Specific CD8 T Cells by Ex Vivo PI3K-delta Inhibition. Cancer Res 77, 4135-4145 (2017). https: / / doi.org / 10.1158 / 0008-5472.CAN-16-1925

[0509] 23 Sukumar, M. et al. Inhibiting glycolytic metabolism enhances CD8+ T cell memory and antitumor function. J Clin Invest 123, 4479-4488 (2013). https: / / doi.org / 10.1172 / JCI69589

[0510] 24 Noll, J. H., Levine, B. L., June, C. H. & Fraietta, J. A. Beyond youth: Understanding CAR T cell fitness in the context of immunological aging. Semin Immunol 70, 101840 (2023). https: / / doi.Org / 10.1016 / j.smim.2023.101840

[0511] 25 Vladislav Volarevic et al. Therapeutic Potential of Chimeric Antigen Receptor- Expressing Mesenchymal Stem Cells in the Treatment of Inflammatory and Autoimmune Diseases. Int J Mol Sci. 2025 Aug 12;26(16):7795. doi: 10.3390 / ijms26167795.

[0512] 26 Yan L. et al. The role of MSCs and CAR-MSCs in cellular immunotherapy. Cell

[0513] Communication and Signaling, 21, Article number: 187 (2023). https: / / doi.org / 10.1186 / sl2964-023-01191-4

[0514] 27 Tavoulari S, et al. Fifty years of the mitochondrial pyruvate carrier: New insights into its structure, function, and inhibition. Acta Physiol (Oxf). 2023 Aug;238(4):el4016. https: / / doi: 10.1111 / apha.l4016

Claims

MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00CLAIMS1. An in-vitro cell culture method comprising a step of contacting a cell, or a population of cells, with a mitochondrial pyruvate carrier (MPC) inhibitor, and with a compound selected from a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate -depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HDAC inhibitor, or a combination thereof, wherein the cell is i) an immune cell, or a population of immune cells, selected from the group comprising a T cell, a gamma delta T cell (y8 T cell), a tumor infdtrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR-expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line derived from one of these cells or a pluripotent stem cell giving rise to one of these cells, or ii) a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, selected from the group comprising a mesenchymal stem cell, a multipotential stromal cell, a mesenchymal stromal cell, a mesenchymal progenitor cell, and a multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR).

2. The in-vitro cell culture method of claim 1, wherein the MPC inhibitor is selected from the group comprising a cinnamic acid, a-cyano- 4-hydroxycinnamate (CHC), diones, thiazolidines and thiazolidinediones (TZDs), the cGMP-specific phosphodiesterase (PDE) inhibitor zaprinast, the anti-cancer agent lonidamine, coumarin derivatives, and quinolone antibiotics, or a combination thereof.

3. The in-vitro cell culture method of claim 2, wherein the cinnamic acid is a cyanocinnamate selected from the group comprising CHC, UK5099, BE1976, BE1978, BE1980, BE1984, BE2617, BE2623, JXL020, JXL069, JXL050, JXL051, JXL069, 2-cyano-3-(5- phenyl-2-furyl)acrylic acid, and 2-cyano-3-[5-(2-nitrophenyl)-2-furyl] acrylic acid, or aMITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 derivative, a tautomer, a geometrical isomer, an optically active form of any one of these compounds.

4. The in-vitro cell culture method of claim 2, wherein the dione, thiazolidine, and thiazolidinedione MPC inhibitor is selected from the group comprising GW604714X, GW450863X, Pioglitazone, Rosiglitazone, MSDC-0160, Carsalam, Nitrofurantoin, (E)-5-(4- Hydroxybenzylidene) thiazolidine-2, 4-dione, and (E)-5-(3-Hydroxy-4- methoxybenzylidene) thiazolidine -2, 4-dione or a derivative, a tautomer, a geometrical isomer, an optically active form of any one of these compounds.

