Improvement of cellular immunotherapy through the use of bacterial pentanoates

Abstract

This invention relates to the short-chain fatty acid pentanoate and its use as enhancer for cellular immune therapy and anti-tumor therapy. The present invention specifically relates to the metabolite pentanoate and its use in the production of antigen-specific or genetically engineered immune cells. The invention also relates to pharmaceutical compositions comprising such immune cells, as well as their uses in cellular immune therapy (adoptive immune therapy), in particular to an improvement in the treatment of cancer, infectious diseases and immune cell-mediated diseases by means of cellular immune therapy.

Description

[0001] IMPROVEMENT OF CELLULAR IMMUNOTHERAPY THROUGH THE USE OF BACTERIAL PENTANOATES

[0002] FIELD OF THE INVENTION

[0003] This invention relates to the short-chain fatty acid pentanoate and its use as enhancer for cellular immune therapy and anti-tumor therapy. The present invention specifically relates to the metabolite pentanoate and its use in the production of antigen-specific or genetically engineered immune cells. The invention also relates to pharmaceutical compositions comprising such immune cells, as well as their uses in cellular immune therapy (adoptive immune therapy), in particular to an improvement in the treatment of cancer, infectious diseases and immune cell-mediated diseases by means of cellular immune therapy.

[0004] BACKGROUND

[0005] Advances in genetic engineering have allowed the design of immune cells that specifically recognize surface antigens on target cells. The introduction of transgenic T cell receptors (TCRs) and chimeric antigen receptors (CARs) to patient-derived T cells has revolutionized cancer immunotherapy. Especially CAR T cell therapy has shown to be capable of mediating high response rates and long-lasting remissions in the context of hematologic malignancies such as acute lymphoblastic leukemia (ALL) and multiple myeloma (MM).

[0006] Although approval of CAR T cell therapies has marked a major breakthrough in the therapy of hematologic neoplasms, many patients with advanced malignancies and non-hematologic cancers in particular do not benefit from these, in part due to T cell-intrinsic dysfunctionality or suppressive factors within the tumor microenvironment (TME) (Narayan V et al., 2022). Moreover, the immune cell-intrinsic fitness is a major prerequisite for their reactivity and persistence.

[0007] Different approaches to equip immune cells with transcription factors, homing and co-stimulatory receptors and knock-outs for inhibitory pathways have been investigated over the years which increase the genetic burden on the cell. Alternatively, epigenetic and metabolic modulation using physiologic small molecules can be considered as a broadly applicable approach to enhance efficacy and in vivo stability.

[0008] In addition, the host's immune environment determines the success of immunotherapies which can be influenced by the intestinal microbiome both under homeostatic and pathophysiological conditions (Kespohl M et al., 2017; Smith PM et al., 2013; Vetizou M et al., 2015) Its impact on cancer immunotherapy was first recognized in the context of immune checkpoint inhibitory therapy (I Cl) when several studies demonstrated that response and resistance to PD-1 and CTLA-4 blockade depends on the gut microbial composition (Vetizou M et al., 2015; Sivan A et al., 2015). Colonization with Akkermansia muciniphila and some Bifidobacterium strains facilitates the improvement of ICI via activation of endogenous T and antigen-presenting cells (Sivan A et al., 2015; Routy B et al., 2018). These cells also translocate from the intestine to the tumor site, thereby inspiring the design of commensal consortia to modulate T cell response favorably (Tanaoue T et al., 2019). Recently, the importance of the microbiome in chimeric antigen receptor (CAR) T cell therapy was highlighted by correlation of antibiotic treatment prior to CAR T cell infusion with worse response and progression free survival (PFS) (Smith A et al., 2022; Luu M et al., 2019). While microbial metabolites and producer strains influence anti-tumor response, their modes of action often remain obscure. In this regard, short-chain fatty acids (SCFAs; a major group of commensal molecules) have been investigated in inflammation, autoimmunity and cancer (Luu M et al., 2019; Coutzac C et al., 2020; Arpaia N et al., 2013). The inventors have previously demonstrated that pentanoate is a commensal metabolite that augments the anti-cancer efficacy of CD8 T cells (Luu et al., 2021, WO 2021 / 058811).

[0009] The contradictory findings in the influences of SCFAs highlight their context- and cell type-dependent effects raising a need for strategies to apply commensal metabolites for clinical use. Hence, despite all current efforts, hostile influences in solid tumors are diminishing the efficacy of CAR T cells in patients, underscoring the necessity for new, interdisciplinary sources of improvement.

[0010] Consequently, the field requires new strategies to improve the engineering and function of genetically modified immune cell to overcome these hurdles.

[0011] DESCRIPTION OF THE INVENTION

[0012] The present invention aims to improve immune cell-based therapies by providing a method of by reprogramming immune cells. In particular, the inventors have found that the metabolite pentanoate can be used to reprogramming of immune cells and thus increases their ability to secrete effector cytokines, cytotoxic function and persistence in vivo via preservation of a naive-like phenotype and reduction of exhaustion. The inventors have therefore developed a protocol which integrates pentanoate-mediated epigenetic-metabolic reprogramming into the immune cell manufacturing workflow in order to confer superior in vivo function, metabolic fitness and a naive-like cell state, overcoming and modulating the detrimental TME in immunocompetent solid tumor models. Hence, the implementation of pentanoate into immune cell manufacturing workflow has the potential to optimize immune cell-based therapies.

[0013] Accordingly, the invention relates to the following preferred embodiments:

[0014] 1 . A method for producing recombinant immune cells, comprising the following steps: (A) Isolating immune cells, (B) Incubating the immune cells in the presence of at least one short-chain fatty acid, and

[0015] (C) Introducing into said immune cells of step (B) a nucleic acid encoding an immunoreceptor or a set of nucleic acids encoding an immunoreceptor.

[0016] 2. The method according to item 1, wherein the immune cells are mammalian immune cells.

[0017] 3. The method according to items 1 or 2, wherein the immune cells are human immune cells.

[0018] 4. The method according to any one of items 1 to 3, wherein said immune cells are T cells, B cells, NK cells,

[0019] NKT cells, macrophages and / or stem cells.

[0020] 5. The method according to any one of items 1 to 4, wherein said immune cells are T cells.

[0021] 6. The method according to any one of items 1 to 5, wherein said immune cells are CD4+ and / or CD8+ T cells.

[0022] 7. The method according to any one of items 1 to 6, wherein the immunoreceptor is a B-cell receptor (BCR), T- cell receptor (TCR), or chimeric antigen receptor (CAR).

[0023] 8. The method according to any one of items 1 to 7, wherein the immunoreceptor is a chimeric antigen receptor (CAR).

[0024] 9. The method according to any one of items 1 to 8, wherein the at least one short-chain fatty acid comprises pentanoate, butyrate, propionate, acetate and / or pharmaceutically acceptable derivatives thereof.

[0025] 10. The method according to any one of items 1 to 9, wherein the at least one short-chain fatty acid comprises pentanoate or a pharmaceutically acceptable derivative thereof.

[0026] 11. The method according to any one of items 1 to 10, wherein the at least one short-chain fatty acid is pentanoate or a pharmaceutically acceptable derivative thereof.

[0027] 12. The method according to any one of items 1 to 11, wherein in step (C), the nucleic acid or set of nucleic acids is introduced by viral gene transfer, preferably wherein the nucleic acid is introduced by retroviral gene transfer. 13. The method according to item 12, wherein in step (C), the viral gene transfer uses a viral vector which comprises a gene cassette encoding said immunoreceptor.

[0028] 14. The method according to item 12 or 13, wherein in step (C), the cells are transduced with retroviral supernatant by spin-infection.

[0029] 15. The method according to any one of items 1 to 14, wherein in step (B), the incubation is performed for more than 24 hours, such as 24 to 72 hours, preferably 36 to 60 hours, more preferably for 48 hours.

[0030] 16. The method according to any one of items 1 to 15, wherein in step (B) the total concentration of the at least one short-chain fatty acid is more than 0.25 mM, preferably between 0.25 mM and 20 mM, more preferably between 0.5 and 10 mM, even more preferably between 1 and 5 mM, even more preferably between 1 and 2.5 mM, even more preferably 2.0 mM.

[0031] 17. The method according to any one of items 1 to 16, wherein in step (B) the at least one short-chain fatty acid is added directly to the culture medium.

[0032] 18. The method according to any one of items 1 to 17, wherein the method further comprises a step (B1) prior to step (B), wherein in step (B1) the immune cells are activated.

[0033] 19. The method according to item 18, wherein in step (B1), the activation is performed by the addition of activating agents of the immune cells, such as an antibody and / or a cytokine.

[0034] 20. The method according to item 19, wherein the antibody is a o-CD3 antibody, or an O-CD28 antibody, and / or wherein the cytokine is an IL-2.

[0035] 21. The method according to any one of items 18 to 20, wherein in step (B1) an o-CD3 antibody, an O-CD28 antibody, and IL-2 are added.

[0036] 22. The method according to any one of items 1 to 21 , wherein step (A) comprises a sorting step to isolate the immune cells of step (A).

[0037] 23. The method according to item 22, wherein the sorting step is carried out via magnetic beads. 24. The method according to any one of items 1 to 23, further comprising a step (D) of expanding the recombinant immune cells obtained in step (C).

[0038] 25. The method according to item 24, wherein expansion is performed for 1 to 5 days.

[0039] 26. The method according to any one of the preceding items, comprising the steps of:

[0040] (A) Isolating immune cells; preferably wherein the immune cells are T-cells;

[0041] (B1) Activating the immune cells; preferably wherein the immune cells are activated by the addition of an o-CD3 antibody, an O-CD28 antibody, an anti-IL-4 antibody and the cytokine IL-2;

[0042] (B) Incubating the immune cells obtained in step (B1) in the presence of pentanoate; preferably wherein the incubation is performed for 48 hours;

[0043] (C) Introducing into said immune cells of step (B) at least one nucleic acid or set of nucleic acids encoding an immunoreceptor; preferably wherein the immune receptor is a CAR; and

[0044] (D) Expanding the recombinant immune cells obtained in step (C), preferably wherein expansion is performed for 1 to 5 days.

[0045] 27. The method according to any one of the preceding items, wherein the immunoreceptor is an immunoreceptor capable of binding to an antigen selected from the group consisting of CD 19, CD20, CD22, CD27, CD30, CD33, CD38, CD44v6, CD52, CD64, CD70, CD72, CD 123, CD 135, CD 138, CD220, CD269, CD319, R0R1, R0R2, SLAMF7, BCMA, ovp3-lntegrin, o4p1-lntegrin, EpCAM-1, MUC-1, MUC-16, L1-CAM, c-kit, NKG2D, NKG2D- Ligand, PD-L1, PD-L2, Lewis-Y, CAIX, CEA, c-MET, EGFR, EGFRvlll, ErbB2, Her2, FAP, FR-a, EphA2, GD2, GD3, GPC3, IL-13Ra, Mesothelin, PSMA, PSCA, and VEGFR, preferably R0R1 or CD19.

[0046] 28. The method according to any one of items 27, wherein the immunoreceptor is a R0R1 -specific CAR or a CD 19 specific CAR.

[0047] 29. The method according to any one of preceding items, wherein the at least one short-chain fatty acid is produced by at least one species of bacteria.

[0048] 30. The method according to item 29, wherein the at least one short-chain fatty acid is produced by the bacterium Megasphaera massiliensis.

[0049] 31. Method according to item 29 or 30, wherein the at least one short-chain fatty acid is produced by a composition of bacteria comprising at least the bacteria Megasphaera massiliensis, Megasphaera elsdenii, Faecalibacterium prausnitzii and Anaerostipes hadrus. 32. Use of the short-chain fatty acid pentanoate to reprogram immune cells, wherein the immune cells are incubated in the presence of pentanoate or a pharmaceutical acceptable derivative thereof.

[0050] 33. The use according to item 32, wherein the immune cells are antigen-specific immune cells or genetically engineered immune cells.

[0051] 34. The use according to items 32 or 33, wherein the reprogramming is metabolic reprogramming and / or epigenetic reprogramming.

[0052] 35. The use according to item 34, wherein the metabolic reprogramming and / or epigenetic reprogramming is characterized by an increased metabolic fitness and a naive-like cell state of the immune cells.

[0053] 36. The use according to item 34 or 35, wherein the metabolic reprogramming and / or epigenetic reprogramming is characterized by an increased ability to secrete effector cytokines such as IFN-y, IL-2 and TNF-o of the immune cells.

[0054] 37. The use according to any one of items 32 to 36, wherein the immune cells are incubated in the presence of pentanoate for more than 24 hours, such as 24 to 72 hours, even more preferably for 48 hours.

[0055] 38. The use according to any one of items 32 to 37, wherein the immune cells are T cells, B cells, NK cells, NKT cells, macrophages and / or stem cells.

[0056] 39. The use according to any one of items 32 to 38, wherein said immune cells are T cells, preferably wherein said immune cells are CD4+ and / or CD8+ T cells.

[0057] 40. The use according to any one of items 32 to 39, wherein said immune cells are mammalian immune cells, preferably wherein said immune cells are human immune cells.

[0058] 41. The use according to any one of items 33 to 40, wherein said genetically engineered immune cells are immune cells engineered to express an immunoreceptor, preferably wherein the immunoreceptor is a B-cell receptor (BCR), T-cell receptor (TCR), or chimeric antigen receptor (CAR).

[0059] 42. The use according to item 41, wherein said genetically engineered immune cells are CAR T cells. 43. The method or use according to any one of the preceding items, wherein all the steps of the method or use are carried out in vitro.

[0060] 44. The method or use according to any one of the preceding items, wherein said method or use does not comprise a method for treatment of the human or animal body by surgery or therapy or a diagnostic method practiced on the human or animal body.

[0061] 45. The method or use according to any one of the preceding items, wherein said method or use does not comprise a process for modifying the germ line genetic identity of a human being.

[0062] 46. A recombinant immune cell obtainable by the method according to any one of items 1 to 45.

[0063] 47. A pharmaceutical composition comprising the recombinant immune cell of item 46, the composition optionally further comprising a pharmaceutically acceptable carrier and / or excipient.

[0064] 48. A recombinant immune cell according to item 46, or a pharmaceutical composition according to item 47, for use in medicine.

[0065] 49. A recombinant immune cell according to item 46, or a pharmaceutical composition according to item 47, for use in the treatment of cancer by cancer immunotherapy.

[0066] 50. A recombinant immune cell according to item 46, or a pharmaceutical composition according to item 47, for use in the treatment of cancer by cancer immunotherapy, wherein the immunoreceptor of the immune cell is capable of binding to a cancer antigen on the cell surface of a cell of the cancer.

[0067] 51. The recombinant immune cell and or a pharmaceutical composition for use according to items 49 or 50, wherein the cancer is haematological or solid cancer, optionally wherein the cancer is selected from the group consisting of lymphoma and myeloma.

[0068] 52. A method for predicting and / or determining the efficacy of an immunotherapy in a subject comprising the steps of: a) determining a pentanoate concentration in a sample isolated from said subject; and b) predicting or determining the efficacy of the immunotherapy based on the concentration determined in step a). 53 The method of item 52, wherein in step b), the efficacy of the immunotherapy is predicted and / or determined to be increased when the determined pentanoate concentration in the sample is at or above a threshold, compared to when the pentanoate concentration in the sample is below said threshold.

[0069] 54. The method according to items 52 or 53, wherein the sample is a fecal sample, and / or wherein the immunotherapy is a chimeric antigen receptor (CAR) T-cell therapy.

[0070] 55. The method according to items 53 or 54, wherein the threshold is: a) A weight concentration of 200 pig / g or more, more preferably 300 pig / g or more, even more preferably 400 pig / g or more, even more preferably 500 pig / g, such as 285.6 pig / g, and / or b) A molar concentration of 2 pimol / g or more, preferably 3 pimol / g or more, more preferably 4 pimol / g or more, even more preferably 5 pimol / g or more, such as 6 pimol / g.

