Method for suppressing car-t cells unresponsiveness for improving cancer immunotherapy

Abstract

The invention relates to methods for identifying unresponsive T cells and unresponsive chimeric antigen receptor-bearing T cells (CAR-T cells), methods of rejuvenation thereof and methods for improving CAR-T cell therapies.

Description

[0001] METHOD FOR SUPPRESSING CAR-T CELLS UNRESPONSIVENESS FOR IMPROVING CANCER IMMUNOTHERAPY

[0002] FIELD OF THE INVENTION

[0003] The invention relates to methods for identifying unresponsive T cells and unresponsive chimeric antigen receptor-bearing T cells (CAR-T cells), methods of rejuvenation thereof and methods for improving CAR-T cell therapies.

[0004] BACKGROUND OF THE INVENTION

[0005] Cancer immunotherapy, such as CAR-T cell therapy, has emerged as a promising strategy for enlisting and strengthening the power of a patient’s immune system to attack tumors. One immunotherapy approach involves collecting and engineering patients’ own T cells to treat their cancer. Two exemplary approaches for engineering T cells include engineering patients’ T cells to express a specific T-cell receptor (TCR) or engineering T cells to express chimeric antigen receptors (CARs). TCRs use naturally occurring receptors that can also recognize antigens that are inside tumor cells. Small pieces of these antigens are shuttled to the cell surface and “presented” to the immune system as part of a collection of proteins called the MHC complex. CARs, on the other hand, comprise portions of an antibody that can recognize a specific antigen on the surface of cancer cells. Despite their potential as a therapy, CAR-T cells also become unresponsive (exhausted) and can require additional strategies so that they can restore their functionality (be rejuvenated).

[0006] The success of CD19-directed CAR-T cells on haematological malignancies has generated a wide interest in the expansion of this approach to solid tumors. However, the clinical outcomes in this context have been unexpectedly poor due to both extrinsic and intrinsic suppressive factors that drive T cell dysfunction within the tumor microenvironment. A fundamental characteristic that contributes to anti-tumor T cell functionality and that determines CAR-T cell efficacy is the maintenance of mitochondrial fitness, which has been shown to sustain memory / stem-like properties and provide prolonged T cell responses.

[0007] Thus, there is a significant unmet need to reduce and / or reverse CAR-T cell unresponsiveness in order to improve CAR-T cell therapy. SUMMARY OF THE INVENTION

[0008] An aspect of the present invention provides a method of suppressing CAR-T cells unresponsiveness, the method comprising contacting unresponsive CAR-T cells with a NAD booster and with one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator, wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN).

[0009] Another aspect of the present invention provides a method for improving a CAR-T cell therapy in a subject, the method comprising i. determining the NAD level and / or the expression level of CD38 in T cells obtained from the subject or in CAR-T cells generated from T cells obtained from the subject; ii. comparing the NAD level determined at step i) with a predetermined reference value of NAD and / or comparing the expression level of CD38 determined at step i) with a predetermined reference value of CD38; iii. when the NAD level determined at step i) is lower than its predetermined reference value and / or the expression level of CD38 determined at step i) is higher than its predetermined reference value, administering to the subject an effective amount of rejuvenated CAR-T cells, wherein the NAD level is increased and / or CD38 is inhibited or deficient in the rejuvenated CAR-T cells.

[0010] A further aspect of the present invention provides a method of identifying unresponsive T cells or unresponsive CAR-T cells, the method comprising: i. determining the NAD level and / or the expression level of CD38 in T cells or in CAR-T cells; ii. comparing the NAD level determined at step i) with a predetermined reference value of NAD and / or comparing the expression level of CD38 determined at step i) with a predetermined reference value of CD38; wherein when the NAD level determined at step i) is lower than its predetermined reference value and / or the expression level of CD38 determined at step i) is higher than its predetermined reference value indicates that T cells or CAR-T cells are unresponsive T cells or unresponsive CAR-T cells. A further aspect of the present invention provides a method for predicting the efficacy of a CAR-T cell therapy in a subject, the method comprising i. determining the NAD level and / or the expression level of CD38 in T cells obtained from the subject or in CAR-T cells generated from T cells obtained from the subject; ii. comparing the NAD level determined at step i) with a predetermined reference value of NAD and / or comparing the expression level of CD38 determined at step i) with a predetermined reference value of CD38; wherein when the NAD level determined at step i) is equal or higher than its predetermined reference value and / or the expression level of CD38 determined at step i) is equal or lower than its predetermined reference value is indicative for good efficacy of the CAR-T cell therapy when applied to the subject, and when the NAD level determined at step i) is lower than its predetermined reference value and / or the expression level of CD38 determined at step i) is higher than its predetermined reference value is indicative for poor efficacy of the CAR-T cell treatment when applied to the subject.

[0011] A further aspect of the present invention provides a method for predicting the efficacy of immune checkpoint blockade (ICB) therapy in a subject, the method comprising i. determining the NAD level and / or the expression level of CD38 in tumor infiltrating lymphocytes (TILs) obtained from the subject; ii. comparing the NAD level determined at step i) with a predetermined reference value of NAD and / or comparing the expression level of CD38 determined at step i) with a predetermined reference value of CD38; wherein when the NAD level determined at step i) is equal or higher than its predetermined reference value and / or the expression level of CD38 determined at step i) is equal or lower than its predetermined reference value is indicative for good efficacy of the immune checkpoint blockade (ICB) therapy when applied to the subject, and when the NAD level determined at step i) is lower than its predetermined reference value and / or the expression level of CD38 determined at step i) is higher than its predetermined reference value is indicative for poor efficacy of the immune checkpoint blockade (ICB) therapy when applied to the subject.

[0012] A further aspect of the present invention provides an ex vivo method of producing rejuvenated CAR-T cells from unresponsive CAR-T cells, the method comprising i. providing a population of unresponsive CAR-T cells; ii. expanding the population of unresponsive CAR-T cells in the presence of an NAD booster and one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and NAMPT activator, and, thereby obtaining rejuvenated CAR-T cells wherein CD38 is inhibited or deficient and wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN), preferably the NAD booster is nicotinamide mononucleotide (NMN).

[0013] BRIEF DESCRIPTION OF THE FIGURES

[0014] Figure 1 shows that aged CAR-T cells are unable to maintain stem-like properties.

[0015] (A) Schematic representation of the experimental plan performed in Figure 1 (B-G).

[0016] (B) Aged CD8+ T cells display a loss of a stem-like phenotype (CD44+CD62L+T cells) when compared to younger counterparts.

[0017] (C)Bar graph summarizing data shown in (B).

[0018] (D) Aged CD8+ T cells display lower levels of TCF1, transcription factor involved in T cell sternness.

[0019] (E) Aged CD8+ CAR-T cells display higher effector functions, as shown by an increased killing capacity against B 16 tumor cells.

[0020] (F) Aged CD8+ CAR-T cells display higher effector functions, as shown by an increased ability to produce cytokines (ZFNy) against B 16 tumor cells.

[0021] (G)Aged CD8+ CAR-T cells display higher effector functions, as shown by an increased ability to produce cytokines (TNFa) against B 16 tumor cells.

[0022] (H) Schematic representation of the experimental plan performed in Figure 1 (I-L)

[0023] (I) Aged CAR-T cells expanded under memory polarizing conditions are unable to control growth of B16 tumors.

[0024] (J) Tumor size in mice treated with aged CAR-T cells is higher when compared to younger controls.

[0025] (K) Aged CAR- T cells are unable to survive long-term in vivo upon adoptive cell transfer.

[0026] (L) Aged CAR-T cells with stem-like properties are unable to engraft in vivo.

[0027] Figure 2 shows that aged T cells display mitochondrial defects. (A) Aged CD8+T cells display a reduced mitochondrial activity and size when compared to younger controls.

[0028] (B) Summarizing data of mitochondrial activity (TMRM) and size (MTG) in young vs old T cells.

[0029] (C) Mitochondrial profile of young vs old CD8+ T cells as assessed by Seahorse analysis.

[0030] (D-E) Old CD8+ T cells show a decreased basal and maximal oxygen consumption rate (OCR).

[0031] Figure 3 shows that age-associated mitochondrial dysfunction of aged T cells is CD38- dependent.

[0032] (A)Metabolomic analysis of young vs old CD8+T cells show an important depletion of NAD levels.

[0033] (B) Aged T cells are not able to respond to NAD-boosters, as shown by an inability to increase mitochondrial function upon supplementation.

[0034] (C) Aged T cells are not able to respond to NAD-boosters, as shown by an inability to rescue an exhausted (unresponsive) phenotype upon supplementation.

[0035] (D) Aged T cells display higher levels of CD38 in both resting an activated state.

[0036] (E) Higher CD38 levels in aged T cells induce mitochondrial dysfunction.

[0037] (F) Young T cells overexpressing CD38 show a reduced ability to engage central memory formation (CD44+CD62L+phenotype).

[0038] (G) Young T cells overexpressing CD38 show reduced mitochondrial content.

[0039] Figure 4 shows that restoration of NAD levels boosts mitochondrial fitness and efficacy of CAR-T cells.