5. The in-vitro cell culture method of claim 1 or 2, wherein the MPC inhibitor is selected from the group comprising Zaprinast, Lonidamine, 7ACC2, 7ACC1, Entacapone, Quinolone antibiotics (e.g. Pefloxacine), and Clinafloxacin, or a tautomer, a geometrical isomer, an optically active form of any one of these compounds.

6. The in-vitro cell culture method of claim 1 or 2, wherein the MPC inhibitor is of formulaI:wherein Rl is a moiety R3-R4;R2 is selected from H, optionally substituted Ci-Cg alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryl Ci-Cg alkyl, optionally substituted heteroaryl Ci-Cg alkyl, optionally substituted Ci-Cg alkoxy such as optionally substituted Ci-Cg alkoxy substituted Ci-Cg alkyl, optionally substituted Cs-Cs heterocycloalkyl and optionally substituted Cs-Cs cycloalkyl such as optionally substituted cyclopropyl;MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00R3 is selected from a bond, S, and NR5;R4 is a group -(CR7R8)n-R6 wherein n is an integer between 0 and 1 ;R5 is H or optionally substituted Ci-Ce alkyl;R6 is selected from optionally substituted heterocycle, optionally substituted aryl;R7 and R8 are independently selected from is H and optionally substituted Ci-Cg alkyl; or a tautomer, a geometrical isomer, an optically active form, a pharmaceutically acceptable salt or a pharmaceutically active derivative thereof.

7. The in-vitro cell culture method of claim 6, wherein the MPC inhibitor is of formula3-(benzylsulfanyl)-5-propyl[l,2,4]triazolo[4,3-a]pyrimidin-7(8H)-oneMl 10-66 or a tautomer, a geometrical isomer, an optically active form, a pharmaceutically acceptable salt or a pharmaceutically active derivative thereof.

8. The in-vitro cell culture method of any one of the preceding claims, wherein the compound is selected from daprodustat, desidustat, enarodustat, molidustat, roxadustat (FG- 4592), vadadustat, dimethyloxallyl glycine (DMOG), FG-2216,, 10X4, JNJ-42041935, MK- 8617, S-2-hydroxyglutarate, fumarate, succinate, malate, deferasirox, deferoxamine, N- oxaloylglycine, N-oxalyl-2S-alanine, Alahopcin, dihydroxybenzoic acid, diazen-l-ium-1,2- diolate, diethylamine NONOate, Tilorone, a Factor inhibiting HIF (FIH) inhibitors, Von Hippel-Lindau (VHL) inhibitors or HIF allosteric agonists, or a combination thereof.

9. The in-vitro cell culture method of claim 8, wherein the MPC inhibitor is of formulaMITO-66 / IDH2 Application PCT Draft 2 PAT8268PC003-(benzylsulfanyl)-5-propyl[l,2,4]triazolo[4,3-a]pyrimidin-7(8H)-oneMITQ 6 or a tautomer, a geometrical isomer, an optically active form, a pharmaceutically acceptable salt or a pharmaceutically active derivative thereof, and wherein the compound is selected from Roxadustat or FG-2216.

10. The in-vitro cell culture method of any one of the preceding claims, further comprising adding nutrients and / or other supplements selected from the group comprising pyruvate, galactose, nicotinamide adenine dinucleotide (NAD), lactate, any amino acid, nucleic acids, inosine, fatty acids, acetate, ketone bodies, a respiratory chain uncoupler agent (e.g. FCCP), a mitochondrial ATPase inhibitor (e.g. Oligomycin A), a PPAR alpha ligand (e.g. Bezafibrate) and urolithin A, or a combination thereof.

11. The in-vitro cell culture method of any one of the preceding claims, wherein the cell is an autologous, an allogeneic cell or a cell line.

12. The in-vitro cell culture method of any one of the preceding claims, wherein the NK cell line is selected from the group comprising NK-92, YTS and KHYG1 cell lines.