[0071] 56. The method according to any of items 53 to 55, wherein the increased efficacy of the immunotherapy is characterized by an increased survival of the subject receiving immunotherapy compared to the efficacy of the immunotherapy in a subject for which the pentanoate concentration in the sample is determined to be below the threshold, optionally wherein the increased survival is an increased overall survival.

[0072] 57. The method according to any of items 53 to 56, wherein the subject has cancer and the immunotherapy is cancer immunotherapy against said cancer, preferably wherein the immunotherapy is a chimeric antigen receptor (CAR) T-cell therapy capable of targeting a cancer antigen on the cell surface of a cell of the cancer, and the increased efficacy of the cancer immunotherapy is characterized by an increased progression-free survival (PFS) in said subject having cancer compared to the efficacy of the cancer immunotherapy in a subject for which the pentanoate concentration in the sample is determined to be below the threshold.

[0073] 58. The method according to any of items 53 to 57, wherein all the steps of the method are carried out in vitro.

[0074] 59. A method of treating cancer in a subject having the cancer, the method comprising:

[0075] (A) predicting or determining the efficacy of a cancer immunotherapy in a group of subjects having cancer with the method according to any of items 53 to 57,

[0076] (B) identifying a subject as having an increased predicted and / or determined efficacy of the cancer immunotherapy when the determined pentanoate concentration in the sample is at or above a threshold as defined in items 53 or 55; and (c) administering to said subject the cancer immunotherapy, preferably wherein the immunotherapy is a chimeric antigen receptor (CAR) T-cell therapy capable of targeting a cancer antigen on the cell surface of a cell of the cancer.

[0077] 60. A CAR T cell capable of targeting a cancer antigen on the cell surface of a cell of a cancer, for use in a method for the treatment of said cancer in a subject, wherein in the method, the CAR T cell is to be administered to a subject in which the pentanoate concentration in a sample isolated from said subject is at or above a threshold, wherein the threshold is as defined in item 55, optionally wherein the sample is a fecal sample.

[0078] 61. The method according to item 59 or the CAR T cell for use according to item 60, wherein the cancer is haematological or solid cancer, optionally wherein the cancer is selected from the group consisting of lymphoma and myeloma.

[0079] BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 : Pentanoate abundance is a predictor for clinical CAR T cell response, a and b, Probability of progression-free survival (a) and overall survival (b) in a cohort of patients receiving CAR T cells depending on different antibiotic-(ABX)-treatment regimen. PIM (piperacillin / tazobactam, imipenem, meropenem); PFS, progression-free survival, c, Levels of multiple short-chain fatty acids following antibiotic exposure, d-g, Kaplan- Meier curves showing PFS (upper panel) and OS (lower panel) depending on the abundance of pentanoate (d), butyrate (e), propionate (f) and acetate (g). OS, overall survival.

[0081] Figure 2: Microbial metabolites improve engineering and favor CTL-phenotype of murine CD8+CAR T cells, a, Schematic illustration of experimental setup for murine CAR T cell generation with pretreatment and functional analysis, b, Representative contour plots of the transduction marker and bar plots show frequencies of CD8+R0R1- CAR T cells on day 6. Mean ± SEM from n = 3. c, Median fluorescence intensity (MFI) of CAR+T cells. Mean ± SEM from n = 3. d, Total CAR+T cell yield and viability on day 7. Mean ± SEM from n = 3. e, Representative contour plots and bar graphs show transduction efficiency of CD4+CAR T cells on day 6. Mean ± SEM from n = 3. f, Median fluorescence intensity (MFI) for generated CAR+T cells. Mean ± SEM from n = 3. g, Yield and viability of CAR+T cells on day. Mean ± SEM from n = 3. h, Frequency of IFN-y and TNF-o producing R0R1- CAR T cells on day 4 following antigen-independent restimulation for 5 hours. Mean ± SEM from n = 3. i, Flow cytometric analysis of Granzyme B and IL-2 production by CD8+R0R1 CAR T cells after restimulation on day 4. Mean ± SEM from n = 4. j, Specific cytolytic activity of R0R1- CAR T cells against tumor cells at different E:T ratios after 6 h. Mean ± SEM from n = 3. k, Killing capacity of CD19- CAR T cells measured by specific lysis of CD19-expressing tumor cells at different E:T ratios after 4 and 8 h. Mean ± SEM from n = 3. 1 and m, Cytokine secretion of IFN-y, TNF-o and IL-2 after 24-hour co-culture of R0R1- (I) or CD19- (m) specific CAR T cells with target cells. Mean ± SEM from n = 3.

[0082] (b-i) Data represent pooled data from independent experiments, (j-m) n=3 biological replicates; pooled data from n=3 independent experiments; mean ± SEM was calculated for n=3 independent experiments. Statistical analysis was performed using unpaired two-tailed Student's t test (b-i) and two-way analysis of variance (ANOVA) with Tukey's multiple-comparison test (j-m).

[0083] Figure 3: HDAC class I inhibition-mediated hyperacetylation improves CAR T cell function, a, Schematic pathway illustration detailing the HDAC-mediated T cell regulation, b and c, Shown are SCFAs (blue sticks), i.e. acetate, propionate, butyrate, pentanoate and hexanoate docked into AlphaFold 3-predicted structures of zinc cofactor-bound (spheres) HDAC1 (gray cartoons), d, Influence of bacterial SCFAs on the activity of recombinant class I and class II HDAC enzymes, e, Mocetinostat (blue sticks, HDAC class I inhibitor) docked into an AlphaFold 3-predicted model of zinc cofactor-bound (spheres) HDAC1 (gray cartoons), f and g, Histone acetylation status of T cells are measured by the expression of H3 / K9-14 (f) and H3K27 (g) via flow cytometry treated with indicated substances. 1 out of 3 representative experiments is shown, h, Specific cytolytic activity of CD8+CAR T cells generated in the presence of indicated substances against R0R1 -expressing tumor cell lines at different E:T ratios after 6 h. Mean ± SEM from n = 3. i Cytokine secretion of CD8+CAR T cells after 24 h coculture with target antigen expressing cell lines, measured for IFN-y, TNF-o and IL-2 by ELISA. Mean ± SEM from n = 3. h and i, biological replicates; pooled data from independent experiments. Statistical analysis was performed using one-way ANOVA (h) or two-way analysis of variance (ANOVA) with Tukey's multiplecomparison test (i).

[0084] Figure 4: Pentanoate synergizes epigenetic and metabolic modulation to boost effector function, a, Schematic pathway representation, b, Flow cytometry analysis and quantification of PGC-1o in CD8+CAR T cells either untreated or treated with pentanoate. Mean ± SEM from n = 3. c, Flow cytometric analysis of MitoFM in CD8+CAR T cells treated with indicated substances. Mean ± SEM from n = 3. d, Combinatorial or single treatment workflow for CD8+T cells, e and f, Cytolytic activity of R0R1- CAR T cells against PancRORI (e) and MC38ROR1 (f) at different E:T ratios after 4 h and 8 h. Mean ± SEM from n = 3. g-j, Secretion of IFN-y (g), IL-2 (h) and TNF-o (I) after 24 hours by CD8+CAR T cells upon co-incubation with R0R1 -expressing tumor cell lines. Mean ± SEM from n = 3. j, Scheme illustrating the different pretreatments of CAR+T cells generated from wildtype or HDAC knockout mice, k, Cytolytic activity of CD8+CAR T cells against target cells with different E:T ratios after 4 h. CAR T cells were generated as shown in j. Mean ± SEM from n = 3. I, Experimental setup of pretreatment with pentanoate or DCA / moceti nostat combination during the CAR+T cell manufacturing process followed by injection in PancRORI tumor bearing mice, m, Tumor growth (left) over time as well as tumor volume (middle) and tumor weight on day 14 (right) after tumor inoculation are shown. Mean ± SEM from n = 10 mice / group until day 7, then n = 5 mice / group until day 14. b, c, e-l and k, independent experiments, e-l, k, biological replicates; pooled data from independent experiments. Statistical analysis was performed using one-way ANOVA (b, c, m) or two-way analysis of variance (ANOVA) with Tukey's multiple-comparison test (e-l and k).

[0085] Figure 5: Pentanoate incorporates into the TCA and becomes part of the epigenetic imprint a, Scheme illustrating pentanoate modulation and metabolism hijacking of T cells, b, Schematic for the generation of CAR+T cells in combination with pretreatment prior to metabolite tracing c, GC-MS isotope tracing of13C-glucose- derived metabolites in CAR T cells engineered in the presence of specified substances. Histograms show fractional enrichment of13C-glucose-derived TCA metabolites. Mean ± SEM from n = 3 biological replicates, d, GC-MS isotope tracing of13C-labeled glutamine in untreated or pentanoate- treated CAR T cells regarding citrate, o-ketoglutarate and malate. Mean ± SEM from n = 3 biological replicates, e, GC-MS tracing of13C- pentanoate in CD8+CAR T cells pretreated with indicated substrates. Histograms show the frequency of pentanoate-derived carbons in the downstream TCA cycle intermediates. n=2 independent T cell pools, f and g, PancRORI (f) or MC38ROR1 (g) tumor killing of CAR+T at different E:T ratios after 6 h. Mean ± SEM from n = 3.

[0086] Figure 6: Epigenetic- metabolic rewiring enhances efficacy and persistence in solid malignancies, a, Scheme illustrating the analysis of R0R1- CAR T cells in a PancRORI tumor model, b, PancRORI tumor growth after treatment with CD8+CAR T cells, c, Tumor weight for mock, CAR T cells or CARpentaT cells over the experimental course, d, Tumor volume at endpoint day 21. Mean ± SEM from n = 5 mice / group for mock, n = 6 mice / group for CAR treated groups, e, Scheme illustrating the analysis of R0R1-CAR T cells in a MC38ROR1 tumor model, f, Survival of MC38ROR1 tumor bearing mice. Mean ± SEM from n = 8 mice for control group, n = 9 for CAR or CARpenta groups, g, Tumor weight at the endpoint. Mean ± SEM from n = 6 for CAR or CARpenta groups, h, Single tumor weight over the course of the experiment. I, Uniform Manifold Approximation and Projection (UMAP) showing the distribution of immune cells collected from mice using scRNAseq data in the LSI space. Each point represents one cell. The cells are marked by color code based on cell annotations. Red indicates T cells, blue non-T cells, j, UMAP showing the distribution of T cells based on their cell states. The cells are marked by color code based on the different cluster they belong to. k and I, Barplots indicating the changes in percentage of the different CD4 (k) and CD8 (I) T cell subsets noticed following the different treatments and time points (day 0, 7 and 14).

[0087] Statistical analysis was performed using one-way ANOVA with Tukey's multiple-comparison test (b, d) and unpaired two-tailed Student's t test (e-h and k). Survival curves were compared by the log-rank Mantel-Cox test 0).

[0088] Figure 7: CAR T cell generation with molecular metabolites, a, Dot blot and bar graphs show expression of transduction marker of CD8+CD19- CAR T cells on day 6. Mean ± SEM from n = 3. b, Representative histogram and median fluorescence intensity (MFI) of the transduction marker. Mean ± SEM from n = 3. c, CAR T cell yield and live cells of CD19+- CAR T cells on day 7. Mean ± SEM from n = 3. d, Dot blots and bar graph show tEGFR expression of CD4+CD19- CAR T cells on day 6. Mean ± SEM from n = 3. e, MFI for generated CAR T cells as histogram representative and bar graph. Mean ± SEM from n = 3. f, Cell number and viability of cells of generated CAR+T cells on day 7. Mean ± SEM from n = 3. g and h, Representative dot plot and bar graph show frequency of IFN-y+TNFo+double positive (g) and GranzymeB+IL-2+double positive (h) CD4 ROR1- CAR+T cells upon restimulation. Mean ± SEM from n = 3. I and j, Cytolytic activity of generated CD4+CAR T cells against antigen-presenting tumor cells PancRORI for ROR1-CAR T cells (i) or Emyc for CD19-CAR T cells (j) at different E:T ratios at indicated timepoints. Mean ± SEM from n = 3. k and I, Secretion of IFN-y, TNF-o and IL-2 was measured by ELISA after 24-hour co-incubation with antigen-presenting tumor cell lines for R0R1- CAR T cells (k) and CD19-CAR T cells (I). Mean ± SEM from n = 3.

[0089] Statistical analysis was performed using unpaired two-tailed Student's t test (a-h and i) or two-way analysis of variance (ANOVA) with Tukey's multiple-comparison test (i, j and k).

[0090] Figure 8: Prediction of HDAC models bound to zinc and palmitate. Shown are superimpositions of AlphaFold 3-predicted structures of HDACs (gray cartoons) bound to zinc (spheres) and palmitate (blue sticks). Five predicted structures were superimposed for each HDAC of classes I (a), HA (b) and II B (c). d, Shown are per-carbon-atom local confidence pLDDT values for the aliphatic chain of palmitate, modeled into HDACs of classes I (HDACs 1, 2, 3 and 8) and Ila (HDACs 4, 5, 7 and 9). Mean ± SD of n = 5 AlphaFold 3 predictions.

[0091] Figure 9: Swissdock SCFAs. a, Free energy AG predictions of SCFAs and mocetinostat docked into an AlphaFold 3-predicted model of zinc bound HDAC1. Ten lowest free energy AG conformations were plotted separately based on the conformation relative to the catalytic center (in and out; active site and channel); Mean ± s.d.. b, Pentanoate was docked into AlphaFold 3-predicted models of zinc-bound HDACs. c, Predicted binding energies for pentanoate into HDAC 1-5 and 7-9 proteins. AG values for the best fitting orientation are shown. The protein model was obtained using AlphaFold 3. Docking was performed using SwissDock. d, Inhibition of HDAC 3 and HDAC 5 for several SCFAs. Mean ± SEM from n = 3.

[0092] Figure 10: Induction of regulatory T cell phenotype by SCFAs. a, Staining for FoxP3 following incubation of CD4+T cells with indicated SCFAs as representative histograms, b, Bar graph of frequency of FoxP3+CD4+T cells. Mean ± SEM from n=3.

[0093] Statistical analysis was performed using two-way analysis of variance (ANOVA) with Tukey's multiplecomparison test (b).

[0094] Figure 11 : Repression of Regulatory T cell phenotype by pentanoate. a, Representative flow cytometry blots of FoxP3 in CD4+T cells following the incubation with pentanoate with several concentrations for 3 days, b, Bar graph for FoxP3 expression for untreated T cells or treated with 2mM pentanoate. Mean ± SEM from n = 3. c, Representative staining for FoxP3+T-bet+of CD4+T cells following iTreg induction, d, Dot blot representative of IL-17A+IFNy+double positive CD4+T cells generated under iTreg conditions from wildtype of Tbx21Amice. Graph shows IFNy+CD4+T cells. Mean ± SEM from n=3.

[0095] Statistical analysis was performed using unpaired two-tailed Student's t test (b) or two-way analysis of variance (ANOVA) with Tukey's multiple-comparison test (d). Figure 12: Modulation of SCFAs for CD8+CAR T cell generation, a, Cytolytic activity at different E:T ratios of R0R1-CAR T cells generated with indicated metabolites. Mean ± SEM from n = 3. b, Cytokine secretion for IFN- y, TNF-a and IL-2 was measured for generated CAR T cells following the co-incubation with antigen-expressing tumor cells at a 5: 1 E:T ratio after 24 h. Mean ± SEM from n = 3.

[0096] Statistical analysis was performed using two-way analysis of variance (ANOVA) with Tukey's multiplecomparison test (a and b).