[0040] (A) Aged T cells treated with CD38 inhibitor (78c) and the NAD-booster NMN rescue their NAD levels.

[0041] (B) Aged T cells treated with CD38 inhibitor (78c) and the NAD-booster NMN rescue their mitochondrial fitness.

[0042] (C) Aged T cells treated with CD38 inhibitor (78c) and the NAD-booster NMN rescue their mitochondrial size.

[0043] (D) Schematic representation of the experimental plan performed in Figure 4 (E-G)

[0044] (E) Aged CAR-T cells expanded using our combination treatment (78c + NMN) are able to control tumor growth in vivo. (F-G) Aged CAR-T cells expanded using our combination treatment (78c + NMN) are able to increase their persistence in vivo upon adoptive cell transfer.

[0045] Figure 5 shows PARP inhibition as an alternative mechanism to rejuvenate aged CAR-T cells

[0046] (A) Aged CAR-T cells present signs of DNA damage.

[0047] (B) Aged CAR-T cells treated with the PARP inhibitor Olaparib in combination with NMN are able to increase NAD levels.

[0048] (C) Aged CAR-T cells treated with the PARP inhibitor Olaparib in combination with NMN are able to improve mitochondrial fitness.

[0049] (D) Aged CAR-T cells treated with the PARP inhibitor Olaparib in combination with NMN are able to boost CD62L levels.

[0050] Figure 6 shows that the combination of NAMPT activator SBI-797812 (SB20) with the NAD booster NAM improves old T cells phenotype

[0051] (A)Expression level of CD62L in old T cells treated with NAM or SB20 or in combination

[0052] (B) Fold change of central memory T cells in old T cells treated with NAM or SB20 or in combination

[0053] Figure 7 shows that the combination of NAMPT activator SBI-797812 (SB20) with the NAD booster NAM improves old T mitochondrial fitness

[0054] (A) Aged T cells treated with SB20 in combination with NAM improved their mitochondrial potential

[0055] (B) Aged T cells treated with SB20 in combination with NAM improved their mitochondrial mass

[0056] (C) Aged T cells treated with SB20 in combination with NAM improved their mitochondrial fitness

[0057] Figure 8 shows CD38 blockade rejuvenates human CAR-T cells.

[0058] (A) Cancer patients providing complete responses (CR) to CAR-T cell therapy display lower levels of CD38.

[0059] (B) CD38 levels are associated to alterations in NAD metabolism in CAR-T cell infusion products of cancer patients. (C)Non-responder patients to ICB in basal cell carcinoma are associated to higher CD38 levels.

[0060] (D)CD38 levels, but not other exhaustion (responsiveness) markers, are upregulated in exhausted (unresponsive) T cells of non-responder melanoma patients to ICB.

[0061] (E) CD38 levels are increased in older cancer patients (>70 years old) when compared to younger patients (<40 years old).

[0062] (F) Increased higher CD38 levels are linked to lower mitochondrial activity in human CD8+ T cells.

[0063] (G)Human CAR-T cells generated from aged donors display an impaired mitochondrial profile when compared to younger controls, which is rescued upon treatment with 78c + NMN.

[0064] (H) Human CAR-T cells generated from aged donors display an impaired spare respiratory capacity (SRC) when compared to younger controls, which is rescued upon treatment with 78c + NMN.

[0065] (I) Human CAR-T cells generated from aged donors display a reduced mitochondrial size when compared to younger controls, which is rescued upon treatment with 78c + NMN.

[0066] DETAILED DESCRIPTION OF THE INVENTION

[0067] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0068] In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

[0069] The term “comprise” is generally used in the sense of include, permitting the presence of one or more features or components. Also as used in the specification and claims, the language "comprising" can include analogous embodiments described in terms of "consisting of “ and / or "consisting essentially of’. The terms "including," "comprising," or "having," and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as "including", "comprising" or "having" certain elements are also contemplated as "consisting essentially of and "consisting of those certain elements.

[0070] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0071] As used in the specification and claims, the term "and / or" used in a phrase such as "A and / or B" herein is intended to include "A and B", "A or B", "A", and "B".

[0072] As used herein the terms "subject" and “patient” are well -recognized in the art, and, are used herein to refer to a mammal, and most preferably a human. These terms do not denote a particular age or sex. Thus, adult and newborn subjects or patients, whether male or female, are intended to be covered. In some embodiments, the subject or the patient is suffering from a cancer at any developmental stage. In some embodiments, the subject or the patient has undergone a cancer immunotherapy. In some embodiments, the subject or the patient is a subject in need of treatment or a subject having a cancer, who is likely to benefit from a treatment of the present invention.

[0073] The term "effective amount" or "therapeutically effective amount" means a sufficient amount of the specified component / composition to have the specified properties under the specified conditions.

[0074] As used herein, the term "immune checkpoint blockade" refers to cancer treatment that uses a class of drugs known as immune checkpoint inhibitors (ICIs). Similarly to other immunotherapies, the goal of immune checkpoint blockade is to strengthen the immune system of the subject or the patient and enhance its ability to fight cancer.

[0075] As used herein, the terms "immune checkpoint inhibitors" (ICIs), "checkpoint inhibitors," and the like refer to compounds that inhibit the activity of negative regulators of the immune response. Immune system checkpoints, or immune checkpoints, are regulatory molecules involved in the control of the duration and amplitude of physiological immune responses as well as maintenance of self-tolerance to minimize collateral tissue damage. Immune checkpoint signaling requires ligand-receptor interaction to send, via receptors, co-stimulatory (positive immune checkpoints) or co-inhibitory (negative immune checkpoints) signals. ICI are molecules disrupting immunosuppressive interaction between immune checkpoint ligand and receptors, reinvigorating thus innate and adaptive immunity to fight cancers. Negative immune checkpoints include, but are not limited to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death- ligand 1 (PD-L1), programmed cell death- ligand 2 (PD- L2), programmed cell death 1 (PD-1), Lymphocyte-activation gene 3 (LAG-3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), V-domain immunoglobulin suppressor of T-cell activation (VISTA), CD276, and V-set domain containing T cell activation inhibitor 1 (VTCN1). As such, ICI include antagonists of, for example, immune checkpoints such as CTLA-4, PD-1, or PD-L1. For example, antibodies that bind to CTLA-4, PD-1, or PD-L1 and antagonize their function are ICI. Moreover, any molecule (e.g., peptide, nucleic acid, small molecule, etc.) that inhibits the inhibitory function of an immune checkpoint is an ICI.

[0076] As used herein, the term "unresponsive" or "unresponsiveness" or "exhaustion" or "exhausted" or "senescent" refers to a state of a cell where the cell does not perform its usual function or activity in response to normal input signals, and includes refractivity of immune cells to stimulation, such as stimulation via an activating receptor or a cytokine. Such a function or activity includes, but is not limited to, proliferation or cell division, entrance into the cell cycle, cytokine production, cytotoxicity, trafficking, phagocytotic activity, or any combination thereof. Normal input signals can include, but are not limited to, stimulation via a receptor, such as T cell receptor, B cell receptor, co-stimulatory receptor, and the like. Unresponsive (or exhausted or senescent) immune cells can have a reduction of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more in cytotoxic activity, cytokine production, proliferation, trafficking, phagocytotic activity, or any combination thereof, relative to a corresponding control immune cell of the same type. In one embodiment, an unresponsive T cell is CD8+ T cell (e.g., an effector CD8+ T cell that is antigen-specific) and / or CD4+ T cell. CD8+ cells normally proliferate (e.g., clonally expand) in response to T cell receptor and / or co-stimulatory receptor stimulation, as well as in response to cytokines such as IL-2. Thus, an unresponsive CD8+ T cell is one which does not proliferate and / or produce cytokines in response to normal input signals. It is well known that the exhaustion of effector functions can be delineated according to several stages, which eventually lead to terminal or full unresponsiveness (exhaustion) and, ultimately, deletion. In the first stage, functional T cells enter a "partial unresponsiveness" or "partial exhaustion" I phase characterized by the loss of a subset of effector functions, including loss of IL-2 production, reduced TNFa production, and reduced capacity for proliferation and / or ex vivo lysis ability, in the second stage, partially unresponsive T cells enter a "partial unresponsiveness" or a "partial exhaustion" II phase when both IL-2 and TNFa production ceases following antigenic stimulation and IFNy production is reduced. "Full unresponsiveness" or "terminal unresponsiveness" ("full exhaustion" or "terminal exhaustion") occurs when CD8+ T cells lose all effector functions, including the lack of production of IL-2, TNFa, and IFNy and loss of ex vivo lytic ability and proliferative potential following antigenic stimulation, A fully unresponsive (exhausted) CD8+ T cell is one which does not proliferate, does not lyse target cells (cytotoxicity), and / or does not produce appropriate cytokines, such as IL-2, TNFa, or IFNy, in response to normal input signals. Such lack of effector functions can occur when the antigen load is high and / or CD4 help is low. This hierarchical loss of function is also associated with the expression of co-inhibitor immune receptors, such as PD-1, TIM-3, LAG-3, and the like. Other molecular markers distinguish the hierarchical stages of immune cell unresponsiveness (exhaustion), such as high eomesodermin (EOMES) and low TBET expression as a marker of terminally unresponsive (exhausted) T cells. Additional markers of unresponsive (exhausted) T cells, such as the reduction of Bci-b and the increased production of BLIMP-1 (Pdrml). Similarly to T cell, CAR-T cells become unresponsive with aging, as described above. An unresponsive CAR-T cell is typically defined as a CAR-T cell unable to proliferate, unable to provide tumor control in vitro and in vivo and unable to persist long-term in vivo. Also, in the present disclosure, it is provided that T cells / CAR-T cells become unresponsive during aging and are thereby identified as "aged" or "old" T cells / CAR-T cells. The present disclosure also provides that unresponsive (aged, old) T cells / CAR-T cells display low levels of NAD, while responsive T cells / CAR-T cells display high levels of metabolite NAD.