13. The in-vitro cell culture method of any one of the preceding claims, wherein the macrophage cell line is a macrophage differentiated from the monocytic THP-1 cell line.MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC0014. The in-vitro cell culture method of any one of the preceding claims, wherein the pluripotent stem cell is a hematopoietic stem and progenitor cell (hPSC) or an induced pluripotent stem cells (iPSC).

15. The in-vitro cell culture method of any one of claims 1 to 13, wherein the immune cell expressing a CAR is selected from the group comprising a CAR-T cell, a TRUCK T cell, an armored CAR-T cell, a CAR-NK cell, and a CAR macrophage.

16. An immune cell, or a population of immune cells, selected from the group comprising a T cell, a gamma delta T cell (y8 T cell), a tumor infiltrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR-expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line or a pluripotent stem cell derived from one of these cells, directly obtained by an in-vitro cell culture method of any one of claims 1 to 15.

17. The immune cell, or a population of immune cells, of claim 16, wherein the immune cell expressing a CAR is selected from the group comprising a CAR-T cell, a TRUCK T cell, an armored CAR-T cell, a CAR-NK cell, and a CAR macrophage.

18. The immune cell, or a population of immune cells, of claims 16 or 17, wherein the immune cell expresses i) a surface marker selected from the group comprising CD45RA+, CCR7+, CD27+, CD62L+, CD127+, CD69+, NKG2D+, DNAM-1+ and NKp46+, or a combination of two or more thereof, and / or ii) a nuclear expression of FOXO1 and TCF1, or a combination thereof.

19. The immune cell, or a population of immune cells, of anyone of claims 16 to 18, wherein the immune cell shows an increased secretion of inflammatory mediators selectedMITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 from the group comprising granzymes, perforins, interferons, TNF, and IL-2, or a combination of two or mor thereof, upon stimulation or repeated stimulation by target cells.

20. A multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, selected from the group comprising a mesenchymal stem cell, a multipotential stromal cell, a mesenchymal stromal cell, a mesenchymal progenitor cell, and a multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR) directly obtained by an in- vitro cell culture method of any one of claims 1 to 15.

21. The multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, of claim 20, wherein the multipotential stem / progenitor cell expresses i) a surface marker selected from the group comprising CD73+, CD90+, and CD105+, or a combination of two or more thereof, and / or ii) a nuclear expression of a transcription factor selected from RUNX2, SOX9, and PPARy, or a combination of two or more thereof, and wherein the multipotential stem / progenitor cell does not express a surface marker selected from the group comprising CD34-, CD45-, and HLA-DR-, or a combination of two or more thereof.

22. A composition comprising an immune cell, or a population of immune cells, of any one of claims 16 to 19 or a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, of claims 20 and 21.

23. A pharmaceutical composition comprising an immune cell, or population of immune cells, of any one of claims 16 to 19 or a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, of claims 20 and 21, and pharmaceutically acceptable carrier, diluent and / or excipient.

24. The immune cell, or population of immune cells, of any one of claims 16 to 19, for use in the treatment and / or prevention of a disease selected from the group comprising a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease and / or an autoimmune disease.MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC0025. The multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, of claims 20 and 21, for use in the treatment and / or prevention of a disease selected from the group comprising a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease and / or an autoimmune disease.

26. The pharmaceutical composition of claim 23, for use in the treatment and / or prevention of a disease selected from the group comprising a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease and / or an autoimmune disease.

27. An in-vitro cell culture method comprising a step of contacting a cell, or population of cells, with a compound selected from a compound that inhibits alpha-ketoglutarate -dependent enzymes, thereby stabilizing HIF1 and / or HIF2, or a compound that inhibits histone and / or DNA demethylases (e.g. daprodustat, desidustat, enarodustat, molidustat, roxadustat, vadadustat, dimethyloxallyl glycine, FG-2216, FG-4592, IOX4, JNJ-42041935, MK-8617, S- 2 -hydroxyglutarate, fumarate, succinate, malate, deferasirox, deferoxamine, N-oxaloylglycine, N-oxalyl-2S-alanine, Alahopcin, dihydroxybenzoic acid, diazen-l-ium-l,2-diolate, diethylamine NONOate, Tilorone, a Factor inhibiting HIF (FIH) inhibitors, Von Hippel-Lindau (VHL) inhibitors or HIF allosteric agonists).