[0097] Figure 13: AG predictions of ligands docked into HDAC1. Free energy AG predictions of pentanoate and mocetinostat docked into an AlphaFold 3-predicted model of zinc and palmitate bound HDAC1. The ten lowest free energy AG conformations were plotted separately based on the conformation relative to the catalytic center (in and out; active site and channel). Mean ± s.d..

[0098] Figure 14: Histone acetylation of CD4+CAR T cells, a, Staining of H3 (Lys9 / 14) following the treatment of CD4+T cells with pentanoate or mocetinostat. 1 out of 3 performed experiments is shown, b, Specific lysis of CD8+CAR T cells either untreated or treated with TMP-195 at different E:T ratios after 6 h. Control CAR same as Figure 3. Mean ± SEM from n = 3.

[0099] Statistical analysis was performed using two-way analysis of variance (ANOVA) with Tukey's multiplecomparison test (b).

[0100] Figure 15: Pentanoate and DCA induce metabolic changes, a and b, Following incubation with T cells in the presence of pentanoate or DCA, phospho-S6 levels were measured for CD8+(a) and CD4+(b) CAR T cells by flow cytometry. 1 out of 3 performed experiments is shown, c, Measurement of 2-NBDG in CD8+CAR T cells. Mean ± SEM from n = 3. d, BODIPY-staining of CAR T cells generated with indicated metabolites. Mean ± SEM from n = 3. e and f, Increased MFI regarding mitochondrial mass (e) and PGC-1o (f) for CD4+CAR T cells following the generation in pentanoate- or DCA- supplemented medium. Mean ± SEM from n = 3.

[0101] Statistical analysis was performed using unpaired two-tailed Student's t test (c left panel, f) or one-way analysis of variance (ANOVA) with Tukey's multiple-comparison test (e right panel).

[0102] Figure 16: Pentanoate affects CAR T cells with a CD28 costimulatory domain, a, MitoFM staining for R0R1- CAR T cells. Mean ± SEM from n = 3. b, Cytolytic activity of CD8+CAR T cells against antigen-expressing tumor cell lines at different E:T ratios after 4 h. Mean ± SEM from n = 3.

[0103] Statistical analysis was performed using one-way ANOVA (a) or two-way analysis of variance (ANOVA) with Tukey's multiple-comparison test (b). Figure 17: HDAC KO T cells cytokines. Secretion of IFN-y and TNF-o of generated CD8+CAR T cells after co-incubation with PancRORI tumor cells at a 5: 1 E:T ratio for 24 h. Mean ± SEM from n = 3.

[0104] Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey's multiplecomparison test.

[0105] Figure 18: Expansion in vivo of Pentanoate or DCA / Moceti nostat treated CAR T cells, a, Frequency of CD4+R0R1- CAR T cells in the bone marrow and draining lymph node on day 14. b, Frequency of CD8+R0R1- CAR T cells in the bone marrow, blood, draining lymph node, spleen and on day 14. Mean ± SEM from n = 5 mice / group.

[0106] Statistical analysis was performed using two-way analysis of variance (ANOVA) with Tukey's multiplecomparison test (a and b).

[0107] Figure 19: Cytokine secretion for CAR T cells treated with 2-HC. CD8+CAR T cells were co-incubated with antigen-expressing tumor cells for 24 h. Cytokine secretion for IFN-y (a), TNF-o (b) and IL-2 (c) was determined by ELISA. Mean ± SEM from n = 3.

[0108] Statistical analysis was performed using two-way analysis of variance (ANOVA) with Tukey's multiplecomparison test (a-c).

[0109] Figure 20: Organ analysis ex vivo, a, CD8+ROR1-CAR T cells and endogenous CD8+T cells in the tumor on day 21. Mean ± SEM from n = 5 for CAR, n = 6 for CARpenta. b, Frequency of CD8+CAR T cells in the draining lymph node (dLN), spleen, bone marrow and blood on day 21. Mean ± SEM from spleen, BM and blood; Mean ± SEM from n = 4 for CAR, n = 5 for CARpenta for dLN. c-e, Flow cytometric analysis of IFN-y* TNF-o+and GranzymeB+IL-2+-double positive ROR1-CAR T cells from the spleen (c), dLN (d) and tumor (e) following restimulation with PMA / lonomycin for 5 h. Mean ± SEM from n = 5 mice / group and n= 4 mice / group.

[0110] Statistical analysis was performed using unpaired two-tailed Student's t test.

[0111] Figure 21: Polyclonal in vivo expansion, a, Experimental setup of in-vivo experiment with polyclonal T cells with stated treatments, b and c, Percentage and total cell number of CD8+T cells in the lymph node (b) and spleen (c). Mean ± SEM from n = 6 mice / group.

[0112] Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey's multiplecomparison test (b and c).

[0113] Figure 22: In vivo expansion of CAR T cells after late treatment, a, Experimental design for the analysis of in vivo expansion of CAR T cells following Pentanoate treatment after the CAR T cell generation, b and c, Flow cytometry staining identify the percentage of ROR1- CAR T cells and cytokine secretion of TNF-o and IFN-y in the lymph node (b) and spleen (c) following restimulation at the endpoint of the experiment; Mean ± SEM from n = 6 mice / group.

[0114] Statistical analysis was performed using unpaired two-tailed Student's t test (b and c).

[0115] Figure 23: Singe cell sequencing data ex vivo, a, Schematic illustration of in-vivo experiment for the collecting of cells for scRNA seq. b-d, Uniform Manifold Approximation and Projection (UMAP) showing the distribution of immune cells collected from mice using scRNAseq data in the LSI space. Each point represents one cell. The cells are marked by color code based on conditions (b), mouse of origin (c) and day / time points (d). e, Dot plot representing the scRNAseq expression of key genes involved in T cell biology and differentiation (CD3, CD4, CD8, Foxp3, CD28, TcfZ, CCR7, Gzmb, Gzmbk, Eomes, Pdcdl, Havcr2, Tox) measured in each subset of T cells. The dot size represents the percentage of cells with values detected in each subset. The color represents the average gene expression in each subset.

[0116] Figure 24: Hypoxia and low IL-2, a, Cytolytic activity under hypoxia conditions of generated CD8+CAR T cells against PancRORI and MC38ROR1 tumor cells at variable E:T ratios after 6 h. b, Specific lysis of antigen- presenting tumor cells by CD8+CAR T cells generated with low IL-2 conditions of after 24 h. Mean ± SEM from n = 3.

[0117] Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey's multiplecomparison test (a and b).

[0118] Figure 25: Pentanoate abundance is a predictor of clinical CAR T cell, a and b, Probability of PFS (a) and overall survival (b) in a cohort of patients receiving CAR T cell therapy.

[0119] DETAILED DESCRIPTION OF THE INVENTION

[0120] Unless otherwise defined below, the terms used in the present invention shall be understood in accordance with their common meaning known to the person skilled in the art. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. Publications referred to herein may be cited by specifying the full literature reference in the text, or by naming the author and the publication year (e.g., "Luu M et al., 2019”) and by specifying the corresponding full literature reference in the "references” section, or by a reference number and by specifying the corresponding full literature reference in the "references” section.

[0121] Definitions and General Techniques

[0122] Where, in the context of the present invention, reference is made to a method comprising particular method steps (e.g., steps (A), (B), and (C)), it is to be understood that the steps are to be carried out in their indicated order, i.e. , in the order in which they have been listed. An immunoreceptor according to the invention is a transmembrane receptor, which, when expressed by an immune cell, is capable of mediating an immune response. The immunoreceptor can be an endogenous immunoreceptor or a non-natural immunoreceptor, i.e., genetically engineered. Exemplary immunoreceptors in accordance with the invention are B-cell receptors (BCRs), T-cell receptors (TCRs), and chimeric antigen receptor (CARs). The immunoreceptor in its monomeric form may either consist of a single molecule comprising all of its domains or consist of a heterodimer that comprises all of its domains. The immunoreceptor can bind to its antigen either directly, or it can bind indirectly through an adapter.

[0123] The immunoreceptor according to the invention can comprise an antigen-binding domain which comprises a first domain, linker, and optionally a second domain. The first and second domain are not limited to a specific molecular orientation, i.e. both first and second domain can be located N-terminal or C-terminal to each other. Optionally, the second domain can be absent, i.e. the antigen-binding domain can be comprised of the first domain and the linker, in any orientation in respect of N-terminal or C-terminal orientation. An exemplary embodiment of an antigen-binding domain is a single chain variable fragment (scFv). In this case, the first domain can comprise a light chain variable domain or a heavy chain variable domain, and the second domain can comprise a light chain variable domain or a heavy chain variable domain, which are connected by a peptide linker. The first and second domain can both either be located at the N-terminus of the scFv, or at the C-terminus of the scFv.

[0124] In one embodiment, the immunoreceptor is capable of binding to an antigen, preferably a cancer antigen, more preferably a cancer cell surface antigen. In a preferred embodiment, the immunoreceptor is capable of binding to extracellular domain of a cancer antigen. In a preferred embodiment, the immunoreceptor is a chimeric antigen receptor. In a preferred embodiment, the immunoreceptor is a genetically engineered T-cell receptor.

[0125] It will be understood that a CAR and / or a TCR in accordance with the invention binds to an antigen, preferably a cancer cell antigen. While a CAR typically binds to an extracellular domain of a cancer cell antigen, i.e., a cancer cell surface antigen, a TCR typically binds to an intracellular cancer cell antigen presented by a HLA molecule.

[0126] As used in connection with the invention, the terms "binds”, "binding” or "bind” refer to specific binding to the antigen of interest. It is to be understood that where the terms "binds”, "binding” or "bind” are mentioned, they refer to the intrinsic capability of the CAR or TCR to specifically bind to the antigen without further modification, but they do not require that the antigen must actually be present. In a preferred embodiment, the immunoreceptor is expressed in T cells. In a preferred embodiment of the invention, the immunoreceptor is expressed in T cells and allows said T cells to bind specifically to antigenexpressing cancer cells with high specificity to exert a growth inhibiting effect, preferably a cytotoxic effect, on said cancer cells.

[0127] In accordance with the invention, the term "T-cell receptor (TCR)” has the meaning known in the art. Typically, a TCR is understood as a heterodimeric cell surface protein of the immunoglobulin superfamily that participate in the activation of T cells in response to the binding of an antigen. The TCR complex can consist of TCRo / B chains and CD3y / 5 / E / subunits, which can associate through hydrophobic interactions. Somatic VDJ recombination allows to generate distinct TCRo and TCRB chains, and TCRoB heterodimers are generally responsible for antigen recognition by binding to peptide-MHC complexes. CD3 can transmit the TCR-triggered signal through immunoreceptor tyrosine-based activation motifs (ITAMs) in its cytoplasmic tail, but it is generally not directly involved in antigen recognition. ITAMs are tandem duplications of a tyrosine-containing sequence (YXXL / I), and the CD3y / 5 / E chains each contain one ITAM, while the CD3 chain contains three. As a consequence of TCR engagement, ITAM phosphorylation can be induced by protein tyrosine kinases (PTKs), which allow other effector molecules to interact with the TCR complex. A TCR can be found on the surface of a cell or in soluble form. The TCR can be an intact or full-length TCR, including but not restricted to a TCR in the oB form or yd form, as a dimeric TCR (dTCR), a single-chain TCR (scTCR). The TCR is an antigen-binding portion that is less than a full-length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC- peptide complex. In some cases, an antigen-binding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as MHC- peptide complex, to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable a chain and variable B chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of a TCR contain complementarity determining regions involved in recognition of the peptide, MHC and / or MHC-peptide complex. A TCR can contain a constant domain, a transmembrane domain and / or a short cytoplasmic tail. Each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end. A TCR can be associated with invariant proteins of the CD3 complex involved in mediating signal transduction.

[0128] In accordance with the invention, the term "chimeric antigen receptor (CAR)” has the meaning known in the art. A "CAR” according to the invention can be any possible form. Typically, a CAR is understood as a receptor protein that have been engineered to give T cells the new ability to target a specific antigen. The receptor is chimeric in that it combines both antigen-binding and T cell activating functions into a single receptor. CAR T cells can be derived either from T cells in a patient's own blood (autologous) or from the T cells of another, healthy, donor (allogeneic). Once isolated from a person, these T cells can be genetically engineered to express a specific CAR, which programs them to target an antigen, e.g., an antigen that is present on the cancer cell surface. In a preferred embodiment, the chimeric antigen receptor is expressed in immune cells, preferably T cells. In a preferred embodiment of the invention, the chimeric antigen receptor is expressed in T cells and allows said T cells to bind specifically to antigen-expressing cancer cells with high specificity to exert a growth inhibiting effect, preferably a cytotoxic effect, on said cancer cells.

[0129] A CAR is designed to enhance the recognition and targeting of cancer cells or other diseased cells. A CAR according to the invention typically comprises, but is not limited to, three main components: an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular domain, often derived from an antibody fragment, provides specificity for the target antigen. The transmembrane domain anchors the receptor in the lymphocyte (e.g., T cell) membrane, while the intracellular signaling domain initiates activation signals upon antigen binding. When expressed in lymphocytes (e.g., T cells), the CAR can allow the modified lymphocytes (e.g., T cells) to recognize and bind to the target antigen, leading to the activation and killing of the target cells expressing the target antigen.

[0130] It will also be understood that the CAR can be any known type of CAR, e.g., a first-generation CAR, a second- generation CAR, a third-generation CAR, a fourth-generation CAR or a fifth-generation CAR. A first-generation CAR generally has an intracellular signalling domain comprising an intracellular signalling domain of CD3 , FcyRI, or other ITAM-containing activating domain to provide a T cell activation signal. Second and third- generation CARs further comprise a costimulatory signalling domain (e.g., a costimulatory signalling domain from an endogenous T cell costimulatory receptor, such as CD28, 4-1 BB, or ICOS) or two costimulatory signalling domains, respectively. A fourth generation CAR, instead, may express one or two costimulatory molecules together with a constitutive or inducible expression cassette containing a transgenic protein such as a cytokine or enzyme. A fifth generation CAR is known in the art and may comprise an additional intracellular domain compared to the first to fourth generation CAR. The CAR may comprise, but is not limited to, truncated intracellular domains of cytokine receptors (e.g., IL-2R chain fragment) with a motif for binding transcription factors such as STAT-3 / 5.

[0131] For CAR T cell therapy, T cells are usually manipulated and expanded ex vivo. However, in accordance with the invention, there is also the option to conduct gene transfer in vivo. One way to program immune cells such as T cells within the body is the gene transfer with DNA-carrying nanoparticles. This has, for instance, been described by Smith et al., 2017. A second strategy is the in vivo CAR immune cell (e.g. CAR T cell) generation with viral vectors. This has, for instance, been described by Agarwal et al., 2019. In a preferred embodiment in accordance with the invention, immune cells are isolated from a healthy donor or a patient having cancer, transduced with a gene transfer vector encoding an immunoreceptor.

[0132] "Immune cells” as used in the invention are not particularly limited and include, for example, T cells, B cells, NK cells, NKT cells, Peripheral Blood Mononuclear Cell (PMBCs) macrophages and stem cells. In a preferred embodiment, the T cells are CD8+ T cells or CD4+ T cells. In accordance with the invention, immune cells such as T cells, NK cells or PBMCs can be isolated from a patient, genetically modified (e.g. transduced) with a gene transfer vector encoding a chimeric antigen receptor according to the invention and administered to the patient in accordance with the methods and uses of the invention. In a preferred embodiment, the T cells are CD8+T cells or CD4+T cells. Alternatively, allogenic immune cells such as T cells, NK cells or PBMCs, from donors, preferably healthy donors, can be used. They can be genetically modified (e.g. transduced) with a gene transfer vector encoding a chimeric antigen receptor according to the invention and administered to the patient in accordance with the methods and uses of the invention. In a preferred embodiment, the T cells are CD8+T cells or CD4+T cells.