[0077] As used herein, the term "rejuvenated" T cells / CAR-T cells are identified as responsive T cells / CAR-T cells that have restored functionality, typically can proliferate and / or produce cytokines in response to normal input signals. In rejuvenated T cells / CAR-T cells, NAD levels are increased compared to unresponsive (aged, old) T cells / CAR-T cells. Typically, the rejuvenated CAR-T cells have increased CAR-T cell activity, responsiveness, and / or ability or receptiveness, with regards to activation comparing to the unresponsive (aged, old) CAR-T cells. Methods of measuring CAR-T cell activity are well known in the art. Modulation of one or more of any of the immune cell unresponsiveness (exhaustion) parameters described above can be assayed. For example, CAR-T cell activity can be measured by contacting CAR-T cells with recall antigen, anti-CD3 in the absence of costimulation, and / or ionomycin. Also, proliferation of CAR-T cells can be measured in the presence of a relevant antigen assayed, e.g. by a 3H-mymidinc incorporation assay or cell number. Markers of CAR- T cell activation after exposure to the relevant antigen can also be assayed, e.g. flow cytometry analysis of cell surface markers indicative of CAR-T cell activation (e.g., CD30, CD25, and HLA-DR) and / or CAR-T cell unresponsiveness (exhaustion). In some embodiments, the assays can be in vivo assays, such as through challenging immune cells with antigen, in vivo.

[0078] The present disclosure provides methods for identifying unresponsive T cells or unresponsive chimeric antigen receptor-bearing T cells (CAR-T cells), methods of rejuvenation thereof, methods of suppressing unresponsiveness in T-cells and / or CAR T-cells, methods of producing rejuvenated CAR T-cells and methods for improving CAR-T cell therapies. In some embodiments, the unresponsive T cells and the unresponsive CAR-T cells are exhausted T cells and exhausted CAR-T cells; or senescent T cells and senescent CAR-T cells.

[0079] A CAR-T cell is a T cell which expresses a chimeric antigen receptor. The T cell expressing a CAR molecule may be a helper T cell, a cytotoxic T cell, a viral- specific cytotoxic T cell, a memory T cell, or a gamma delta (gd) T cell.

[0080] A chimeric antigen receptor (CAR) is a recombinant fusion protein comprising: 1) an extracellular ligand-binding domain, i.e., an antigen-recognition domain, 2) a transmembrane domain, and 3) a signaling transducing domain. CARs have the ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner. The non- MHC-restricted antigen recognition gives CAR-T cells the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape. Moreover, when expressed on T-cells, CARs advantageously do not dimerize with endogenous T-cell receptor (TCR) alpha and beta chains. The extracellular ligand-binding domain is an oligo- or polypeptide that is capable of binding a ligand. Preferably, the extracellular ligand-binding domain will be capable of interacting with a cell surface molecule which may be an antigen, a receptor, a peptide ligand, a protein ligand of the target, or a polypeptide of the target. The extracellular ligand-binding domain can specifically bind to an antigen with an affinity constant or affinity of interaction (KD) between about 0.1 pM to about 10 pM, to about 0.1 pM to about 1 pM, or to about 0.1 pM to about 100 nM. Methods for determining the affinity constant or affinity of interaction (KD) are well- known in the art. In some instances, the extracellular ligand-binding domain is chosen to recognize a ligand that acts as a cell surface marker on target cells associated with particular disease states.

[0081] In one embodiment, the extracellular ligand-binding domain comprises a single chain antibody fragment (scFv) comprising the light (VL) and the heavy (VH) variable fragment joined by a linker (e.g., poly-G) and confers specificity for either a T cell antigen or an antigen that is not specific to a T cell. In one embodiment, the chimeric antigen receptor of a CAR-T cell may bind to an T cell-specific antigen expressed or overexpressed on a malignant T cell for which a CAR-T cell is deficient in the antigen (e.g., a genome-edited CAR-T cell). Non-limiting examples of CAR-targeted antigens expressed on malignant T cells include CD5, CD7, CD2, CD4, and CD3. Non-limiting examples of CAR-targeted antigens expressed on the surface of leukemia cells (e.g., abnormal myeloblasts, red blood cells, or platelets) include CD123 (IL3RA), CD371 (CLL-1; CLEC12A), CD117 (c-kit), and CD135 (FLT3), CD7, and Tim3. A CAR may be constructed with an extracellular ligand-binding domain to target these antigens for treatment of leukemia, i.e., acute myeloid leukemia (AML). Non-limiting examples of CAR-targeted antigens expressed on the surface of a multiple myeloma cell (e.g., a malignant plasma cell) include BCMA, CS1, CD38, CD79A, CD79B, CD138, and CD 19. A CAR may be constructed with an extracellular ligand-binding domain to target these antigens for treatment of multiple myeloma. In another embodiment, the CAR may be constructed with a portion of the APRIL protein, targeting the ligand for the B-Cell Maturation Antigen (BCMA) and Transmembrane Activator and CAML Interactor (TACI), effectively co-targeting both BCMA and TACI for the treatment of multiple myeloma. A signal peptide directs the transport of a secreted or transmembrane protein to the cell membrane and / or cell surface to allow for correct localization of the polypeptide. Particularly, the signal peptide of the present disclosure directs the appended polypeptide, i.e., the CAR receptor, to the cell membrane wherein the extracellular ligand -binding domain of the appended polypeptide is displayed on the cell surface, the transmembrane domain of the appended polypeptide spans the cell membrane, and the signaling transducing domain of the appended polypeptide is in the cytoplasmic portion of the cell. In one embodiment, the signal peptide is the signal peptide from human CDBcx. In one embodiment, the signal peptide is a functional fragment of the CD8a signal peptide. A functional fragment is defined as a fragment of at least 10 amino acids of the CD8a signal peptide that directs the appended polypeptide to the cell membrane and / or cell surface.

[0082] Chimeric antigen receptor (CAR) T-cell therapy uses genetically modified T cells to more specifically and efficiently target and kill cancer cells. After T cells have been collected from the blood, the cells are engineered to include CARs on their surface. The CARs may be introduced into the T cells using CRISPR / Cas9 gene editing technology or via a virus, such as lentivirus. When these allogeneic CAR-T cells are injected into a patient, the receptors enable the T cells to kill cancer cells.

[0083] Chimeric antigen receptor (CAR) T cell therapies are adoptive T cell therapeutics used to treat human malignancies. Although CAR-T cell therapy has led to tremendous clinical success, including durable remission in relap sed / refractory non-Hodgkin lymphoma (NHL) and pediatric acute lymphoblastic leukemia (ALL), the approved products are autologous and require patient-specific cell collection and manufacturing. Because of this, some patients have experienced disease progression or death while awaiting treatment. Allogeneic CAR-T cell therapy, comprising disrupted MHC-1 complexes, presents an attractive off-the-shelf option to autologous CAR-T cell therapy. The disrupted MHC class I in the allogeneic T cells, however, renders the CAR-T cells susceptible to elimination by the host immune system, for example, by natural killer (NK) cell-mediated immune response. Accordingly, there remains a need for improved CAR-T cell therapy.

[0084] Suitable methods to produce CAR-T cells (genetically engineered T cells expressing the CAR) are known in the art, such as WO 2019 / 097305 and WO 2019 / 215500, the relevant disclosures of each of the prior applications are incorporated by reference herein for the purpose and subject matter referenced herein.

[0085] The present disclosure provides that CAR-T cells from old mice display remarkable defects in mitochondrial activity and mass. Furthermore, aged CAR-T cells are unable to reach an appropriate stem-like phenotype, as shown by a lower proportion of CD44+CD62L+TCF1+ CAR-T cells. As a result, aged CAR-T cells are unable to survive in vivo and control tumor growth upon adoptive cell transfer. The inventors have discovered that aged CAR-T cells decrease NAD levels which is linked to the upregulation of CD38, a cell membrane protein with NADase properties. It was further observed that aged CAR-T are not able to respond to NAD boosters and, importantly, that higher CD38 levels are associated with lower mitochondrial activity. Indeed, blockade of CD38 activity using the small molecule 78c (CD38 inhibitor 78c) restores NAD levels and mitochondrial function when administered in combination with NAD boosters. Thus the present disclosure provides that the age-associated upregulation of CD38 drives NAD decline and mitochondrial dysfunction in CAR-T leading to limited acquisition of mem ory / stem -like characteristics. CD38 was identified as a target to restore stem-like properties of CAR-T cells.