28. A cell culture medium comprising an MPC inhibitor and a compound selected from a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate -depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor and an HDAC inhibitor , or a combination thereof.

29. A method of treating and / or preventing a disease, comprising (i) providing a cell, or population of cells, (ii) expanding and culturing ex vivo or in vitro into a larger population of cells according to the in-vitro cell culture method of any one of claims 1 to 15, and (iii)MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC00 introducing (i.e. administering) said cell, or population of cells, into the patient or subject in need thereof.

30. The method of claim 29, wherein the cell is a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, selected from the group comprising a mesenchymal stem cell, a multipotential stromal cell, a mesenchymal stromal cell, a mesenchymal progenitor cell, and a multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR).

31. The method of claim 29, wherein the cell is an immune cell, or a population of immune cells, selected from the group comprising a T cell, a gamma delta T cell (y8 T cell), a tumor infiltrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR- expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line derived from one of these cells or a pluripotent stem cell giving rise to one of these cells.

32. The method of any one of claims 29 or 31, wherein the immune cell, or the population of immune cells is genetically engineered with one recombinant construct encoding a chimeric antigen receptor (CAR).

33. The method of any one of claims 29 to 32, wherein the disease is selected from a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease.

34. A method of treating and / or preventing a disease in a patient or subject in need thereof, comprising (i) providing cells isolated from the patient or subject in need thereof, (ii) expanding and culturing ex vivo or in vitro into a larger population of cells according to the in-vitro cell culture method of any one of claims 1 to 15 and (iii) reintroducing (i.e. administering) said immune cells into the patient or subject in need thereof.MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC0035. The method of claim 34, wherein the cell is a multipotential stem / progenitor cell, or a population of multipotential stem / progenitor cells, selected from the group comprising a mesenchymal stem cell, a multipotential stromal cell, a mesenchymal stromal cell, a mesenchymal progenitor cell, and a multipotential stem / progenitor cell expressing a chimeric antigen receptor (CAR).

36. The method of claim 34, wherein the cell is an immune cell, or a population of immune cells, selected from the group comprising a T cell, a gamma delta T cell (y3 T cell), a tumor infiltrating lymphocyte (TIL), an NK cell, a regulatory T cell (Treg cell), a macrophage, a TCR- expressing cell, an eosinophil, a basophil, a neutrophil, myeloid cells, a B cell, a plasma cell, a regulatory B cell (Breg), an innate lymphoid cell 1 (ICL1), an ILC2, an ICL3, a dendritic cell, an NK-T cell, an immune cell expressing a chimeric antigen receptor (CAR), and a cell line derived from one of these cells or a pluripotent stem cell giving rise to one of these cells.

37. The method of any one of claims 34 or 36, wherein the immune cell, or the population of immune cells is genetically engineered with one recombinant construct encoding a chimeric antigen receptor (CAR).

38. The method of any one of claims 34 to 37, wherein the disease is selected from a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease.

39. A kit for treating and / or preventing a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease, the kit comprising a pharmaceutical composition of claim 23.

40. A kit for in-vitro cell culture, the kit comprising a cell culture medium of claim 28.MITO-66 / IDH2 Application PCT Draft 2 PAT8268PC0041. The kit of claim 40, further comprising one or more vials, wherein one vial comprises the cell culture medium of claim 28, wherein one vial comprises an MPC inhibitor and / or wherein one vial comprises a compound selected from a prolyl hydroxylase inhibitor, a small molecule that stabilizes hypoxia-inducible factor 1 and 2 (HIF1 / HIF2) protein, a small molecule or metabolite inhibiting alpha-ketoglutarate-depending enzymes, an isocitrate dehydrogenase 2 (IDH2) inhibitor, and an HDAC inhibitor, or a combination thereof.

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