[0133] The term antibody as used herein refers to any functional antibody that is capable of specific binding to the antigen of interest. Without particular limitation, the term antibody encompasses antibodies from any appropriate source species, including avian such as chicken and mammalian such as mouse, goat, non-human primate and human. Preferably, the antibody is a humanized or human antibody. Humanized antibodies are antibodies which contain human sequences and a minor portion of non-human sequences which confer binding specificity to an antigen of interest (e.g. human FLT3). The antibody is preferably a monoclonal antibody which can be prepared by methods well-known in the art. The term antibody encompasses an lgG-1, -2, -3, or -4, IgE, IgA, IgM, or IgD isotype antibody. The term antibody encompasses monomeric antibodies (such as IgD, IgE, IgG) or oligomeric antibodies (such as IgA or IgM). The term antibody also encompasses— without particular limitations— isolated antibodies and modified antibodies such as genetically engineered antibodies, e.g., chimeric antibodies or bispecific antibodies.

[0134] An antibody fragment or fragment of an antibody as used herein refers to a portion of an antibody that retains the capability of the antibody to specifically bind to the antigen. This capability can, for instance, be determined by determining the capability of the antigen-binding portion to compete with the antibody for specific binding to the antigen by methods known in the art. Without particular limitation, the antibody fragment can be produced by any suitable method known in the art, including recombinant DNA methods and preparation by chemical or enzymatic fragmentation of antibodies. Antibody fragments may be Fab fragments, F(ab') fragments, F(ab')2 fragments, single chain antibodies (scFv), single-domain antibodies, diabodies or any other portion(s) of the antibody that retain the capability of the antibody to specifically bind to the antigen. In accordance with the invention, the term "cancer cell antigen” has the meaning known in the art. In those embodiments of the invention where the lymphocyte contains and expresses a recombinant nucleic acid or a set of recombinant nucleic acids encoding a chimeric antigen receptor, the cancer cell antigen in accordance with the invention to which the chimeric antigen receptor binds is preferably a cancer cell surface antigen. In those embodiments of the invention where the lymphocyte contains and expresses a recombinant nucleic acid or a set of recombinant nucleic acids encoding a T-cell receptor, the cancer cell antigen in accordance with the invention to which the T-cell receptor binds is preferably an intracellular cancer cell antigen presented by a HLA molecule.

[0135] The term "capable of binding” as used herein refers to the capability to form a complex with a molecule that is to be bound (e.g. CD19, FLT3, BCMA, or R0R1). Binding typically occurs non-covalently by intermolecular forces, such as ionic bonds, hydrogen bonds and Van der Waals forces and is typically reversible. Various methods and assays to determine binding capability are known in the art. Binding is usually a binding with high affinity, wherein the affinity as measured in KD values is preferably is less than 1 pM, more preferably less than 100 nM, even more preferably less than 10 nM, even more preferably less than 1 nM, even more preferably less than 100 pM, even more preferably less than 10 pM, even more preferably less than 1 pM.

[0136] The term "reprogramming” or "metabolic reprogramming” as used herein, refers to a variety of cellular alterations in bioenergetic pathways to adapt to the cellular metabolic needs. Metabolic reprogramming is a hallmark of the immune cells in response to inflammatory stimuli. In particular, in the present application reprogramming of an immune cells is characterized by increases their ability to secrete effector cytokines, cytotoxic function and persistence in vivo via preservation of a naive-like phenotype and / or reduction of exhaustion.

[0137] In accordance with the methods for obtaining a modified immune cells of the invention, the step of introducing the recombinant nucleic acid or set of recombinant nucleic acids into an immune cell may be performed by using any appropriate standard techniques as known in the art, for example, electroporation, electro-injection, microinjection, calcium phosphate co-precipitation, a calcium chloride / rubidium chloride method, retroviral and lentiviral infection, DEAE-dextran, a cationic liposome method, polyethylene glycol-mediated uptake, gene guns, etc., but is not limited thereto. The nucleic acid or set of recombinant nucleic acids encoding the positive regulator of autophagy may be introduced prior to, simultaneously with, or after introduction of the nucleic acid or set of recombinant nucleic acids encoding and expressing the T-cell receptor or chimeric antigen receptor.

[0138] The term "vector” is known in the art and encompasses, for instance, a plasmid, a retroviral vector, a lentiviral vector, an adeno-virus vector, an adeno-associated virus vector or a transposon vector. It is understood that a vector as used in connection with the present invention is a vector which is suitable for therapeutic applications in humans.

[0139] Methods for obtaining a modified human immune cell

[0140] The methods for isolating / obtaining a human immune cell according to the invention are as defined herein, including the claims. They may be performed ex vivo, e.g., using isolated human immune cells as starting material. That is, preferably, the method for obtaining a human immune cell according to the invention is not a method for treatment of the human or animal body by surgery or therapy and is not a diagnostic method practiced on the human or animal body.

[0141] Methods of Treatments and Uses of Lymphocytes in such Treatments

[0142] Terms such as "treatment of cancer” or "treating cancer” according to the present invention refer to a therapeutic treatment. An assessment of whether or not a therapeutic treatment works can, for instance, be made by assessing whether the treatment inhibits cancer growth in the treated patient or patients. Preferably, the inhibition is statistically significant as assessed by appropriate statistical tests which are known in the art. Inhibition of cancer growth may be assessed by comparing cancer growth in a group of patients treated in accordance with the present invention to a control group of untreated patients, or by comparing a group of patients that receive a standard cancer treatment of the art plus a treatment according to the invention with a control group of patients that only receive a standard cancer treatment of the art. Such studies for assessing the inhibition of cancer growth are designed in accordance with accepted standards for clinical studies, e.g. doubleblinded, randomized studies with sufficient statistical power. The term "treating cancer” includes an inhibition of cancer growth where the cancer growth is inhibited partially (i.e. where the cancer growth in the patient is delayed compared to the control group of patients), an inhibition where the cancer growth is inhibited completely (i.e. where the cancer growth in the patient is stopped), and an inhibition where cancer growth is reversed (i.e. the cancer shrinks). An assessment of whether or not a therapeutic treatment works can be made based on known clinical indicators of cancer progression.

[0143] A treatment of cancer according to the present invention does not exclude that additional or secondary therapeutic benefits also occur in patients. For example, an additional or secondary benefit may be an enhancement of engraftment of transplanted hematopoietic stem cells that is carried out prior to, concurrently to, or after the treatment of cancer. However, it is understood that the primary treatment for which protection is sought is for treating the cancer itself, and any secondary or additional effects only reflect optional, additional advantages of the treatment of cancer growth. The treatment of cancer according to the invention can be a first-line therapy, a second-line therapy, a third-line therapy, or a fourth-line therapy. The treatment can also be a therapy that is beyond fourth-line therapy. The meaning of these terms is known in the art and in accordance with the terminology that is commonly used by the US National Cancer Institute.

[0144] The invention also provides a method of treating cancer, an infectious disease or an autoimmune disease, such as a chronic inflammatory disease or degenerative disease, using the recombinant immune cell, or pharmaceutical composition as described herein, preferably cancer.

[0145] Cancer includes all known malignancies, e.g., hematologic malignancies such as leukemia, lymphoma, multiple myeloma; solid tumors such as breast, ovarian, adrenocortical, thyroid cancer, or lung cancer, or pancreatic cancer. For instance, the cancer may be Renal cell carcinoma (ccRCC), B cell Acute Lymphoblastic Leukaemia (B-ALL), Chronic Lymphocytic Leukaemia (CLL), Diffuse Large B-cell Lymphoma (DLBCL), Follicular Lymphoma (FL), Mantle Cell Lymphoma (MCL), Marginal Zone Lymphoma (MZL), Burkitt Lymphoma, Multiple Myeloma (MM), Acute Myeloid Leukaemia (AML), Hodgkin Lymphoma (HL), T-cell lymphoma, Hairy Cell Leukaemia (HCL), Triple-Negative Breast Cancer (TNBC), Non-small cell Lung Cancer (NSCLC), Small Cell Lung Cancer (SCLC), Ovarian Cancer, Pancreatic Cancer, Gastrointestinal Cancers (Colorectal, Gastric, Oesophageal), Prostate Cancer, Glioblastoma (GBM), Neuroblastoma, Sarcomas (Osteosarcoma, Ewing's Sarcoma, Rhabdomyosarcoma, Chondrosarcoma), or Mesothelioma.

[0146] It is understood that where, in the context of the present invention, reference is made to a substance (e.g., CAR T cell) or composition for use in a treatment (e.g., treatment of a cancer in a subject), the disclosure of the present invention is meant to also encompass the use of said substance or composition in the manufacture of a medicament for said treatment.

[0147] A "subject” in the context of the invention is mammalian, such as human, mouse, rat or macaque. Preferably, it is a human subject. The subject is preferably a (human) subject diagnosed with a disease to be treated using immunotherapy.

[0148] As used herein, each occurrence of terms such as "comprising” or "comprises” may optionally be substituted with "consisting of’ or "consists of’.

[0149] A pharmaceutically acceptable carrier, including any suitable diluent or, can be used herein as known in the art. As used herein, the term "pharmaceutically acceptable” means being approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopia, European Pharmacopia or other generally recognized pharmacopia for use in mammals, and more particularly in humans. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. It will be understood that the formulation will be appropriately adapted to suit the mode of administration.

[0150] Compositions and formulations in accordance with the present invention are prepared in accordance with known standards for the preparation of pharmaceutical compositions and formulations. For instance, the compositions and formulations are prepared in a way that they can be stored and administered appropriately, e.g., by using pharmaceutically acceptable components such as carriers, excipients or stabilizers. Such pharmaceutically acceptable components are not toxic in the amounts used when administering the pharmaceutical composition or formulation to a patient. The pharmaceutical acceptable components added to the pharmaceutical compositions or formulations may depend on the chemical nature of the tyrosine kinase inhibitor present in the composition or formulation (depend on whether the targeting agent is e.g., an antibody or fragment thereof or a cell expressing a chimeric antigen receptor), the particular intended use of the pharmaceutical compositions and the route of administration.

[0151] The administration route of in vitro activated immune cells for use in cellular immune therapy is well known by a skilled person.

[0152] In a preferred embodiment in accordance with the invention, the composition or formulation is suitable for administration to humans, preferably the formulation is sterile and / or non-pyrogenic.

[0153] EXAMPLES

[0154] The present invention is further illustrated by the following non-limiting examples:

[0155] Study design and patients

[0156] The study was designed as a prospective observational study. All patients receiving CAR-T cell therapy were eligible for this study regardless of the target antigen. Participants were enrolled from 05 / 2020 to 12 / 2023 at two centers: the University Medical Center Regensburg and the University Hospital Heidelberg. All patients provided informed written consent. The study was approved by the Institutional Ethics Review Board of the University of Regensburg, vote no. 21-2521-101. Fecal samples were collected from all patients prior to CAR T cell infusion. Fecal samples were prepared and short-chain fatty acids were measured as previously described (Thiele Orberg E. et al. 2024). Briefly, metabolites from 100mg feces were extracted using a 15-ml bead-beater tube (CKMix50, 15 ml, Bertin Technologies) filled with 2.8-mm and 5.0-mm ceramic beads and a bead-beater (Precellys Evolution, Bertin Technologies) at 10,000 rpm (3 rounds of 30s with 15s breaks). Methanol-based dehydrocholic acid extraction solvent (5 ml, c = 1.3 pmol 1-1) was added as an internal standard to account for work-up losses. Short chain fatty acids were measured using the 3-NPH method in a QTRAP 5500 triple quadrupole mass spectrometer (SCI EX) coupled to an ExionLC AD (SCI EX) ultra-highperformance liquid chromatography system. Data were analyzed with MultiQuant 3.0.3 (SCI EX) and MetaboAnalyst.

[0157] An external cohort of 60 patients with large B cell lymphoma or follicular lymphoma, treated with commercial CD19-directed CAR T cells, was used to validate the association between stool pentanoate levels and CAR T therapy outcome. Stool samples collected between day -30 and day 0 (median: day -3 [IQR: -4.3 to -1]) were aliquoted and stored at -80°C. One sample per patient was sent to Metabolon for metabolomic profiling Sample Preparation: Feces samples (fresh / frozen) are analyzed for eight short chain fatty acids: acetic acid (C2), propionic acid (C3), isobutyric acid (C4), butyric acid (C4), 2-methy l-buty ric acid (C5), isovaleric acid (C5), valeric acid (C5) and caproic acid (hexanoic acid, C6) by LC-MS / MS. Samples are spiked with stable labelled internal standards and are homogenized and subjected to protein precipitation with an organic solvent. After centrifugation, an aliquot of the supernatant is derivatized. The reaction mixture is diluted, and an aliquot is injected onto an Agilent 1290 Infinity or Infinity II / Sciex QTrap 5500 or 6500 LC MS / MS system equipped with a C18 reversed phase UHPLC column. The mass spectrometer is operated in negative mode using electrospray ionization (ESI). Sample Analysis: The peak area of the individual analyte product ions is measured against the peak area of the product ions of the corresponding internal standards. Quantitation is performed using a weighted linear least squares regression analysis generated from fortified calibration standards prepared immediately prior to each run. LC-MS / MS raw data are collected and processed using AB SCIEX software Analyst 1.6.3 and processed using SCIEX OS-MQ software. QA / QC: Three levels of QCs are prepared in feces by diluting and / or spiking with stock solutions to obtain the appropriate concentrations for each level (low / med / high). Accuracy will be evaluated using the corresponding QC replicates in the sample runs. Targeted acceptance criteria are at least 50% of QC samples at each concentration level per analyte should be within ±20.0% of a set mean and at least 2 / 3 of all QC samples per analyte should fall within ±20.0% of the corresponding mean.

[0158] Animals

[0159] C57BL / 6 wild-type mice were purchased from Charles River and maintained under specific pathogen free conditions at the Center for Experimental Medicine (ZEMM) at the University of Wurzburg. Mice were kept under a 12 / 12 h light / dark cycle between 20-24 °C in individually ventilated cages. Mice had access to standard chow and autoclaved water ad libitum and the health status of the animals was inspected by the responsible animal caretakers. Male and female mice between 6-12 weeks old at the time of the experiment were used in this study. All animal protocols were approved by government (Approval number: 1457, Regierung von Unterfranken, Bayern, Germany). Tbx21~'~ mice (on C57BL / 6 background) were maintained under specific pathogen free (SPF) conditions at the animal facility of the Philipps-University of Marburg, Germany.

[0160] Hdac1fl / flHdac2fl / fl(HDAC1-2cK0) CD4-Cre mice (Mouse Genome Informatics [MGI] 4440556 for Hdacl; MGI 4440560 for Hdac2) were previously described and kept under specific pathogen free conditions at the Medical University of Vienna (Preglej T et al. 2020; Arapaia N et al., 2013).

[0161] For in-vivo experiments, male or female mice between 6-8 weeks at the time of tumor engraftment were used which were not involved in previous procedures. Mice were monitored daily and euthanized when mice reached limits designated in the approved protocols.

[0162] Cell lines

[0163] The mouse MC38 colon adenocarcinoma cell line was provided by the lab of Tobias Bopp / Toska Bohn. The mouse Panc02 OVA pancreatic tumor cell line was gifted from the Christian Bauer lab. All solid tumor cell lines were cultured in standard DMEM (Gibco) supplemented with 10 % heat-inactivated FCS (Gibco) and 1 % penicillin / streptomycin (Gibco) at 37 °C with 5 % CO2. The mouse lymphoma cell line E -myc was supplied by the lab of Dirk Busch and cultured in standard RPMI 1640 (Gibco) with heat-inactivated 10 % FCS (Gibco) and 1 % penicillin / streptomycin (Gibco) at 37 °C with 5 % CO2

[0164] The Platinum-E retroviral packaging cell line (Cell Biolabs) was cultured in standard DMEM (Gibco) supplemented with 10 % heat-inactivated FCS and 1 % penicillin / streptomycin (both Gibco) at 37 °C with 5 % CO2. Following thawing, PlatinumE cells were selected for 5 days using 1 pig / ml puromycin and 10 pig / ml blasticidin S (both Invivogen) and subsequently cultured. All cell lines were tested for mycoplasma contamination.