[0086] An aspect of the present invention provides a method of identifying unresponsive T cells or unresponsive CAR-T cells, the method comprising: i. determining the NAD level and / or the expression level of CD38 in T cells or CAR-T cells; ii. comparing the NAD level determined at step i) with a predetermined reference value of NAD and / or comparing the expression level of CD38 determined at step i) with a predetermined reference value; wherein when the NAD level determined at step i) is lower than its predetermined reference value and / or the expression level of CD38 determined at step i) is higher than its predetermined reference value indicates that T cells or CAR-T cells are unresponsive T cells or unresponsive CAR-T cells.

[0087] In some embodiments of the method of identifying the unresponsive T cells or the unresponsive CAR-T cells, the unresponsive T cells and the unresponsive CAR-T cells are exhausted T cells and exhausted CAR-T cells; or senescent T cells and senescent CAR-T cells.

[0088] In one embodiment of the method of identifying the unresponsive T cells or the unresponsive CAR-T cells, the unresponsive T cells are CD4+ and / or CD8+ T cells; or the unresponsive CAR-T cells are CD4+ and / or CD8+ CAR-T cells.

[0089] In some preferred embodiments, the method of identifying the unresponsive T cells or the unresponsive CAR-T cells is an in vitro method. In further preferred embodiments, the NAD level and / or the expression level of CD38 is determined in isolated T cells from a blood sample obtained from a subject (i.e. T cells before being genetically modified to generate CAR-T cells) or in CAR-T cells generated from T cells obtained from a subject (which are genetically modified T cells as disclosed in the present disclosure and which are ready to be administered to the patient).

[0090] The term “isolated T cells" as used herein refers to a population of T cells that is not comprised within the body. An isolated population of T cells may have been previously removed from a subject. An isolated population of cells may be cultured and manipulated ex vivo or in vitro using standard techniques known in the art. An isolated population of T cells may later be reintroduced into a subject or may be genetically modified to generate a population of CAR-T cells which is then reintroduced into a subject. Said subject may be the same subject from which the T cells were originally isolated or a different subject.

[0091] In some embodiments, the NAD level determined at step i) is lower than its predetermined reference value is when the NAD level is 5%, 10%, 20%, 30%, 40%, 50%, or 60% lower than the predetermined reference value. In other embodiments, the NAD level determined at step i) is lower than its predetermined reference value when the expression level is more than 5%, more than 10%, more than 20%, more than 30%, more than 40%, more than 50% or more than 60% lower than the predetermined reference value. In some preferred embodiments, the NAD level determined at step i) is lower than its predetermined reference value when the NAD level is 30 % or more than 30% lower than the predetermined reference value.

[0092] In some embodiments, the expression level of CD38 determined at step i) is higher than its predetermined reference value is when the expression level is 5%, 10%, 20%, 30%, 40%, 50%, or 60% higher than the predetermined reference value. In other embodiments, the expression level determined at step i) is higher than its predetermined reference value when the expression level is more than 5%, more than 10%, more than 20%, more than 30%, more than 40%, more than 50% or more than 60% higher than the predetermined reference value. In some preferred embodiments, the expression level determined at step i) is higher than its predetermined reference value when the expression level is 30 % or more than 30% higher than the predetermined reference value. A further aspect of the present invention provides a method for predicting the efficacy of a CAR-T cell therapy in a subject, the method comprising i. determining the NAD level and / or the expression level of CD38 in T cells obtained from the subject or in CAR-T cells generated from T cells obtained from the subject; ii. comparing the NAD level determined at step i) with a predetermined reference value of NAD and / or comparing the expression level of CD38 determined at step i) with a predetermined reference value of CD38; wherein when the NAD level determined at step i) is equal or higher than its predetermined reference value and / or the expression level of CD38 determined at step i) is equal or lower than its predetermined reference value is indicative for good efficacy of the CAR-T cell therapy when applied to the subject, and when the NAD level determined at step i) is lower than its predetermined reference value and / or the expression level of CD38 determined at step i) is higher than its predetermined reference value is indicative for poor efficacy of the CAR-T cell treatment when applied to the subject.

[0093] In some embodiments, the NAD level and / or the expression level of CD38 is determined either before or after generation of CAR-T cells. Typically, a blood sample is obtained from a subject, T cells are isolated therefrom and then CAR-T are generated ex -vivo from these isolated T cells as disclosed in the present disclosure. The CAR-T cells (CAR-T cell products) are then administered (re-injected) to the same or different subject.

[0094] Another aspect of the present invention provides a method for predicting the efficacy of immune checkpoint blockade (ICB) therapy in a subject, the method comprising i. determining the NAD level and / or the expression level of CD38 in tumor infiltrating lymphocytes (TILs) obtained from the subject; ii. comparing the NAD level determined at step i) with a predetermined reference value of NAD and / or comparing the expression level of CD38 determined at step i) with a predetermined reference value of CD38; wherein when the NAD level determined at step i) is equal or higher than its predetermined reference value and / or the expression level of CD38 determined at step i) is equal or lower than its predetermined reference value is indicative for good efficacy of the immune checkpoint blockade (ICB) therapy when applied to the subject, and when the NAD level determined at step i) is lower than its predetermined reference value and / or the expression level of CD38 determined at step i) is higher than its predetermined reference value is indicative for poor efficacy of the immune checkpoint blockade (ICB) therapy when applied to the subject.

[0095] In preferred embodiments, TILs are extracted from tumor biopsies obtained from a subject.

[0096] Measuring the NAD level can be done by measuring the level / quantity of the nicotinamide adenine dinucleotide (NAD) in a sample and can be performed by a variety of techniques well known in the art.

[0097] The NAD levels determine T cell and CAR-T cell functionality and the NAD level can be affected either by CD38 up regulation, enhanced PARP activity or decreased NAMPT activity.

[0098] Measuring the expression level of CD38 can be done by measuring the gene expression level of CD38 or by measuring the level of the protein CD38 in a sample and can be performed by a variety of techniques well known in the art. In some embodiments, the level of CD38 is determined at nucleic acid level. Typically, the level of a gene may be determined by determining the quantity of mRNA. Methods for determining the quantity of mRNA are well known in the art. For example, the nucleic acid contained in the samples (e.g., T cell prepared from the subject or CAR-T cells prepared and ready to be administered to the subject) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and / or amplification (e.g., RT-PCR). Other methods of Amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence-based amplification (NASBA).

[0099] CD38 amino acid sequence (SEP ID NO: 1)

[0100] MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQWSGPGTTK RFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGT QTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQ SCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEV HNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQ CVKNPEDSSCTSEI CD38 nucleic acid sequence is provided in SEQ ID NO: 2.

[0101] The methods of the present invention comprise comparing the NAD level with a predetermined reference value of NAD and / or the expression level of CD38 with a predetermined reference value of CD38 wherein detecting a difference between the NAD level and the predetermined reference value of NAD and / or a difference between the expression level of CD38 and the predetermined reference value of CD38 indicates whether the T cells or the CAR-T cells are unresponsive (exhausted or senescent). In some embodiments, the predetermined reference value of NAD is the level (quantity) of NAD in T cells derived from population studies, typically from population of young subjects, preferably young healthy subjects. In some embodiments, the predetermined reference value of CD38 is the expression level of CD38 in T cells derived from population studies, typically from population of young subjects, preferably young healthy subjects. In the context of the present invention, the young subjects are less than 30 years old. Indeed, the NAD level in T cells and CAR-T cells obtained from young subjects, preferably healthy young subjects, is higher than in elderly population, and the expression level of CD38 in T cells and CAR-T cells obtained from young subjects, preferably healthy young subjects, is lower than in elderly population, as shown in the present disclosure. In preferred embodiments, the predetermined reference value of NAD is the NAD level in T cells derived from population of young healthy subjects being less than 30 years old and the predetermined reference value of CD38 is the expression level of CD38 in T cells derived from population of young healthy subjects being less than 30 years old. Such predetermined reference values can be derived from statistical analyses and / or risk prediction data of populations obtained from mathematical algorithms and computed indices. In some embodiments, retrospective measurement of the level of the marker in properly banked historical subject samples may be used in establishing these predetermined reference values. Accordingly, in some embodiments, the predetermined reference value is a threshold value or a cut-off value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the level of the marker in a group of reference, one can use algorithmic analysis for the statistic treatment of the measured levels of the marker in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate. It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

[0102] The present disclosure provides that in vivo, the combination treatment of unresponsive (aged, old) CAR-T cells with a NAD booster and with one or more compounds selected from a group consisting of a CD38 inhibitor, aPARP inhibitor and aNAMPT activator, restores functionality of unresponsive (aged, old) CAR-T cells, promoting long-term CAR-T cell survival and controlling tumor growth as its younger (not unresponsive) CAR-T cells. Furthermore, the inventors have also demonstrated that CAR-T cells generated from old human donors display an impaired mitochondrial fitness, which is rescued upon combination treatment of these CAR- T cells with a NAD booster and with one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator. In some embodiments, the unresponsive (aged, old) CAR-T cells are generated from old human donors being more than 40 years old, more than 50 years old or more than 60 years.

[0103] Thus another aspect of the present invention provides a method of suppressing CAR-T cells unresponsiveness or suppressing T cells unresponsiveness, the method comprising contacting unresponsive CAR-T cells with a NAD booster and with one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator, wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN), or contacting the unresponsive T cells with a NAD booster and with one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator, wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN). In some embodiments, the unresponsive CAR-T cells are CD4+ and / or CD8+ CAR-T cells, and the unresponsive T cells are CD4+ and / or CD8+ T cells.

[0104] In an embodiment, the present invention provides the method of suppressing CAR-T cells unresponsiveness or suppressing T cells unresponsiveness, the method comprising contacting unresponsive CAR-T cells with a NAD booster and with a CD38 inhibitor, wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN), or contacting the unresponsive T cells with a NAD booster and with a CD38 inhibitor, wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN).

[0105] In another embodiment, the present invention provides the method of suppressing CAR-T cells unresponsiveness or suppressing T cells unresponsiveness, the method comprising contacting unresponsive CAR-T cells with a NAD booster and with a PARP inhibitor, wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN), or contacting the unresponsive T cells with a NAD booster and with a PARP inhibitor, wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN).

[0106] In another embodiment, the present invention provides the method of suppressing CAR-T cells unresponsiveness or suppressing T cells unresponsiveness, the method comprising contacting unresponsive CAR-T cells with a NAD booster and with a NAMPT activator, wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN), or contacting the unresponsive T cells with a NAD booster and with a NAMPT activator, wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN).

[0107] In another embodiment, the present invention provides the method of suppressing CAR-T cells unresponsiveness or suppressing T cells unresponsiveness, the method comprising contacting unresponsive CAR-T cells with a NAD booster and with two or three compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator, wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN), or contacting the unresponsive T cells with a NAD booster and with two or three compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator, wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN).

[0108] In some embodiments, the one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator can be only the CD38 inhibitor, only the PARP inhibitor, or only the NAMPT activator, as well as the combination of the CD38 inhibitor and the PARP inhibitor, the combination of the CD38 inhibitor and the NAMPT activator, the combination of the PARP inhibitor and the NAMPT activator, the combination of the CD38 inhibitor, the PARP inhibitor and the NAMPT activator. In one embodiment, the one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator is a combination of the PARP inhibitor and NAMPT activator.

[0109] Suppressing CAR-T cells unresponsiveness or suppressing T cells unresponsiveness according to the method of the present invention provides rejuvenated CAR-T cells or rejuvenated T cells.

[0110] In some embodiments, the unresponsive CAR-T cells are exhausted CAR-T cells or senescent CAR-T cells; or the unresponsive T cells are exhausted T cells or senescent T cells.

[0111] In the context of the present invention, the NAD booster, selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), is alternatively named NAD precursor. The NAD booster (or the NAD precursor) has a different activity and consists of a different group of compounds from the CD38 inhibitor, the PARP inhibitor and the NAMPT activator, which have activity on specific enzymes described herein.

[0112] In some embodiments, the CD38 inhibitor is selected from the group consisting of

[0113] • flavonoids selected from quercetin, apigenin, luteolin, luteolinidin, kuromanin, or Rehin, preferably apigenin, luteolinidin, or kuromanin;

[0114] • chemical compounds selected from CD38 inhibitor 78c or derivatives thereof, CD38 inhibitor lah or derivatives thereof, or CD38 inhibitor lai or derivatives thereof;

[0115] • antibodies selected from Isatuximab, Daratumumab, TAK-079, or MOR-202;

[0116] • NAD analogs selected from Ara-F-NAD, Ara-F-NFM, Ara-F-NMN, or Carba-NAD, preferably Ara-F-NAD, Ara-F-NMN, or Carba-NAD; and

[0117] • Compound of Formula I The above-mentioned CD38 inhibitors have a common mechanism of action of being inhibitors of NADase activity, typically competitive inhibitors of NADase activity.

[0118] In preferred embodiments, the CD38 inhibitor is selected from the group consisting of

[0119] • flavonoids selected from apigenin, luteolinidin, or kuromanin;

[0120] • chemical compounds selected from CD38 inhibitor 78c or derivatives thereof, CD38 inhibitor lah or derivatives thereof, or CD38 inhibitor lai or derivatives thereof;

[0121] • NAD analogs selected from preferably Ara-F-NAD, Ara-F-NMN, or Carba-NAD.

[0122] In other preferred embodiments, the CD38 inhibitor is CD38 inhibitor 78c.

[0123] As used herein, the term "CD38" refers to the protein CD38 (cluster of differentiation 38), which is a multifunctional enzyme (NADase enzyme) that degrades NAD and modulates cellular NAD homeostasis.

[0124] In some embodiments, the PARP inhibitor is selected from the group consisting of Olaparib, Rucaparib and Niraparib, preferably the PARP inhibitor is Olaparib.

[0125] As used herein, the term “PARP inhibitor”, such as Olaparib, Rucaparib and Niraparib, refers to a small molecule able to inhibit the enzymatic activity of poly ADP ribose polymerase (PARP), a critical enzyme involved in processes such as DNA repair, genomic stability, and programmed cell death. By inhibiting PARPs, these compounds increase NAD+ production, which plays a vital role in cellular energy metabolism, DNA repair, and cell survival.

[0126] In other embodiments, the NAMPT activator is selected from the group comprising

[0127]

[0128] Nampt activator-4

[0129] Nampt activator-2 Nampt activator-3

[0130]

[0131] Nampt activator-5

[0132] Nampt activator-6

[0133] Nampt activator-7 Nampt activator-8

[0134] Quercitrin

[0135] Preferably, the NAMPT activator is l-[4-(8-Oxa-3-azabicyclo[3.2.1]octane-3-sulfonyl)- phenyl]-3-pyridin-4-ylmethylurea (SBI-797812) or analogs thereof.

[0136] SBI-797812

[0137] As used herein, the term “NAMPT activators”, such as SBI-797812, analogs thereof and other compounds disclosed above, refers to small molecule able to enhance the enzymatic activity of nicotinamide phosphoribosyltransferase (NAMPT), a critical enzyme in the NAD+ biosynthetic pathway. By activating NAMPT, these compounds increase NAD+ production, which plays a vital role in cellular energy metabolism, DNA repair, and cell survival.

[0138] In preferred embodiments, the CAR-T cells or the T cells are simultaneously contacted with the NAD booster and with the one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator, wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN).

[0139] In a further embodiment, the method of suppressing CAR-T cells unresponsiveness or suppressing T cells unresponsiveness is an ex vivo method. In a preferred embodiment, the ex vivo method comprises providing a population of unresponsive CAR-T cells or a population of unresponsive T cells and contacting the unresponsive CAR-T cells or the unresponsive T cells with the NAD booster and with the one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator, wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN), that consists in expanding the population of the unresponsive CAR-T cells or the population of the unresponsive T cells in presence of the NAD booster and the one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator thereby obtaining responsive CAR-T cells or responsive T cells wherein CD38 is inhibited or deficient.

[0140] In some embodiments, the one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator can be only the CD38 inhibitor, only the PARP inhibitor, or only the NAMPT activator, as well as the combination of the CD38 inhibitor and the PARP inhibitor, the combination of the CD38 inhibitor and the NAMPT activator, the combination of the PARP inhibitor and the NAMPT activator, the combination of the CD38 inhibitor, the PARP inhibitor and the NAMPT activator. In one embodiment, the one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator is a combination of the PARP inhibitor and NAMPT activator.

[0141] In some embodiments, the unresponsive CAR-T cells are CAR-T cells generated from T cells obtained from a subject and the unresponsive T cells are obtained from a subject.

[0142] The term "suppressing unresponsiveness" (or "reducing unresponsiveness"), "suppressing exhaustion" (or "reducing exhaustion") or "suppressing senescence" (or "reduction of senescence") refers to a given treatment or set of conditions that leads to rejuvenation of CAR- T cells, increased CAR-T cell activity, responsiveness, and / or ability or receptiveness, with regards to activation. Methods of measuring CAR-T cell activity are well known in the art. Modulation of one or more of any of the immune cell unresponsiveness (exhaustion) parameters described above can be assayed. For example, CAR-T cell activity can be measured by contacting CAR-T cells with recall antigen, anti-CD3 in the absence of costimulation, and / or ionomycin. Also, proliferation of CAR-T cells can be measured in the presence of a relevant antigen assayed, e.g. by a 3H-mymidinc incorporation assay or cell number. Markers of CAR- T cell activation after exposure to the relevant antigen can also be assayed, e.g. flow cytometry analysis of cell surface markers indicative of CAR-T cell activation (e.g., CD30, CD25, and HLA-DR) and / or CAR-T cell unresponsiveness (exhaustion). In some embodiments, the assays can be in vivo assays, such as through challenging immune cells with antigen, in vivo.