[0165] Vector construction

[0166] The R11-ROR1-specific CAR containing the lgG4 hinge-CH2-CH3, CD28 transmembrane domain, and a signaling module comprising the cytoplasmic domains of 4-1 BB and CD3z was described earlier (Luu M et al., 2021). As a transduction marker, a murine truncated CD19 marker separated from the CAR by a T2A ribosomal skip element was used. A similar ROR1 CAR containing the murine CD28 co-stimulatory domain was cloned creating MP71_R11_CD28_mCD19t. The CD19- specific CAR incorporates a CD8a hinge and transmembrane domain, as well a CD28 costimulatory domain followed by CD3 . A human truncated EGFR is placed as a transduction marker downstream of the CAR. The sequence was cloned into the MP71 retroviral vector creating MP71-CD19-hEGFRt. For the generation of a construct containing only truncated CD 19, the CAR segment in the MP71_R11_mCD19t construct was deleted via site-directed mutagenesis using the Q5 Site-Directed Mutagenesis Kit (NEB).

[0167] Virus production Platinum-E cells for retroviral packaging were co-transfected with the desired construct in addition with retroviral packaging construct pCL-10A1, using the Effectene transfection reagent (QIAGEN) according to the manufacturer's instructions. The retroviral supernatant was collected 48 and 72 h after transfection, pooled and stored at -80 °C until use.

[0168] Generation of ROR1- / CD19-expressing tumor cell lines

[0169] All cell lines were lentivirally transduced to express GFP and firefly luciferase. Additionally, MC38 and Panc02 - OVA were transduced to co-express murine ROR1 . E-myc cells were lentivirally transduced to express murine CD19. After harvesting the cells, 1x105cells were seeded in 500 pl medium with 5 pg / ml polybrene in a 48-well plate in addition with the stated Lentivirus at a final MOI of 5. The cells were washed 48 h after transduction and expanded for 10 days before being sorted with the MACSQuant® Tyto® Cell sorter.

[0170] In vitro T cell differentiation and culture

[0171] For in-vitro cultures, murine T cells from male or female mice were isolated from single cell suspension of spleen and lymph nodes. Isolation of CD8+T cells was performed by positive selection (Miltenyi Biotech), followed by a negative selection for CD4+T cells (Invitrogen). T cells were cultured unless stated otherwise in modified RPMI 1640 medium (Gibco) supplemented with 10 % heat-inactivated FCS (Gibco), 50 pM 2-mercaptoethanol (Gibco), 1 % penicillin / streptomycin and 1 % GlutaMAX-l (Gibco) at 37 °C with 5 % CO2.

[0172] Culture plates were pre-coated with 10 pg / ml polyclonal anti-hamster IgG (MP Biomedicals) for 2 h and washed once with PBS (Gibco). For Th1 differentiation, 0.5x106CD4+T cells were activated in the presence of 1 pg / ml anti-CD3 (Biolegend, 145-2C11), 1 pg / ml anti-CD28 (Biolegend, 37.51), 1 pg / ml anti-IL-4 (Biolegend, BVD4- 1D11), 50 U / ml recombinant human (rh) IL-2 (Miltenyi Biotech) and 10 ng / ml IL-12 (Miltenyi Biotech).

[0173] For supoptimal CTL differentiation, 0.5x106CD8+T cells were cultured with 1 pg / ml anti-CD3, 1 pg / ml anti- CD28, 1 pg / ml anti- IFN-y (Invitrogen, XMG1.2), 1 pg / ml anti- IL-4 and 50 U / ml rh IL-2.

[0174] For iTreg differentiation, CD4+T cells were activated with 1 pg / ml anti-CD3, 0.5 pg / ml anti-CD28, 100U / ml rh IL- 2, 2 g / ml anti- IFN-y, anti- IL-4 and 2 ng / ml rhTGF-p1 (R&D Systems).

[0175] In some experiments, medium of cells was added with 2 mM sodium Pentanoate (Ambeed), 5 mM dichloroacetate (Merck), 100 nM mocetinostat (Biomol), 1 pM TMP-195 (Biomol), 1 mM Butyrate (Sigma), 2 mM Propionate (Merck), 10 mM Acetate (Merck) or a combination of DCA and mocetinostat once 2 hours after activation. Furthermore, 5 mM 2-hydrocycitrate (2-HC) (Sigma) was added to cell culture at indicated time points. All exogenous metabolites were dissolved in DMSO solutions according to the manufacturer's recommendation.

[0176] Generation of murine CAR T cells

[0177] 48 hours after activation, the medium of the cells was removed and stored at 4°C. Cells were transduced with retroviral supernatant in addition with 10 pg / ml polybrene by spin-infection at 800 rpm for 2 hours at 32 °C and

[0178] T1 placed in an incubator for 4 hours. Afterwards, the supernatant was replaced by the stored medium. The next day, T cells were put into new culture-plates. On day 4, fresh medium including 50 U / ml rh IL-2, 10 ng / ml IL-7 and 10 ng / ml IL-15 (all Miltenyi) was added. Cells were maintained between 0.5x106and 2x106cells / ml and expanded until day 7.

[0179] Flow cytometry

[0180] T cells were harvested, washed once with PBS and stained for surface antigens with fluorophore-conjugated antibodies for 20 min in PBS at 4 °C in the dark. The following antibodies were used for the detection of cell surface proteins: mouse CD3 (Pacific blue™ / PE-Cyanine7, 17A2, BioLegend), mouse CD8a (APC-Cyanine7 / Pacific blue™ / PE-Cyanine7, 53-6.7, Biolegend), mouse CD4 (FITC / APC-Cyanine7, GK1.5, Biolegend), mouse CD19 (PE / FITC / PerCP-Cyanine5.5, 1D3 / CD19, BioLegend), human EGFR (APC, Rituximab / Erbitux, selfconjugated).

[0181] For staining of intracellular cytokines, cells were stimulated with 100 ng / ml phorbol-12-myristat-acetate (PMA) and 1 pig / ml lonomycin in the presence of 5 pig / ml brefeldin A for 4-5 hours at 37 °C. To detect intracellular cytokines, cells were fixed with 2 % paraformaldehyde (Invitrogen) for 20 min at 4°C. Following antibodies were used for intracellular cytokines: mouse IFN-y (APC / APC-Cyanine7 / Pacific blue™, XMG1.2, Biolegend), mouse TNF-a (PE, MP6-XT22, Biolegend), mouse IL-2 (PE-Cyanine7, JES6-5H4, Biolegend), Anti-human / mouse recombinant Granzyme B (PerCP-Cyanine5.5, APC, QA16A02).

[0182] For the detection of transcription factors, activated T cells were fixed with the Foxp3 I Transcription Factor Staining buffer set following manufacturer's instructions (eBioscience). Intracellular staining for PGC1a (Alexa Fluor® 647, D-5, Santa Cruz) was conducted for 30 min in Saponinbuffer at 4 °C. To assess neutral lipid content, cells were stained with BODIPY 493 / 503 (Invitrogen) for 20 min at RT.

[0183] For the quantification of mitochondrial volume, T cells were labeled with 200 nM MitoTracker Deep Red (Invitrogen) and stained for 30 min at 37 °C.

[0184] Glucose uptake by T cells was measured by the fluorescent glucose analog 2-NBDG (Cayman Chemicals). After starving cells for 30 min in glucose-free medium, T cells were incubated with 200 piM 2-NBDG for 20 min at 37 °C, washed and analyzed.

[0185] To assess phosphorylation of intracellular STAT5, cells were stained as described before (Luu M et al., 2021). In brief, pretreated T cells were washed with RPMI without supplements and starved for 4 hours at 37 °C. Afterwards, medium containing 50 U / ml rh IL-2 was added and cells were fixed with 2 % PFA before fixation of the cells at different timepoints. Samples were put on ice and permeabilized by the addition of methanol. Cells were washed three times with phospho-washing buffer (PBS, 2 % FCS, 0.2 % Tween-20) before being stained with 0.5 ug of p-STAT5 for 45 min at RT.

[0186] To evaluate histone modifications, stimulated T cells were fixed with the Foxp3 / TF staining buffer set (eBioscience) for 30 min at RT and stained with anti-mouse acetyl-histone H3 (Lys27) or anti-mouse acetyl- histone H3 (Lys97Lys14) (both Cell Signaling) for 30 min at 4 °C. After washing, anti-histone antibodies were detected using a donkey polyclonal anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 546 (Invitrogen) in Saponin buffer for 30 min.

[0187] For exclusion of dead cells, all stainings were performed with either Zombie aqua (Biolegend) or 7AAD (Miltenyi). All data was collected on a BD Canto II and analyzed using FlowJo. MFI marks median fluorescence intensity calculated by FlowJo.

[0188] Cytotoxicity assay

[0189] To determine antigen-specific tumor cell lysis, 5 x 103ffLuc-transduced tumor cells were co-cultured with CAR- transduced or untransduced (UTD) T cells at effectortarget (E:T)-ratios of 10:1 , 5:1 , 2.5: 1 or 1.25: 1 in triplicates in RPMI medium supplemented with 150 ng / ml D-Luciferin. For experiments under hypoxia conditions, cells were plated in an incubator with 2 % O2. The bioluminescence signal was measured on a Tecan Infinite 200 PRO plate reader after 4, 6, 8 and 24 h. Specific lysis was determined in reference to the corresponding untransduced T cells.

[0190] Cytokine secretion

[0191] Secretion of cytokines for murine CAR T cells was detected by ELISA kits for mouse IFN-y, IL-2, Granzyme B (all Biolegend) and TNF-o (Invitrogen) according to the manufacturer's instructions. Cells were co-incubated with 5:1 or 2.5: 1 E:T ratio with antigen-presenting tumor cells in triplicates for 24 h. Absorbance was measured using a Tecan Infinite 200 PRO plate reader.

[0192] HDAC docking methods

[0193] Structure predictions of HDACs 1-5, 7-9 with zinc ions (one zinc for HDACs 1 and 2, two zinc ions for HDACs 3-5 and 7-9) with and without palmitate were performed using AlphaFold 3 (Abramson J et al., 2024). SCFAs and mocetinostat were docked into AlphaFold 3-generated HDAC1 models using the Swissdock attracting cavities docking engine (Bugnon M et al., 2024; Grosdidier A et al., 2011; Zoete V et al., 2016). Medium sampling exhaustivity and buried cavity prioritization parameters were used for all docking jobs. Random initial conditions (RIC) were set to 3 for docking SCFAs with HDAC1. RIC were set to 1 for docking mocetinostat into HDAC1 and pentanoate into HDAC4. Models were visualized and analyzed using UCSF ChimeraX (Meng EC et al., 2023). Palmitate molecules in the AlphaFold 3 models were truncated to C5 using UCSF ChimeraX and used for free energy calculations alongside models with palmitate. Free energy calculations were performed using PRODIGYLIGAND (Honorato RV et al., 2021 ; Vangone A et al., 2019) .

[0194] HDAC activity assays For the impact of SCFAs on specific HDAC isoforms, the fluorogenic assay for each HDAC enzyme (HDAC1-3, HDAC5) was used (BPS Bioscience). Assays were conducted according to the manufacturer's instructions, with measurements taken in triplicates using the FLUOstar Omega plate reader.

[0195] Metabolomic profiling

[0196] To analyze polar intracellular metabolites, CD8+and CD4+CAR T cells were generated as described above. After removal from the antibody on day 3, cells were put into medium containing 1 g / l13C- glucose / reglutamine for 24 hours. Per condition, 1x106T cells were washed with 0.9 % Natriumchloride. Metabolite extraction was conducted by incubation with ice-cold 80 % methanol including internal standards. Following an incubation time of 20 min at 4 °C, cells were pelleted and the supernatant containing the polar metabolites was transferred into a new tube and stored at -80 °C until further processing. For the tracking of pentanoate, 2 mM of13C-labeled pentanoate was added at day 0 following activation and samples were taken from day 1 until day 4 after activation and isolated for metabolites as described above.

[0197] Metabolites were automatically derivatized using a Gerstel MPS. Derivatization was done with 15 pi I of 2 % (w / v) methoxyamine hyprochloride (Thermo Scientific) in pyridine and 15 pl N-tertbutyldimethylsilyl-N- methyltrifluoroacetamide with 1 % tert-butyldimethylchlorosilane (Regis Technologies). Measurement was carried out by GC / MS with a 30 m DB-35MS + 5 m Duraguard capillary column (0.25 mm inner diameter, 0.25 pim film thickness) equipped in an Agilent 7890B gas chromatograph (GC) connected to an Agilent 5977A mass spectrometer (MS).

[0198] The GC oven temperature was held at 80 °C for 6 min and steadily adjusted at 6 °C per min until reaching 280 °C where the temperature was held for 10 min. The quadropole was set to 150 °C. The MS source operated under electron impact ionization mode at 70 eV and was held at 230°C.

[0199] Targeted single ion chromatogram measurements were conducted for pyruvate (174, 175, 176, 177, 178, 179; 10 scans per second), lactate (261, 262, 263, 264, 265, 266, 267; 10 scans per second), citrate (591, 592, 593, 594, 595, 596, 597, 598, 599, 600; 10 scans per second), o-ketoglutarate (346, 347, 348, 349, 350, 351, 352, 353, 354; 10 scans per second), fumarate (287, 288, 289, 290, 291, 292, 293; 10 scans per second), glutamate (432, 433, 434, 435, 436, 437, 438, 439, 440; 10 scans per second) and malate (419, 420, 421, 422, 423, 424, 425, 426; 10 scans per second).

[0200] All chromatograms were subsequently analyzed with the MetaboliteDetector software (Hiller K et al., 2009).

[0201] Preparation of cells for RNA-single cell sequencing

[0202] Cells of draining lymph nodes (dLN) and tumor-infiltrating lymphocytes (TILS) were isolated from tumor-bearing mice at day 7 and 14 post T cell infusion. Following incubation with TruStain FcX™ PLUS (anti-mouse CD16 / 32) antibody (Biolegend) to prevent unspecific binding, cells were stained with 2 pl of TotalSeqTM-C hashing antibodies (Biolegend) in wash buffer for 30 min at 4 °C. Afterwards, 6 samples were pooled in one and suspended in 65 l of PBS / 0.04 % BSA.

[0203] Single cell RNA-sequencing

[0204] Chromium™ X / iX Controller was used for partitioning single cells into nanoliter-scale Gel Bead-1 n-EMulsions (GEMs) and Chromium GEM-X Single Cell 5' Kit v3 kits for reverse transcription, cDNA amplification and library construction for all gene expression, TCR and hashtag libraries (10x Genomics), following manufacturer's instructions. A SimpliAmp Thermal Cycler was used for amplification and incubation steps (Applied Biosystems). Libraries were quantified by a QubitTM 3.0 fluorometer (Thermo Fisher Scientific) and quality was checked using a 2100 Bioanalyser with High Sensitivity DNA kit (Agilent). Libraries were pooled and sequenced using the NextSeq 2000 platform (Illumina) in paired-end mode for gene expression as well as for the T-cell receptor repertoire and hashtags. Demultiplexed FASTQ files were generated with bcl-convert v4.0.3 (Illumina). Data were analyzed using the Cell Ranger 7.2.0 software suite pipelines available on the lOxGenomics website.

[0205] The Cell Ranger output of each pool (P1 to P9) was converted to a Seurat object using the Seurat package (ReadlOX and CreateSeuratObject). After adding the HTO data as an independent assay, cells were demultiplexed using the HTODemux function with kmeans clustering and positive.quantile =0.99. Only singlets were selected for further analysis. The percentage of mitochondrial genes (MT-) was determined for each cell using the PercentageFeatureSet function. The inventors filtered out low quality cells based on the number of features (< 200 or > 6000) and the percentage of mitochondrial genes (> 20 %) using the Subset function. The 9 Seurat objects were merged into a single object regrouping a total of 16908cells. Normalization and integration were performed using NormalizeData and STACAS. RunPCA, runUMAP, FindNeighbors and FindClusters with 30dimensions were used to identify 21 clusters of T cells. T cells were then annotated using scGate with the predefined gating mouse model, and only pure T cells were subset and used for downstream analysis. A total of 4774 pure T cells were annotated for the different CD4 and CD8 T cell subsets using ProjecTILs and the human reference atlas to characterize cell states.

[0206] In vivo models

[0207] For all experiments, group sizes were determined based on experience by previously published models. Tumorbearing mice were randomly assigned to receive CAR T cell infusions, to ensure similar tumor sizes across groups before treatment. Tumor engraftment and T cell infusions were performed by blinded technicians. Generated CAR T cells were not sorted but additional non-transduced cells were added, so that the same amount of total T cell count was applied to each animal.

[0208] Panc02 tumor models For adoptive transfer experiments with Panc02 mR0R1 tumor cells, mice were subcutaneously (s.c.) injected with 2x106tumor cells. On day 5 after tumor injection, 2x106CD8+CAR T cells were transferred intraperitoneally (i.p) into BI6 mice.

[0209] For the analysis of transferred CAR T cells by scRNA sequencing, mice were subcutaneously (s.c.) injected with 1x106tumor cells. After an inoculation time of 7 days, 1x106CAR T cells with a 1 :1 mixture of CD4:CD8 CAR T cells were transferred into tumor bearing mice. On day 7 and day 14 after T cell injection, 5 mice per group were sacrificed and samples of dLN and TILs were sent for RNA single cells sequencing.

[0210] MC38 tumor model

[0211] For adoptive transfer experiments with MC38 mR0R1 tumor cells, mice were subcutaneously (s.c.) injected with 2x106tumor cells in PBS with matrigel. Mice were treated with 2x106CD8+CAR T cells on day 7.

[0212] For all in-vivo experiments, tumor progression was monitored by caliper every second day. At the endpoint of the experiments, peripheral blood was obtained by tail vein puncture in a tube containing Alsever's solution. Cell pellets of blood were resuspended in ACK lysis (Thermo Fisher) buffer for 10min. Spleen, bone marrow, draining lymph node and tumors were collected. After determination of the weight, tumors were dissociated using the tumor dissociation kit (Miltenyi Biotech) and a GentleMACS™ Dissociator (Miltenyi Biotech) according to manufacturer's constructions. All organs were passed through a 70 piM strainer to aquire single-cell suspension. Tumor-infiltrating lymphocytes were isolated using CD45 (TIL) microbeads (Miltenyi Biotech). All samples were washed twice and stained with live / dead marker (Biolegend) for 20 min at RT. After washing, samples were incubated with TruStain FcX™ (anti-mouse CD16 / 32) Antibody (Biolegend) to prevent unspecific binding for 15min at RT. In the following, cells were stained with corresponding antibodies: mouse CD45 (Pacific blue™, 30- F11, Biolegend), mouse CD8a (APC-Cyanine7, 53-6.7, Biolegend), mouse CD4 (FITC, GK1.5, Biolegend), mouse CD19 (PE, 1 D3 / CD19, Biolegend). For staining of intracellular cytokines, cells were stimulated with 100 ng / ml phorbol-12-myristat-acetate (PMA) and 1 pig / ml lonomycin in the presence of 5 pig / ml brefeldin A for 5 hours. To detect intracellular cytokines, cells were fixed with 2 % paraformaldehyde (Invitrogen). Following antibodies were used for intracellular cytokines: mouse IFN-y (APC, XMG1.2, Biolegend), mouse TNF-o (PerCP- Cyanine5.5, MP6-XT22, Biolegend), mouse IL-2 (PE-Cyanine7, JES6-5H4, Biolegend), anti-human / mouse recombinant Granzyme B (Pacific blue™, QA16A02, Biolegend).

[0213] Quantification and statistical analysis

[0214] The results are shown as mean ± standard error of the means (SEM). To determine the statistical significance of the differences between two experimental groups unpaired Student's t tests were performed using Prism 9 software (GraphPad). To identify the statistical significance of the difference between more than to experimental groups one-way or two-way analysis of variance (ANOVA) with Tukey's multiple-comparison test were performed using Prism 9 software (GraphPad). For in-vivo tumor growth curves, significance was determined at indicated timepoints on the blot by unpaired Student's t-test comparing control group with the treatment group. Significance for the survival data was calculated using the log-rank Mantel-Cox test. Samples sizes were based on experience and complexity of the experiment but no methods were used to determine normal distribution of the samples. Differences reached significance with p- values <0.05, p < 0.0.1, p < 0.001 or < 0.0001. The figure legends contain the number of independent experiments or mice per group that were used in the respective experiment.

[0215] For the clinical study, statistical analyses were conducted using RStudio version 2023.12.1+402 (Posit, Boston, MA, USA) and R version 4.3.3 (The R foundation, Vienna, Austria). The level of significance was set at a two- sided p < 0.05 with 95% confidence intervals. Grouped data are presented as violin plots. To compare two groups, based on the distribution of the data, the Wilcoxon-Mann-Whitney-Test (for non-normally distributed data) or the t-test (for normally distributed data) were conducted and adjusted for false discovery rate (FDR) using the rstatix package (version 0.7.2). Progression-free survival (PFS) was defined as the time from CAR T cell infusion to disease progression or death, whichever occurred first. Overall survival (OS) is defined as the timespan between the CAR T cell infusion and the patient's death irrespective of the cause. Metabolite cutoffs were determined utilizing surv_cutpoint () function from the survminer package (version 0.4.9). Survival data are depicted as Kaplan-Meier curves. Differences between two groups were assessed with the log-rank test via the ggsurvfit () function and package (version 1.0.0). For comparisons of more than one group, a cox regression was performed using the coxph() function from the survival package (version 3.5-8).

[0216] Example 1 : PIM exposure confers adverse progression-free and overall survival

[0217] The inventors recently showed the human microbiome strain Megasphaera massiliensis as a potent producer of the SCFAs pentanoate. Its strong capacity to induce antitumor features in T cells prompted us to investigate its role in a clinical real-life context.

[0218] The inventors evaluated the relationship between faecal SCFA levels and PFS of patients receiving CAR-T cells in a German cohort of lymphoma and myeloma patients (n=66). Patients with high levels of pentanoate exhibited significantly better PFS compared to those with low levels of pentanoate (Hazard ratio (HR) 6.9, estimated 1- year PFS 90% vs. 41.5%; median PFS not reached vs. 310 days, Fig. 1a). These patients also exhibited a trend towards improved overall survival (HR 3.0, estimated 1-year overall survival (OS) 80% vs. 71.7%, median OS not reached vs. 749 days, Fig. 1a). Notably, other SCFAs such as butyrate, propionate and acetate could not predict PFS and OS to a similar degree (Fig. 1c-d). In a second cohort of US patients (n=60) with non-Hodgkin lymphoma treated with CD19-directed CAR T cells, the pre-CAR T infusion fecal pentanoate concentrations were categorized by terciles. The higher terciles had numerically higher 2-year PFS (p=0.4) and 2-year OS (p=0.4, Fig. 25a, b). Thus, pentanoate abundance constitutes a predictive marker of CAR T-cell response. Previous reports highlighted the impact of broad-spectrum antibiotics on anti-CD 19 CAR T cell therapy outcomes (Smith A et al., 2022; Luu M et al., 2019). Consistently, in the present cohort, exposure to PIM group antibiotics (piperacillin / tazobactam, imipenem, meropenem) prior to (any target) CAR T cell therapy resulted in worse PFS (HR 2.66, Fig. 1a) and OS (HR 4.11, Fig. 1b) compared to patients not exposed to antibiotics. Conversely, patients receiving non-PIM antibiotics showed similar PFS (HR 1.55, Fig. 1a) and only a trend towards adverse OS (HR 3.95, Fig. 1b) compared to those not treated with antibiotics.

[0219] Example 2: Patients with PIM exposure display reduced fecal pentanoate levels

[0220] Examination of the association between SOFA levels and antibiotic exposure revealed that pentanoate levels were significantly reduced only in patients with PIM exposure (Fig. 1c). No significant reduction was observed with non-PIM antibiotics. Similar effect could be observed with the odd-chain family member propionate. However, levels of the even-chain fatty acid butyrate, were already decreased with non-PIM antibiotics. Acetate showed no significant reduction with either PIM or non-PIM antibiotics. These correlations suggest that PIM exposure crucially affects pentanoate-producing commensal and thereby PFS.

[0221] Example 3: Pentanoate levels prior to CAR T cell infusion associate with progression-free survival

[0222] Next, the inventors evaluated the relationship between fecal SCFA levels and PFS of CAR T cell patients. Patients with high levels of pentanoate exhibited significantly better PFS compared to those with low levels of pentanoate (HR 6.9, estimated 1-year PFS 90% vs. 41.5%; median PFS not reached vs. 310 days, Fig. 1d). These patients also exhibited a trend towards improved overall survival (HR 3.0, estimated 1 year OS 80% vs. 71.7%, median OS not reached vs. 749 days, Fig. 1d). Notably, other SCFAs, such as butyrate, propionate and acetate could not predict PFS and OS to a similar degree (Fig. 1e-g). Thus, pentanoate abundance might serve as a predictive parameter for CAR T cell response in patients.

[0223] Example 4: Implementation of pentanoate in the CAR T cell manufacturing improves product characteristics

[0224] Based on the results of the correlative analysis, the inventors hypothesized that the superior PFS might be a consequence of pentanoate-mediated immunostimulation improving host environment and CAR T cell function. To investigate the latter and develop an approach to exploit pentanoate's benefits for clinical application facing hostile, immunosuppressive TME, the inventors designed a protocol featuring a short in vitro expansion phase designed to better retain sternness and fitness of the CAR T cell product in the host after infusion (Fig. 2a).

[0225] Isolated T cells from murine spleens and lymph nodes were activated with o-CD3 and O-CD28 antibodies in presence of pentanoate for 2 days, prior to retroviral gene transfer of the CAR transgene. Between 3 to 7 days post transduction, the inventors characterized phenotypic and functional changes both in vitro and in vivo (Fig. 2a-m). Generation of CD4+ and CD8+ ROR1 -specific CAR T cells showed that both transduction efficacy and transduction marker expression were elevated in the pentanoate-engineered CAR T cells (CARpenta) as compared to the untreated ones (CAR), further accompanied by increased cell yield without impairment of viability (Fig. 2b-g). Similar data were obtained when the workflow was applied to the production of CD4+ and CD8+ CD19 CAR T cells (Fig. 7a-f). Moreover, both R0R1 and CD19 CARpenta T cells showed strong upregulation of the effector cytokines IFN-y, and TNF-o as well as granzyme B and IL-2 (Fig. 2h, I and Fig. 7g, h) in an antigen-independent manner. Prior to assessment of cytotoxic activity, CAR T cells were cultured for 4 days without pentanoate to explore the longevity of the modulation. Co-cultures of R0R1 -specific CARpenta T cells with the R0R1 -expressing murine pancreatic ductal adenocarcinoma cells Panc02 (PancRORI) and colorectal carcinoma cells MC38 (MC38ROR1), respectively, as well as of CD19-specific CARpenta T cells with CD 19+ Epi-myc cells showed significant improvements in cytotoxic activity and antigen-dependent cytokine release as compared to conventionally engineered CAR T cells (Fig. 2j-m and Fig. 7I-I). These results suggest that short-term pentanoate treatment leads to a sustained phenotype in different CAR products.

[0226] Example 5: HDAC class l-inhibition-mediated hyperacetylation enhances CAR T cell function

[0227] The sustained increase in effector function after removal of the commensal metabolite suggests a lasting effector of pentanoate. This might be attributed to SCFA-mediated inhibition of histone deacetylases (HDACs) and consequently histone hyperacetylation, thereby altering the epigenetically programmed fate of eukaryotic cells (Fig. 3a, b). To predict how HDACs interact with fatty acids, the inventors used AlphaFold 3 to model zinc and palmitate bound structures of classes I and II HDACs (Fig. 8a-c). The structural prediction revealed that HDACs of both classes coordinate palmitate in their reactive centers with high confidence up to C5 (pLDDT >90 for class I and 70-90 for class II; Fig. 8d). Docking of C2-C6 SCFAs (acetate to hexanoate) into an AlphaFold 3-predicted structure of zinc cofactor-bound HDAC1 suggested that the aliphatic chains might bind in an inward facing orientation up to a chain length of C4 and in an outward facing conformation for C6 SCFAs (Fig. 3b, c and Fig. 9a). Notably, docking of pentanoate (C5) showed two conformations, with the best inward facing conformation being preferred (predicted AG ~ -8.6 kcal / mol), relative to the best outward facing orientation (predicted AG ~ - 7.6 kcal / mol, Fig. 9a). Docking of pentanoate into HDACs of class I and Ila predicted preferred binding to class I enzymes (Fig. 9b, c). Experimentally, the inventors confirmed the predicted inhibition of HDAC1 and 2 over class Ila by SCFAs (Fig. 3d and Fig. 9d). Although the SCFAs analyzed are all structurally similar and elicit HDAC inhibition, their effects are highly context- and cell type-dependent. Differentiation of CD4 T cells towards regulatory T cells (Tregs) has long been linked to the HDAC-inhibitory activity of SCFAs (Kespohl M et al.2017; Coutzac C et al., 2020; Smith PM et al., 2013). Interestingly, only propionate and butyrate, but not pentanoate, were able to induce the Treg master regulator Foxp3 (Fig. 10). Haradhvala and colleagues have reported that patient relapse is associated with the presence of CAR Tregs, which favored the use of pentanoate over other SCFAs (Haradhvala NJ et al., 2022). Of note, treatment of inducible Tregs (ITregs) with pentanoate suppressed Foxp3 and reciprocally induced the CTL / Th1-related transcription factor T-bet in a concentration-dependent manner (Fig. 11 a-d). Tregs derived from T-bet-deficient (Tbx21- / -) animals were unable to express the same level of IFN-Y as compared to their WT counterparts. Moreover, engineering of CAR T cells in the presence of different SCFAs highlighted pentanoate as the most potent modulator of anti-tumor activity (Fig. 12a, b). These findings suggest that use of pentanoate could repress CAR Treg development and favor CTL / Th 1 polarization. To assess whether clinically used HDAC class I inhibitors could mimic pentanoate's modulatory benefits, the inventors engineered R0R1 CAR T cells in presence of mocetinostat (CARMOC). AlphaFold 3-modeling of zinc- bound HDAC1 and docking of mocetinostat into the reactive center suggests a binding mode similar to that of pentanoate, with a predicted AG of approximately -8.1 kcal / mol (Fig. 3e and Fig. 13). Intracellular flow cytometry staining for histone post-translational modifications revealed an increase in acetylation within CD8 and Th1 T cells at H3 Lys9 / 14 and H3 Lys27, respectively, both prominent marks of open chromatin and transcriptional activity (Fig. 3f, g and Fig. 14a). Next, the inventors analyzed the consequences of pentanoate- and mocetinostat-mediated hyperacetylation for the CD4 and CD8 CAR T cell phenotype. Surprisingly, and despite mocetinostat's strong HDAC-inhibitory activity, CARMOC T cells elicited less potent lysis of target cells and antigen-specific cytokines secretion compared to CARpenta T cells, although both were superior in killing relative to untreated CAR T cells (Fig. 3h, I). Engineering in the presence of HDAC class II inhibitor TMP-195 did not improve CAR T cell features (Fig. 14b). These results suggest that epigenetic remodeling affects the CTL phenotype; however, a standalone implementation of a clinical HDAC! such as mocetinostat is not sufficient to fully reconstitute CARpenta T cells features.