[0143] Another aspect of the present invention provides a method for improving a CAR-T cell therapy in a subject, the method comprising administering to the subject an effective amount of rejuvenated CAR-T cells, wherein CD38 is inhibited or deficient in the rejuvenated CAR-T cells.

[0144] Another aspect of the present invention provides a method for improving a CAR-T cell therapy in a subject, the method comprising i. determining the NAD level and / or the expression level of CD38 in T cells obtained from the subject or in CAR-T cells generated from T cells obtained from the subject; ii. comparing the NAD level determined at step i) with a predetermined reference value of NAD and / or comparing the expression level of CD38 determined at step i) with a predetermined reference value of CD38; iii. when the NAD level determined at step i) is lower than its predetermined reference value and / or the expression level of CD38 determined at step i) is higher than its predetermined reference value, administering to the subject an effective amount of rejuvenated CAR-T cells, wherein the NAD level is increased and / or CD38 is inhibited or deficient in the rejuvenated CAR-T cells.

[0145] In an embodiment, the present invention provides rejuvenated CAR-T cells, wherein the NAD level is increased and / or CD38 is inhibited or deficient in the rejuvenated CAR-T cells, for use in a method for improving a CAR-T cell therapy in a subject, the method comprising i. determining the NAD level and / or the expression level of CD38 in T cells obtained from the subject or in CAR-T cells generated from T cells obtained from the subject; ii. comparing the NAD level determined at step i) with a predetermined reference value of NAD and / or comparing the expression level of CD38 determined at step i) with a predetermined reference value of CD38; iii. when the NAD level determined at step i) is lower than its predetermined reference value and / or the expression level of CD38 determined at step i) is higher than its predetermined reference value, administering to the subject an effective amount of rejuvenated CAR-T cells, wherein the NAD level is increased and / or CD38 is inhibited or deficient in the rejuvenated CAR-T cells.

[0146] In some embodiments, the NAD level and / or the expression level of CD38 are determined either before or after generation of CAR-T cells. Typically, a blood sample is obtained from a subject, T cells are isolated therefrom and then CAR-T are generated ex -vivo from these isolated T cells as disclosed in the present disclosure. The CAR-T cells (CAR-T cell products) are then administered (re-injected) to the same or different subject.

[0147] In some embodiments of the method for improving a CAR-T cells therapy, the CAR-T cell therapy is for the treatment of cancer. In some embodiments, cancer is a solid cancer or a haematological cancer. In further embodiments, an haematological cancer is selected from acute lymphoblastic leukaemia (ALL), non-Hodgkin’s lymphoma (NHL), mantle cell lymphoma (MCL) or diffuse large B-cell lymphoma (DLBCL). In other embodiments, a solid cancer is selected from breast cancer, glioblastoma, non-small-cell lung cancer (NSCLC), liver cancer (e.g. HCC), or colorectal cancer (CRC).

[0148] In some embodiments, a CAR-T cells therapy can be as follows: CD19-directed CAR-T cells for the treatment of a haematological cancer, HER2-directed or EGFR-directed CAR-T cells for treating breast cancer; GD2 CAR-T cells for treating glioblastoma; MUC1 -targeted CAR- T for treating non-small-cell lung cancer.

[0149] Another aspect of the present invention provides an ex -vivo method of producing rejuvenated CAR-T cells from unresponsive CAR-T cells, the method comprising i. providing a population of unresponsive CAR-T cells; ii. expanding the population of unresponsive CAR-T cells in the presence of a NAD booster and one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator, thereby obtaining rejuvenated CAR-T cells wherein CD38 is inhibited or deficient, and wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN). In some embodiments, the NAD level is increased in the rejuvenated CAR-T cells comparing to the unresponsive CAR-T cells.

[0150] In some embodiments, the one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator can be only the CD38 inhibitor, only the PARP inhibitor, or only the NAMPT activator, as well as the combination of the CD38 inhibitor and the PARP inhibitor, the combination of the CD38 inhibitor and the NAMPT activator, the combination of the PARP inhibitor and the NAMPT activator, the combination of the CD38 inhibitor, the PARP inhibitor and the NAMPT activator. In one embodiment, the one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator is a combination of the PARP inhibitor and NAMPT activator.

[0151] In another embodiment of the ex-vivo method of producing rejuvenated CAR-T cells from unresponsive T cells, the method comprises i. isolating unresponsive T cells from a blood sample from a subject, preferably from an old subject; ii. activating the isolated T cells, preferably activating the isolated T cells in the presence of IL2 during 24 hours; iii. generating CAR-T cells, preferably generating CAR-T cells by lentiviral transduction; iv. contacting the generating CAR-T cells of step iii) with a NAD booster and with one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator, preferably 3 days after generation of CAR-T cells, contacting the generating CAR-T cells of step iii) with a NAD booster and with one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator, during 10 days, wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN).

[0152] In some embodiments, the one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator can be only the CD38 inhibitor, only the PARP inhibitor, or only the NAMPT activator, as well as the combination of the CD38 inhibitor and the PARP inhibitor, the combination of the CD38 inhibitor and the NAMPT activator, the combination of the PARP inhibitor and the NAMPT activator, the combination of the CD38 inhibitor, the PARP inhibitor and the NAMPT activator. In one embodiment, the one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator is a combination of the PARP inhibitor and NAMPT activator.

[0153] In some embodiments of the method, the unresponsive T cells and the unresponsive CAR-T cells are exhausted T cells and exhausted CAR-T cells; or senescent T cells and senescent CAR- T cells.

[0154] In the context of the present invention, the old subject is a subject being more than 40 years old, more than 50 years old or more than 60 years old.

[0155] In some embodiments, the CD38 inhibitor is selected from the group consisting of

[0156] • flavonoids selected from quercetin, apigenin, luteolin, luteolinidin, kuromanin, or Rehin, preferably apigenin, luteolinidin, or kuromanin;

[0157] • chemical compounds selected from CD38 inhibitor 78c or derivatives thereof, CD38 inhibitor lah or derivatives thereof, or CD38 inhibitor lai or derivatives thereof;

[0158] • antibodies selected from Isatuximab, Daratumumab, TAK-079, or MOR-202;

[0159] • NAD analogs selected from Ara-F-NAD, Ara-F-NFM, Ara-F-NMN, or Carba-NAD, preferably Ara-F-NAD, Ara-F-NMN, or Carba-NAD; and

[0160] • Compound of Formula I

[0161] I

[0162] The above-mentioned CD38 inhibitors have a common mechanism of action of being inhibitors of NADase activity, typically competitive inhibitors of NADase activity. In preferred embodiments, the CD38 inhibitor is selected from the group consisting of

[0163] • flavonoids selected from apigenin, luteolinidin, or kuromanin;

[0164] • chemical compounds selected from CD38 inhibitor 78c or derivatives thereof, CD38 inhibitor lah or derivatives thereof, or CD38 inhibitor lai or derivatives thereof;

[0165] • NAD analogs selected from preferably Ara-F-NAD, Ara-F-NMN, or Carba-NAD.

[0166] In other preferred embodiments, the CD38 inhibitor is CD38 inhibitor 78c.

[0167] As used herein, the term "CD38" refers to the protein CD38 (cluster of differentiation 38), which is a multifunctional enzyme (NADase enzyme) that degrades NAD and modulates cellular NAD homeostasis.

[0168] In some embodiments, the PARP inhibitor is selected from the group consisting of Olaparib, Rucaparib and Niraparib, preferably the PARP inhibitor is Olaparib.

[0169] As used herein, the term “PARP inhibitor”, such as Olaparib, Rucaparib and Niraparib, refers to a small molecule able to inhibit the enzymatic activity of poly ADP ribose polymerase (PARP), a critical enzyme involved in processes such as DNA repair, genomic stability, and programmed cell death. By inhibiting PARPs, these compounds increase NAD+ production, which plays a vital role in cellular energy metabolism, DNA repair, and cell survival.

[0170] In other embodiments, the NAMPT activator is selected from the group comprising

[0171]

[0172] SBI-797812

[0173] SBI-796950

[0174]

[0175] Nampt activator-4

[0176] Nampt activator-2 Nampt activator-3

[0177]

[0178] Nampt activator-5

[0179] Nampt activator-6

[0180] Nampt activator-7 Nampt activator-8

[0181] Quercitrin

[0182] Preferably, the NAMPT activator is l-[4-(8-Oxa-3-azabicyclo[3.2.1]octane-3-sulfonyl)- phenyl]-3-pyridin-4-ylmethylurea (SBI-797812) or analogs thereof.

[0183] SBI-797812

[0184] As used herein, the term “NAMPT activator”, such as SBI-797812 and analogs thereof, refers to a small molecule able to enhance the enzymatic activity of nicotinamide phosphoribosyltransferase (NAMPT), a critical enzyme in the NAD+ biosynthetic pathway. By activating NAMPT, these compounds increase NAD+ production, which plays a vital role in cellular energy metabolism, DNA repair, and cell survival.

[0185] Suitable conditions and media for the culture and the expansion of CAR-T cells are known in the art. For example, expansion of CAR-T cells may be achieved by culturing in the presence of IL-2 or IL-7 / IL-15.

[0186] In preferred embodiments, the NAD booster and the one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator, are simultaneously present during the expanding step ii).

[0187] In other embodiments of the present invention, the rejuvenated CAR-T cells can be obtained by disrupting CD38 gene. Thus the rejuvenated CAR-T cells can have a disrupted CD38 gene. The disruption of the CD38 locus results in loss of expression of CD38, which is a NADase that degrades both NAD and NAD-precursor and is intended to reduce or to eliminate NAD decline in T cells. The disruption of the CD38 gene increases efficacy and activity of the resultant rejuvenated CAR-T cells.

[0188] As used herein, the term “a disrupted gene” refers to a gene containing one or more mutations (e.g., insertion, deletion, or nucleotide substitution, etc.) relative to the wild-type counterpart so as to substantially reduce or completely eliminate the activity of the encoded gene product. The one or more mutations may be located in a non-coding region, for example, a promoter region, a regulatory region that regulates transcription or translation; or an intron region. Alternatively, the one or more mutations may be located in a coding region (e.g., in an exon). In some instances, the disrupted gene does not express or expresses a substantially reduced level of the encoded protein. In other instances, the disrupted gene expresses the encoded protein in a mutated form, which is either not functional or has substantially reduced activity. In some embodiments, a disrupted gene is a gene that does not encode functional protein. In some embodiments, a cell that comprises a disrupted gene does not express (e.g., at the cell surface) a detectable level (e.g. by antibody, e.g., by flow cytometry) of the protein encoded by the gene. A cell that does not express a detectable level of the protein may be referred to as a knockout cell. For example, a cell having a CD38 gene edit may be considered a CD38 knockout cell if CD38 protein cannot be detected in the cell. In some embodiments, a disrupted gene may be described as comprising a mutated fragment relative to the wild-type counterpart. The mutated fragment may comprise a deletion, a nucleotide substitution, an addition, or a combination thereof. In other embodiments, a disrupted gene may be described as having a deletion of a fragment that is present in the wild-type counterpart. In some instances, the 5' end of the deleted fragment may be located within the gene region targeted by a designed guide RNA such as those disclosed herein (known as on-target sequence) and the 3' end of the deleted fragment may go beyond the targeted region. Alternatively, the 3' end of the deleted fragment may be located within the targeted region and the 5' end of the deleted fragment may go beyond the targeted region. Alternatively or in addition, the disrupted CD38 gene in the rejuvenated CAR- T cells disclosed herein may be generated using the CRISPR / Cas technology.

[0189] Another aspect of the present invention provides a rejuvenated CAR-T cell, which comprises one or more chimeric antigen receptors (CARs) targeting one or more antigens, wherein CD38 is inhibited or deficient in the rejuvenated CAR-T cell. Such rejuvenated CAR-T cells can be obtained by the method of the present invention or by disrupting CD38 gene in CAR-T cell, as disclosed above.

[0190] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

[0191] The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practicing the present invention and are not intended to limit the application and the scope of the invention.

[0192] EXAMPLES

[0193] CAR-T cells from aged mice are unable to sustain stem-like properties (Figure 1).

[0194] To decipher how aging affects CAR-T cell function, Her2-directed CAR-T cells were generated from young vs old C57B16-derived splenocytes and tested their differentiation capacity when cultured under effector or memory polarising conditions (i.e. IL-2 or IL-7 / IL-15, respectively) (Fig. 1 A). Strikingly, old CAR-T cells are unable to reach an appropriate stem-like phenotype, as shown by a lower proportion of CD44+CD62L+ T cells (Tcm) (Fig. IB and C) and decreased levels of TCF1, a key transcription factor associated to sternness (Fig. ID). Functionally, ‘memory’ old CAR-T cells exhibit higher killing capacity against Her2+ B16 tumor cells in vitro (Fig. IE), as well as an increased capacity to produce cytokines (IFNy and TNFa) (Fig. IF and G), comparably behaving like ‘effector’ CAR-T cells. These results suggest that aged CAR-T cells are unable to efficiently respond to mitochondrial / memory-inducer stimulus (i.e., IL-7 and IL- 15) and acquire stem -like properties, which consequently might dampen the efficacy of CAR-T cell therapies. To further test this, these Her2-directed CAR-T cells were challenged in an in vivo murine tumor model. Specifically, Her2+ B16 melanoma cells were injected subcutaneously in mice. Upon tumor formation, sublethal whole body irradiation was performed followed by cell transfer of young or old CAR-T cells (generated as described in Fig. 1 A). It was observed that only CAR-T cells derived from young mice and expanded with IL-7 / IL-15, but not those derived from aged mice, can control the growth of Her2+ B16 tumors. Accordingly, it was shown that young 'memory' CAR-T cells are able to persist long-term in vivo, generating a pool of CD44+CD62L+TCF1+ CAR-T cells in the spleen. On the contrary, very few aged CAR-T cells were found in spleen 20 days post-transfer, highlighting their poor ability to persist in vivo, key feature associated to CAR-T cell therapy success.

[0195] Aged CAR-T cells display remarkable mitochondrial defects.

[0196] The development and maintenance of stem-like properties relies on the cells’ capacity to boost mitochondrial metabolism. It has been extensively studied that a hallmark of aging is mitochondrial dysfunction. Thus, the study was carried out to verify that aged T cells would also present features of mitochondrial dysfunction, ultimately leading to the loss of sternness.

[0197] To demonstrate a link between aging, mitochondrial dysfunction and sternness, it was first explored the mitochondrial fitness of CD8+ T cells derived from aged mice and compared to younger counterparts. Analysis on mitochondrial profiles (TMRM: mitochondrial membrane potential; MitoTracker Green-MTG: mitochondrial mass) of CD8+ T cells derived from old mice revealed an age-dependent drop in mitochondrial potential, which reflects a diminished mitochondrial fitness and activity (Fig 2A and B). Accordingly, 'old' T cells display a reduced basal and maximal oxygen consumption rate (OCR) upon TCR-stimulation (Fig 2C-E), as analyzed using the Seahorse XFe96 analyzer.

[0198] To show that mitochondrial dysfunction is a driver of loss of sternness, aged T cells were treated with Ml and Mdivi-1, which promote the fusion and prevent the fission of mitochondria, respectively. Ml+Mdivi-1 treatment has been shown to significantly increase mitochondrial size in T cells, leading to a better mitochondrial performance. Here, it was observed that Ml+Mdivi-1 treatment can significantly increase TCF1 expression in aged T cells. These data suggest that there is a tight association between the loss of mitochondrial fitness and sternness, and that targeting mitochondrial metabolism in immunotherapy could be particularly beneficial in the context of aging. NAD-decline associated to CD38 upregulation drives mitochondrial dysfunction in aged CAR-T cells.

[0199] To demonstrate mechanisms driving mitochondrial dysfunction and CAR-T cell failure during aging, metabolomic analysis of young and old CD8+ T cells isolated from splenocytes was performed. Interestingly, it was found that NAD was one of the key metabolites diminished in aged T cells (Fig 3A). NAD decline and its association to mitochondrial dysfunction was observed. Thus, it seemed that reestablishing NAD levels would boost and rejuvenate the mitochondrial and functional performance of aged T cells. To this end, aged T cells were treated with the NAD booster nicotinamide mononucleotide (NMN). However, it was found that while NMN was able to improve mitochondrial function of young T cells (Fig. 3B) and prevent their exhaustion (unresponsiveness) upon chronic stimulation (Fig. 3C), aged T cells were irresponsive to NMN treatment.

[0200] To further investigate this unresponsiveness, the inventors investigated how CD38 levels change during T cell aging and, indeed, it was found that CD38 expression accumulates in old T cells (Fig. 3D). Furthermore, it was determined that, in aged T cells, higher CD38 levels are associated to a decreased mitochondrial activity (Fig. 3E). Moreover, induction of CD38 overexpression in young T cells leads to an impaired mitochondrial profile that is accompanied by a lower generation of CD44+CD62L+ T cells (Tcm) (Fig. 3F and G). These results suggest that CD38 has an important role in the modulation of T cell metabolism and functionality, creating a direct link between aging, mitochondrial fitness, and maintenance of T cell sternness.

[0201] Restoration of NAD levels via CD38 blockade restores mitochondrial fitness and functionality of murine aged CAR-T cells.

[0202] A further study was performed to verify that the upregulation of CD38 in aged T cells was causing the loss of responsiveness to NMN. Indeed, combining NMN treatment with the small molecule 78c (an inhibitor of CD38) increased NAD levels in aged CD8+ T cells (Fig. 4A) and restored its mitochondrial fitness, as shown by an increased SRC and mitochondrial mass (Fig. 4B and C). Importantly, the effects of the CD38 inhibitor 78c and NMN treatment were only observed in aged T cells but not in younger controls, suggesting that this combination strategy is age specific. To assess whether the recovery of the mitochondrial fitness is sufficient to also restore CAR-T cell functionality in vivo, young and old CAR-T cells treated ± CD38 inhibitor 78c and NMN were re-infused in mice bearing Her2+ B 16 tumors (Fig. 4D). It was observed that, whereas the untreated aged CAR-T cells were unable to control tumor growth, aged CAR-T cells supplemented with CD38 inhibitor 78c and NMN were impeding tumor growth at the same rate as the younger controls (Fig. 4E). Importantly, treated aged CAR-T cells were able to prolong their long-term survival in vivo, as shown by a significant increase in the number of CD44+CD62L+TCF1+ CAR-T cells found in spleens 20 days post- ACT (Fig. 4F and G).