[0228] Example 6: Pentanoate synergizes epigenetic-metabolic modulation to boost effector function

[0229] The inventors next investigated whether pentanoate influences cellular metabolism to augment HDAC! function (Fig. 4a). CD4 and CD8 CARpenta T cells showed increased phosphorylation of the central metabolic regulator S6, a downstream target of mTOR (Fig. 15a, b). By staining metabolic key functions, the inventors observed an increased glucose and fatty acid uptake (Fig. 15c, d). Moreover, CD4 and CD8 CARpenta T cells increased the expression of the master regulator of mitochondrial biogenesis PGC-1 a and mitochondrial mass, compared to control CAR T cells (Fig. 4b, c and Fig. 15e, f). Thus, an mTOR-mediated shift towards glucose oxidation might contribute to the CARpenta T cell phenotype. To probe this effect in a more isolated manner, the inventors next generated CAR T cells in presence of the clinically used drug dichloroacetate (DCA, CARDCAT cells), according to the inventors' initial concept. Of note, DCA is a pharmacological inhibitor of mitochondrial pyruvate dehydrogenase kinase (PDK) and is currently under clinical investigation for cancer therapy. PDK activates pyruvate dehydrogenase (PDH), which itself acts as a gatekeeper enzyme for pyruvate flux into the TCA cycle. Consequently, DCA redirects metabolism from lactate fermentation to glucose oxidation in mitochondria. Similar to pentanoate, engineering of CARDCA T cells increased fatty acid uptake and mitochondrial mass (Fig. 4c and Fig. 15d). Thus, the inventors decided to use DCA to mimic the glucose oxidative enhancement during the engineering process. Next, the inventors tested whether the simultaneous HDAC inhibition and metabolic reprogramming synergistically improved CARpenta T cell effector function. To do so, the inventors engineered CAR T cells in the absence or presence of pentanoate, DCA, mocetinostat or a combination of the latter, respectively (Fig. 4d). The engineered T cells were subjected to co-cultures using PancRORI or MC38ROR1 tumor cells to assess cytotoxicity (Fig. 4e, f). Interestingly, while CARDCA T cells did not show a significant improvement in killing, combined administration of DCA and mocetinostat during manufacturing (CARMOC+DCAT cells) resulted in an additive effect which was also reflected in antigen-dependent secretion of TNF-o, IFN-y+ and IL-2 (Fig. 4g-i). Noteworthy, varying the co-stimulatory domain within CAR constructs alters the cellular metabolism and therefore CAR T cell function. The inventors thus performed a set of experiments using a CD28-based R0R1 CAR. This resulted in similar effects upon treatment with the previously mentioned drugs with regards to cytotoxicity and mitochondrial mass (Fig. 16a, b).

[0230] To understand a potential contribution of mocetinostat off-target effects, the inventors probed HDAC class I involvement by using mice T cells with homozygous HDAC1- (HDAC1 - / -) and heterozygous HDAC2-(HDAC1- / -) deficiency (Fig. 4j). HDAC-deficient CAR T cells showed superior specific lysis compared to the WT CAR control, mimicking the trends observed in CARMOC T cells (Fig. 4k). Engineering in the presence of DCA further boosted lytic activity and cytokine secretion, comparable to levels of CARpenta T cells (Fig. 4k and Fig. 17). This demonstrates a synergistic relationship between HDAC inhibition and metabolic enhancement that appears to be responsible for the CARpenta T cell phenotype.

[0231] To evaluate whether the in vitro results could be recapitulated in vivo, the inventors applied suboptimal doses of CAR, CARpenta and CARMOC+DCAT cells in an immunocompetent pancreatic tumor model characterized by a cold TME, without lymphodepletion (Fig. 4I). Surprisingly, differences in tumor mass and a lack of peripheral expansion indicated that the clinical drugs were not able to reproduce the efficacy of pentanoate, although improved tumor control was observed in both groups as compared to the control CAR T cells (Fig. 4m and Fig. 18a, b). Thus, though mocetinostat and DCA mimic some key features of pentanoate, other mechanisms might be involved that are not impacted by these drugs.

[0232] Example 7: Pentanoate becomes incorporated into the TCA and part of the epigenetic imprint

[0233] While DCA and pentanoate both influence mitochondrial activity, they might differ in their effect on glycolytic and oxidative pathway intermediates (Fig. 5a). To trace the pathway intermediates, the inventors pulsed their engineered CAR T cells with either13C-labeled glucose or13C -glutamine tracers, prior to analysis via GC-MS (Fig. 5b). Tracing of13C-glucose revealed that less glucose was metabolized to lactate upon DCA treatment, confirming its activity as PDK inhibitor. No significant changes in pyruvate were observed for any of the small molecules used (Fig. 5c). Of note, the inventors detected a strong decrease in glucose-derived citrate in CARpenta T cells that was not observed for mocetinostat or DCA (Fig. 5c). However, the impact on the glycolysis end product was rather minor with a reduction of approximately 25%. This trend was more pronounced with a reduction of approximately 50% glucose-derived labeling for downstream TCA intermediates such as a- ketoglutarate, fumarate and malate, as compared to control CAR T cells. In contrast, although DCA reduced cellular lactate levels, its effects on glucose oxidation did not change the overall glucose-dependent citrate production. These data highlight pentanoate's capacity to reprogram the TCA in T cells which cannot be mimicked by conventional drugs.

[0234] The inventors next addressed which alternative source was used by CARpenta T cells to replenish the citrate carbons. Upon butyrate treatment, T cells may fuel the TCA via glutamine anaplerosis to uncouple it from glycolysis (Bachem A et al., 2019). By contrast, pulsing of CARpenta T cells with13C-glutamine did not show a preference for incorporation into the TCA intermediates (Fig. 5d). Hence, fatty acid oxidation and metabolization of pentanoate might contribute to citrate generation. To understand how pentanoate is metabolized, the inventors next administered13C-pentanoate during the CAR manufacturing. GC-MS analysis revealed the incorporation of pentanoate-derived carbons into citrate via the acetyl-CoA entry point. Additionally, the labeled carbons entered the TCA through a second entry point via the succinyl-CoA route (Fig. 5e). This is a unique feature of pentanoate attributed to the C5 aliphatic chain, which cannot be emulated by acetate, propionate or butyrate. The combined citrate amounts derived from both entry points correspond to the gap detected in the previous glucose tracing experiment which underlines a limited preference for pentanoate over glucose (Fig. 5c, e). Besides TCA intermediates being required for energy homeostasis, conversion of citrate into acetyl-CoA by the nuclear ATP-citrate lyase (ACLY) serves as a source for histone acetylation, thereby linking metabolism and epigenetic regulation. To test whether ACLY directs the pentanoate-derived citrate flux, the inventors inhibited ACLY via 2-hydroxy citrate (2-HC) in the presence of the13C-pentanoate tracer (Fig. 5e). Interestingly, 2-HC supplementation during CARpenta T cell manufacturing not only led to a 2-fold increase in pentanoate labeling of glutamate, fumarate and malate downstream of citrate from the first entry point, but to an additional 4-fold increase of pentanoate-derived citrate from the second one. This suggests that the latter might be used for histone acetylation in the nucleus and could be accumulated upon ACLY blockade in the TCA. To investigate the functional outcome of this crosstalk, the inventors evaluated CARpenta T and control CAR T cells that were engineered in the absence and presence of 2-HC in killing assays. Specific lysis and antigen-specific cytokine analysis showed that 2-HC administration reduced the functionality of CARpenta T cell significantly emphasizing the importance of the epigenetic-metabolic crosstalk engaged by pentanoate (Fig. 5f, g and Fig. 19a-c).

[0235] Example 8: Epigenetic-metabolic rewiring enhances CAR T cell efficacy, persistence and resistance to TME factors

[0236] To assess the therapeutic potential of CARpenta T cells, the inventors established syngeneic tumor models in fully immunocompetent mice, accounting for hostile TME conditions that are lacking in conventional xenogeneic NSG models. CARpenta T cells were transferred into mice bearing 5-day-old PancRORI tumors, prior to endpoint analysis on day 21 post transfer (Fig. 6a). The Panc02 model develops a cold, immunosuppressive and T cell- excluding TME. Treatment of mice with pentanoate-engineered R0R1 CAR T cells led to significantly improved tumor control, approaching mass clearance in some mice starting 5-7 days after administration. In contrast, the conventionally engineered T cell group showed inferior anti-tumor activity and signs of losing efficacy towards the end of the experiment (Fig. 6b, c). Analysis at the endpoint showed low residual tumor mass and an increase in both CARpenta T cells as well as endogenous CD8+ tumor infiltrating lymphocytes (TILs) (Fig. 6d and Fig. 20a). Further, CARpenta T cells in the draining lymph node (dLN), spleen, bone marrow and blood were more persistent (Fig. 20b). Intracellular cytokine staining revealed elevated frequencies of TNF-o + IFN-y+ and Granzyme B + IL- 2 + CAR T cells in the spleen and to a lesser extent in the dLN. Significant differences were found in the tumor which reflected preservation of the cytokine profile in vitro (Fig. 2h, I and Fig. 20c-e). Interestingly, in vivo transfer of a polyclonal T cell population showed an antigen-independent expansion and persistence in the periphery upon pentanoate but not butyrate treatment in a similar fashion (Fig. 21). By contrast, pentanoate treatment for 3 days after CAR engineering limited persistence upon in vivo transfer into immunocompetent hosts (Fig. 22a-c). This emphasizes the importance of timing the modulation within an early activation window when chromatin is primed to adopt an open state.

[0237] The inventors next assessed the impact of CARpenta T cells on mouse survival in an aggressive MC38ROR1 model with hot TME characteristics (Fig. 6e). Monitoring over 34 days post transfer revealed superior tumor control and survival as compared to the control group with conventionally engineered CAR T cells (Fig. 6f-h). While the control group delayed tumor growth before the experimental cut-off size was reached, pentanoate- engineered CAR T cells showed homogeneous, synchronized response and tumor control. Two mice were taken out of the experiment due to skin irritation at the tumor site.

[0238] To better understand at transcriptomic level the effects of the drug treatments on the immune microenvironment, scRNA-seq was performed on ex vivo isolated immune cells obtained from tumor-bearing mice treated with CAR, CARDCA+MOC and CARpenta T cells on day 7 and day 14 post transfer, respectively, and the pre-infusion products (n=24, n=4 mice / treatment / point in time, Fig. 23a). After performing the QC steps a total of 16908 cells were identified. The UMAP representing the distribution of the cells based on conditions, time points and mouse of origin are presented in Fig. 23b-d. By using scGate the inventors were able to annotate these cells and identify 4774 of pure T cells that were further divided in 14 clusters based on their cell state (Figure 6i, j and Fig. 23e). Of interest, following treatment with pentanoate the inventors observed an enrichment of CD4 and CD8 naive-like T cells (TCF7+, CCR7+, PD1-, TOX-) while a decreased of CD8 TEX (GZMK-, TCF7-, TOX+, HAVCR2+, PD1 +) was noted (Figure 6k, I). Taken together these data confirm the ability of pentanoate to increase the fitness of T cells creating a more active immune microenvironment that supports T cell anti-tumor activity and reduce T cell exhaustion.

[0239] Conceivably, pentanoate-mediated epigenetic-metabolic reprogramming might allow also for enhanced resistance to hostile TME factors present in the solid tumors. Thus, the inventors challenged CAR and CARpenta T cells for tumor elimination under hypoxic conditions. CARpenta T cells maintained their superior lytic activity, suggesting better preservation of effector functions despite the oxygen-reduced environment (Fig. 24a). The inventors next engineered CAR T cells under low IL-2 conditions mimicking the IL-2-depleted environment characteristic of Treg-rich TMEs. Analysis of tumor cell survival showed that under conditions typically unfavorable to T cell functionality CARpenta T cells retained superior lytic activity, potentially due their ability to produce autocrine IL-2 (Fig. 21, m and Fig. 24b).

[0240] The inventors' results demonstrate that introducing the commensal metabolite pentanoate into CAR manufacturing confers long-lasting improvements in effector function, in vivo persistence and resistance to immunosuppressive factors in hot and cold TMEs, generating enhanced cell products with improved activity against hard-to-treat malignancies.

[0241] INDUSTRIAL APPLICABILITY

[0242] The method for producing recombinant immune cells, the immune cell obtainable by said method, and products for use in the invention are industrially applicable. For example, they can be used in the manufacture of, or as, pharmaceutical products.

[0243] REFERENCES

[0244] 1. Narayan V, Barber-Rotenberg JS, Jung l-Y et al. (2022) PSMA-targeting TGFp-insensitive armored CAR T cells in metastatic castration-resistant prostate cancer: a phase 1 trial. Nat Med 28:724-734. https: / / doi.Org / 10.1038 / S41591-022-01726-1

[0245] 2. Kespohl M, Vachharajani N, Luu M et al. (2017) The Microbial Metabolite Butyrate Induces Expression of Th 1- Associated Factors in CD4+T Cells. Front Immunol 8. https: / / doi.org / 10.3389 / fimmu.2017.01036

[0246] 3. Smith PM, Howitt MR, Panikov N et al. (2013) The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science 341 :569-573. https: / / doi.org / 10.1126 / science.1241165

[0247] 4. Vetizou M, Pitt JM, Daillere R et al. (2015) Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. Science 350:1079-1084. https: / / doi.org / 10.1126 / science.aad1329

[0248] 5. Sivan A, Corrales L, Hubert N et al. (2015) Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy. Science 350:1084-1089. https: / / doi.org / 10.1126 / science.aac4255

[0249] 6. Routy B, Le Chatelier E, Derosa L et al. (2018) Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors. Science 359:91-97. https: / / doi.org / 10.1126 / science.aan3706

[0250] 7. Tanoue T, Morita S, Plichta DR et al. (2019) A defined commensal consortium elicits CD8 T cells and anti-cancer immunity. Nature 565:600-605. https: / / doi.org / 10.1038 / s41586-019-0878-z Stein-Thoeringer CK, Saini NY, Zamir E et al. (2023) A non-antibiotic-disrupted gut microbiome is associated with clinical responses to CD19-CAR-T cell cancer immunotherapy. Nature Medicine:1— 11 . https: / / doi.Org / 10.1038 / S41591-023-02234-6 Smith M, Dai A, Ghilardi G et al. (2022) Gut microbiome correlates of response and toxicity following anti-CD19 CAR T cell therapy. Nat Med 28:713-723. https: / / doi.org / 10.1038 / s41591-022-01702-9 Luu M, Pautz S, Kohl V et al. (2019) The short-chain fatty acid pentanoate suppresses autoimmunity by modulating the metabolic-epigenetic crosstalk in lymphocytes. Nat Commun 10:760. https: / / d0i.0rg / l 0.1038 / S41467-019-08711 -2 Coutzac C, Jouniaux J-M, Pad A et al. (2020) Systemic short chain fatty acids limit antitumor effect of CTLA-4 blockade in hosts with cancer. Nat Commun 11 :2168. https: / / doi.org / 10.1038 / s41467-020- 16079-x Arpaia N, Campbell C, Fan X et al. (2013) Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature 504:451-455. https: / / doi.org / 10.1038 / nature12726 Luu M, Riester Z, Baldrich A et al. (2021) Microbial short-chain fatty acids modulate CD8+ T cell responses and improve adoptive immunotherapy for cancer. Nat Commun 12:4077. https: / / d0i.0rg / l 0.1038 / S41467-021-24331 -1 Smith PM, Howitt MR, Panikov N et al. (2013) The Microbial Metabolites, Short-Chain Fatty Acids, Regulate Colonic Treg Cell Homeostasis. Science 341:569-573. https: / / d0i.0rg / l 0.1126 / science.1241165 Haradhvala NJ, Leick MB, Maurer K et al. (2022) Distinct cellular dynamics associated with response to CAR-T therapy for refractory B cell lymphoma. Nature Medicine 28:1848-1859. https: / / d0i.0rg / l 0.1038 / S41591-022-01959-0 Bachem A, Makhlouf C, Binger KJ et al. (2019) Microbiota-Derived Short-Chain Fatty Acids Promote the Memory Potential of Antigen-Activated CD8+ T Cells. Immunity 51:285-297.e5. https: / / doi.Org / 10.1016 / j.immuni.2019.06.002 Thiele Orberg E, Meedt E, Hiergeist A et al. (2024) Bacteria and bacteriophage consortia are associated with protective intestinal metabolites in patients receiving stem cell transplantation. Nat Cancer 5:187-208. https: / / doi.org / 10.1038 / s43018-023-00669-x Preglej T, Hamminger P, Luu M et al. (2020) Histone deacetylases 1 and 2 restrain CD4+ cytotoxic T lymphocyte differentiation. JCI Insight 5. https: / / doi.org / 10.1172 / jci.insight.133393 Abramson J, Adler J, Dunger J et al. (2024) Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630:493-500. https: / / doi.org / 10.1038 / s41586-024-07487-w Bugnon M, Rbhrig UF, Goullieux M et al. (2024) SwissDock 2024: major enhancements for smallmolecule docking with Attracting Cavities and AutoDock Vina. Nucleic Acids Res 52:W324-W332. https: / / d0i.0rg / l 0.1093 / nar / gkae300 Grosdidier A, Zoete V, Michielin 0 (2011) SwissDock, a protein-small molecule docking web service based on EADock DSS. Nucleic Acids Res 39:W270-7. https: / / doi.org / 10.1093 / nar / gkr366 Zoete V, Daina A, Bovigny C et al. (2016) SwissSimilarity: A Web Tool for Low to Ultra High Throughput

[0251] Ligand-Based Virtual Screening. J Chem Inf Model 56:1399-1404. https: / / doi.org / 10.1021 / acs.jcim.6b00174 Meng EC, Goddard TD, Pettersen EF et al. (2023) UCSF ChimeraX: Tools for structure building and analysis. Protein Sci 32:e4792. https: / / doi.org / 10.1002 / pro.4792 Honorato RV, Koukos PI, Jimenez-Garcia B et al. (2021) Structural Biology in the Clouds: The WeNMR- EOSC Ecosystem. Front Mol Biosci 8:729513. https: / / doi.org / 10.3389 / fmolb.2021.729513 Vangone A, Schaarschmidt J, Koukos P et al. (2019) Large-scale prediction of binding affinity in proteinsmall ligand complexes: the PRODIGY-LIG web server. Bioinformatics 35:1585-1587. https: / / doi.Org / 10.1093 / bi oi nform ati cs / bty 816 Hiller K, Hangebrauk J, Jager C et al. (2009) MetaboliteDetector: comprehensive analysis tool for targeted and nontargeted GC / MS based metabolome analysis. Analytical chemistry 81 :3429-3439. https: / / d0i.0rg / l 0.1021 / ac802689c T.T. Smith, et. al, In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers, Nat Nanotechnol. 2017 Aug; 12(8): 813-820. Published online 2017 Apr 17. doi: 10.1038 / nnano.2017.57 Agarwal S,et. al, Oncoimmunology. 2019 Oct 10;8(12):e1671761. In vivo generated human CAR T cells eradicate tumor cells, doi: 10.1080 / 2162402X.2019.1671761]

Claims

CLAIMS1 . A method for producing recombinant immune cells, comprising the following steps:(A) Isolating immune cells,(B) Incubating the immune cells in the presence of at least one short-chain fatty acid, and(C) Introducing into said immune cells of step (B) a nucleic acid encoding an immunoreceptor or a set of nucleic acids encoding an immunoreceptor.

2. The method according to claim 1, wherein the immune cells are mammalian immune cells.

3. The method according to claims 1 or 2, wherein the immune cells are human immune cells.

4. The method according to any one of claims 1 to 3, wherein said immune cells are T cells, B cells, NK cells, NKT cells, macrophages and / or stem cells.

5. The method according to any one of claims 1 to 4, wherein said immune cells are T cells.

6. The method according to any one of claims 1 to 5, wherein said immune cells are CD4+ and / or CD8+T cells.

7. The method according to any one of claims 1 to 6, wherein the immunoreceptor is a B-cell receptor (BCR), T- cell receptor (TCR), or chimeric antigen receptor (CAR).

8. The method according to any one of claims 1 to 7, wherein the immunoreceptor is a chimeric antigen receptor (CAR).

9. The method according to any one of claims 1 to 8, wherein the at least one short-chain fatty acid comprises pentanoate, butyrate, propionate, acetate and / or pharmaceutically acceptable derivatives thereof.

10. The method according to any one of claims 1 to 9, wherein the at least one short-chain fatty acid comprises pentanoate or a pharmaceutically acceptable derivative thereof.

11. The method according to any one of claims 1 to 10, wherein the at least one short-chain fatty acid is pentanoate or a pharmaceutically acceptable derivative thereof.4312. The method according to any one of claims 1 to 11, wherein in step (C), the nucleic acid or set of nucleic acids is introduced by viral gene transfer, preferably wherein the nucleic acid is introduced by retroviral gene transfer.

13. The method according to claim 12, wherein in step (C), the viral gene transfer uses a viral vector which comprises a gene cassette encoding said immunoreceptor.

14. The method according to claim 12 or 13, wherein in step (C), the cells are transduced with retroviral supernatant by spin-infection.

15. The method according to any one of claims 1 to 14, wherein in step (B), the incubation is performed for more than 24 hours, such as 24 to 72 hours, preferably 36 to 60 hours, more preferably for 48 hours.

16. The method according to any one of claims 1 to 15, wherein in step (B) the total concentration of the at least one short-chain fatty acid is more than 0.25 mM, preferably between 0.25 mM and 20 mM, more preferably between 0.5 and 10 mM, even more preferably between 1 and 5 mM, even more preferably between 1 and 2.5 mM, even more preferably 2.0 mM.

17. The method according to any one of claims 1 to 16, wherein in step (B) the at least one short-chain fatty acid is added directly to the culture medium.

18. The method according to any one of claims 1 to 17, wherein the method further comprises a step (B1) prior to step (B), wherein in step (B1) the immune cells are activated.

19. The method according to claim 18, wherein in step (B1), the activation is performed by the addition of activating agents of the immune cells, such as an antibody and / or a cytokine.

20. The method according to claim 19, wherein the antibody is an o-CD3 antibody, or an O-CD28 antibody, and / or wherein the cytokine is an IL-2.

21. The method according to any one of claims 18 to 20, wherein in step (B1) an o-CD3 antibody, an O-CD28 antibody, and IL-2 are added.

22. The method according to any one of claims 1 to 21, wherein step (A) comprises a sorting step to isolate the immune cells of step (A).4423. The method according to claim 22, wherein the sorting step is carried out via magnetic beads.

24. The method according to any one of claims 1 to 23, further comprising a step (D) of expanding the recombinant immune cells obtained in step (C).

25. The method according to claim 24, wherein expansion is performed for 1 to 5 days.

26. The method according to any one of the preceding claims, comprising the steps of:(A) Isolating immune cells; preferably wherein the immune cells are T-cells;(B1) Activating the immune cells; preferably wherein the immune cells are activated by the addition of an o-CD3 antibody, an O-CD28 antibody, an anti-IL-4 antibody and the cytokine IL-2;(B) Incubating the immune cells obtained in step (B1) in the presence of pentanoate; preferably wherein the incubation is performed for 48 hours;(C) Introducing into said immune cells of step (B) at least one nucleic acid or set of nucleic acids encoding an immunoreceptor; preferably wherein the immune receptor is a CAR; and(D) Expanding the recombinant immune cells obtained in step (C), preferably wherein expansion is performed for 1 to 5 days.

27. The method according to any one of the preceding claims, wherein the immunoreceptor is an immunoreceptor capable of binding to an antigen selected from the group consisting of CD19, CD20, CD22, CD27, CD30, CD33, CD38, CD44v6, CD52, CD64, CD70, CD72, CD 123, CD 135, CD 138, CD220, CD269, CD319, R0R1, R0R2, SLAMF7, BCMA, ovp3-lntegrin, o4p1 -Integrin, EpCAM-1, MUC-1, MUC-16, L1-CAM, c- kit, NKG2D, NKG2D-Ligand, PD-L1, PD-L2, Lewis-Y, CAIX, CEA, c-MET, EGFR, EGFRvlll, ErbB2, Her2, FAP, FR-a, EphA2, GD2, GD3, GPC3, IL-13Ra, Mesothelin, PSMA, PSCA, and VEGFR, preferably R0R1 or CD19.

28. The method according to any one of claims 27, wherein the immunoreceptor is a R0R1 -specific CAR or a CD 19 specific CAR.

29. The method according to any one of preceding claims, wherein the at least one short-chain fatty acid is produced by at least one species of bacteria.

30. The method according to claim 29, wherein the at least one short-chain fatty acid is produced by the bacterium Megasphaera massiliensis.4531. Method according to claim 29 or 30, wherein the at least one short-chain fatty acid is produced by a composition of bacteria comprising at least the bacteria Megasphaera massiliensis, Megasphaera elsdenii, Faecalibacterium prausnitzii and Anaerostipes hadrus.

32. Use of the short-chain fatty acid pentanoate to reprogram immune cells, wherein the immune cells are incubated in the presence of pentanoate or a pharmaceutical acceptable derivative thereof.

33. The use according to claim 32, wherein the immune cells are antigen-specific immune cells or genetically engineered immune cells.

34. The use according to claims 32 or 33, wherein the reprogramming is metabolic reprogramming and / or epigenetic reprogramming.

35. The use according to claim 34, wherein the metabolic reprogramming and / or epigenetic reprogramming is characterized by an increased metabolic fitness and a naive-like cell state of the immune cells.

36. The use according to claim 34 or 35, wherein the metabolic reprogramming and / or epigenetic reprogramming is characterized by an increased ability to secrete effector cytokines such as IFN-y, IL-2 and TNF-o of the immune cells.

37. The use according to any one of claims 32 to 36, wherein the immune cells are incubated in the presence of pentanoate for more than 24 hours, such as 24 to 72 hours, even more preferably for 48 hours.

38. The use according to any one of claims 32 to 37, wherein the immune cells are T cells, B cells, NK cells, NKT cells, macrophages and / or stem cells.

39. The use according to any one of claims 32 to 38, wherein said immune cells are T cells, preferably wherein said immune cells are CD4+ and / or CD8+ T cells.

40. The use according to any one of claims 32 to 39, wherein said immune cells are mammalian immune cells, preferably wherein said immune cells are human immune cells.

41. The use according to any one of claims 33 to 40, wherein said genetically engineered immune cells are immune cells engineered to express an immunoreceptor, preferably wherein the immunoreceptor is a B-cell receptor (BCR), T-cell receptor (TCR), or chimeric antigen receptor (CAR).

42. The use according to claim 41, wherein said genetically engineered immune cells are CAR T cells.

43. The method or use according to any one of the preceding claims, wherein all the steps of the method or use are carried out in vitro.

44. The method or use according to any one of the preceding claims, wherein said method or use does not comprise a method for treatment of the human or animal body by surgery or therapy or a diagnostic method practiced on the human or animal body.

45. The method or use according to any one of the preceding claims, wherein said method or use does not comprise a process for modifying the germ line genetic identity of a human being.

46. A recombinant immune cell obtainable by the method according to any one of claims 1 to 45.

47. A pharmaceutical composition comprising the recombinant immune cell of claim 46, the composition optionally further comprising a pharmaceutically acceptable carrier and / or excipient.

48. A recombinant immune cell according to claim 46, or a pharmaceutical composition according to claim 47, for use in medicine.

49. A recombinant immune cell according to claim 46, or a pharmaceutical composition according to claim 47, for use in the treatment of cancer by cancer immunotherapy.

50. A recombinant immune cell according to claim 46, or a pharmaceutical composition according to claim 47, for use in the treatment of cancer by cancer immunotherapy, wherein the immunoreceptor of the immune cell is capable of binding to a cancer antigen on the cell surface of a cell of the cancer.51 . The recombinant immune cell and or a pharmaceutical composition for use according to claims 49 or 50, wherein the cancer is haematological or solid cancer, optionally wherein the cancer is selected from the group consisting of lymphoma and myeloma.

52. A method for predicting and / or determining the efficacy of an immunotherapy in a subject comprising the steps of: a) determining a pentanoate concentration in a sample isolated from said subject; andb) predicting or determining the efficacy of the immunotherapy based on the concentration determined in step a).53 The method of claim 52, wherein in step b), the efficacy of the immunotherapy is predicted and / or determined to be increased when the determined pentanoate concentration in the sample is at or above a threshold, compared to when the pentanoate concentration in the sample is below said threshold.

54. The method according to claims 52 or 53, wherein the sample is a fecal sample, and / or wherein the immunotherapy is a chimeric antigen receptor (CAR) T-cell therapy.

55. The method according to claims 53 or 54, wherein the threshold is: a) A weight concentration of 200 pig / g or more, more preferably 300 pig / g or more, even more preferably 400 pig / g or more, even more preferably 500 pig / g, such as 285.6 pig / g, and / or b) A molar concentration of 2 pimol / g or more, preferably 3 pimol / g or more, more preferably 4 pimol / g or more, even more preferably 5 pimol / g or more, such as 6 pimol / g.

56. The method according to any of claims 53 to 55, wherein the increased efficacy of the immunotherapy is characterized by an increased survival of the subject receiving immunotherapy compared to the efficacy of the immunotherapy in a subject for which the pentanoate concentration in the sample is determined to be below the threshold, optionally wherein the increased survival is an increased overall survival.

57. The method according to any of claims 53 to 56, wherein the subject has cancer and the immunotherapy is cancer immunotherapy against said cancer, preferably wherein the immunotherapy is a chimeric antigen receptor (CAR) T-cell therapy capable of targeting a cancer antigen on the cell surface of a cell of the cancer, and the increased efficacy of the cancer immunotherapy is characterized by an increased progression-free survival (PFS) in said subject having cancer compared to the efficacy of the cancer immunotherapy in a subject for which the pentanoate concentration in the sample is determined to be below the threshold.

58. The method according to any of claims 53 to 57, wherein all the steps of the method are carried out in vitro.

59. A method of treating cancer in a subject having the cancer, the method comprising:(A) Predicting or determining the efficacy of a cancer immunotherapy in a group of subjects having cancer with the method according to any of claims 53 to 57,(B) Identifying a subject as having an increased predicted and / or determined efficacy of the cancer immunotherapy when the determined pentanoate concentration in the sample is at or above a threshold as defined in claims 53 or 55;48and(c) Administering to said subject the cancer immunotherapy, preferably wherein the immunotherapy is a chimeric antigen receptor (CAR) T-cell therapy capable of targeting a cancer antigen on the cell surface of a cell of the cancer.

60. A CAR T cell capable of targeting a cancer antigen on the cell surface of a cell of a cancer, for use in a method for the treatment of said cancer in a subject, wherein in the method, the CAR T cell is to be administered to a subject in which the pentanoate concentration in a sample isolated from said subject is at or above a threshold, wherein the threshold is as defined in claim 55, optionally wherein the sample is a fecal sample.

61. The method according to claim 59 or the CAR T cell for use according to claim 60, wherein the cancer is haematological or solid cancer, optionally wherein the cancer is selected from the group consisting of lymphoma and myeloma.49

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