[0203] This data demonstrated that restoration of NAD levels in aged CAR-T cells using a combination of NAD-booster and CD38 inhibition rejuvenates their mitochondrial profile and stem -like properties, ultimately leading to improved CAR-T cell efficacy.

[0204] To find alternative mechanisms to increase NAD levels, it was investigated how other NAD- consuming enzymes could be exploited to this purpose. Besides CD38, PARPs, which are activated in response to DNA damage, highly consume NAD. First, it was proved that aged CAR-T cells accumulated signs of DNA damage, as assessed by H2AX staining (Fig. 5 A). To assess whether PARP inhibition could be used to substitute CD38 inhibition, old CAR-T cells were expanded in the presence of the PARP inhibitor Olaparib + / - NMN. It was observed that supplementation with Olaparib led to a small but not significant increase of NAD levels. However, when combined with NMN, NAD levels were increased to the same levels observed using the combination of NMN and the CD38 inhibitor 78c (Fig. 5B). Accordingly, aged CAR- T cells supplemented with NMN and Olaparib were able to restore mitochondrial fitness (Fig. 5C) and the levels of CD62L, key marker of CAR-T cell efficacy (Fig. 5D).

[0205] Another alternative mechanism to increase NAD levels is to target NAMPT. NAMPT is the rate limiting enzyme of the NAD synthesis pathway. The ability of the NAMPT activator SBI- 797812 to improve the phenotype and mitochondrial function of old T cells was tested. It was observed that SBI-797812 in combination with the NAD booster NAM improved old the T cell phenotype by increasing CD62L expression (Fig.6 A) resulting in the expansion of the central memory population (Fig.6B). In addition, old T cells cultured in presence of SBI-797812 and NAM displayed better mitochondria as assessed by the increased mitochondrial potential (Fig.7A), mitochondrial mass (Fig.7B) and higher mitochondrial fitness (Fig.7C). Restoration of NAD levels via CD38 inhibitions restores mitochondrial fitness and functionality of human aged CAR-T cells

[0206] To assess the importance of CD38 in the clinical field, multiple databases containing transcriptomic data from cancer patients before treatment were analyzed and the levels of CD38 expression to the responsiveness to the immunotherapeutic treatment were correlated.

[0207] In the context of CAR-T cell therapy, RNAseq data of CAR-T cells before pre-infusion to CLL patients were analyzed and it was observed that complete responders (CR) to the treatment express lower levels of CD38 than partial responders (PR) (Fig 8 A). Importantly, when assessing the metabolic pathways regulated by CD38 levels, it was shown that mainly NAD metabolism is altered by the upregulation of CD38, in accordance with data in murine T cells (Fig 8B).

[0208] In the context of ICB, scRNAseq data of TILs from melanoma patients under treatment with PD-1 blockade were exploited. It was noticed that CD38 expression is significantly upregulated in TILs of non-responder patients suggesting that CD38 could be used as a marker of T cell dysfunctionality and a predictive marker to anti -PD-1 (Fig 8C). CD38 increased levels in non- responder patients were consistent in other cell types too, such as B cells, myeloid cells or DCs (Fig 8C). An interesting observation is that only CD38, but not other exhaustion (unresponsiveness) markers (e.g., Tox, PD1, TIM3 or LAG3), was differentially expressed in terminally unresponsive (exhausted) T cells (Fig 8D), one of the most prominent populations within the tumor microenvironment, highlighting the potential use of CD38 as a predictive marker for ICB.

[0209] Next, another study was performed to demonstrate that CD38 could be used not only as a predictive marker but also as a therapeutic target to rejuvenate human CAR-T cells. First, it was validated in human PBMCs that older cancer patients (>70 years old) accumulate higher levels of CD38 than younger patients (<30 years old) (Fig. 8E). Moreover, it was observed that CD8+ T cells expressing higher levels of CD38 are consistently associated to a lower mitochondrial activity (Fig. 8F). Next, CD19-directed CAR-T cells from young and old healthy donors were generated and compared their mitochondrial fitness. As expected, CAR-T cells generated from older donors displayed a significant reduction on their SRC (Fig. 8G and H), as well as in their mitochondrial size (Fig. 81). Remarkably, old CAR-T cells supplemented with CD38 inhibitor 78c and NMN during ex vivo expansion were able to revert their mitochondrial defects and mimic the mitochondrial profile of younger CAR-T cells (Fig. 8G-I).

[0210] Conclusion Altogether, this data supports the notion that aging induces NAD decline, such as a CD38- dependent NAD decline, that leads to mitochondrial defects and loss of stem-like properties, ultimately resulting in CAR-T cell failure. It is herein shown that restoration of NAD levels (i.e. increase of NAD levels) either by inhibiting CD38 activity, inhibiting PARP activity or activating NAMPT in combination with an NAD-booster rejuvenates mitochondrial activity and functionality of CAR-T cells.

Claims

CLAIMS1. A method of suppressing CAR-T cells unresponsiveness, the method comprising contacting unresponsive CAR-T cells with a NAD booster and with one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and a NAMPT activator, wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN).

2. The method of claim 1, wherein the unresponsive CAR-T cells are CD4+ and / or CD8+CAR-T cells.

3. The method of claim 1 or claim 2, wherein the CD38 inhibitor is selected from the group consisting of• flavonoids selected from quercetin, apigenin, luteolin, luteolinidin, kuromanin, or Rehin; chemical compounds selected from CD38 inhibitor 78c, CD38 inhibitor lah, or CD38 inhibitor lai; antibodies selected from Isatuximab, Daratumumab, TAK-079, or MOR-202;NAD analogs selected from Ara-F-NAD, Ara-F-NFM, Ara-F-NMN, or Carba-NAD; andCompound of Formula II preferably the CD38 inhibitor is CD38 inhibitor 78c.

4. The method of any one of claims 1 to 3, wherein the PARP inhibitor is selected from the group consisting of Olaparib, Rucaparib and Niraparib, preferably the PARP inhibitor is Olaparib.

5. The method of any one of claims 1 to 4, wherein the NAMPT activator is selected from the group comprisingSBI-797812SBI-796850Nampt activator-4Nampt activator-2 Nampt activator-3Nampt activator-5Nampt activator-6Nampt activator-7 Nampt activator-8Quercitrin6. A method for improving a CAR-T cell therapy in a subject, the method comprising i. determining the NAD level and / or the expression level of CD38 in T cells obtained from the subject or in CAR-T cells generated from T cells obtained from the subject; ii. comparing the NAD level determined at step i) with a predetermined reference value of NAD and / or comparing the expression level of CD38 determined at step i) with a predetermined reference value of CD38; iii. when the NAD level determined at step i) is lower than its predetermined reference value and / or the expression level of CD38 determined at step i) is higher than its predetermined reference value, administering to the subject an effective amount of rejuvenated CAR-T cells, wherein the NAD level is increased and / or CD38 is inhibited or deficient in the rejuvenated CAR-T cells.

7. The method of claim 6, wherein the CAR-T cell therapy is for the treatment of cancer.

8. A method of identifying unresponsive T cells or unresponsive CAR-T cells, the method comprising: i. determining the NAD level and / or the expression level of CD38 in T cells or in CAR-T cells; ii. comparing the NAD level determined at step i) with a predetermined reference value of NAD and / or comparing the expression level of CD38 determined at step i) with a predetermined reference value of CD38; wherein when the NAD level determined at step i) is lower than its predetermined reference value and / or the expression level of CD38 determined at step i) is higher thanits predetermined reference value indicates that T cells or CAR-T cells are unresponsive T cells or unresponsive CAR-T cells.

9. The method of claim 8, wherein the unresponsive T cells are CD4+ and / or CD8+ T cells and CAR-T cells are CD4+ and / or CD8+ CAR-T cells.

10. A method for predicting the efficacy of a CAR-T cell therapy in a subject, the method comprising i. determining the NAD level and / or the expression level of CD38 in T cells obtained from the subject or in CAR-T cells generated from T cells obtained from the subject; ii. comparing the NAD level determined at step i) with a predetermined reference value of NAD and / or comparing the expression level of CD38 determined at step i) with a predetermined reference value of CD38; wherein when the NAD level determined at step i) is equal or higher than its predetermined reference value and / or the expression level of CD38 determined at step i) is equal or lower than its predetermined reference value is indicative for good efficacy of the CAR-T cell therapy when applied to the subject, and when the NAD level determined at step i) is lower than its predetermined reference value and / or the expression level of CD38 determined at step i) is higher than its predetermined reference value is indicative for poor efficacy of the CAR-T cell treatment when applied to the subject.

11. A method for predicting the efficacy of immune checkpoint blockade (ICB) therapy in a subject, the method comprising i. determining the NAD level and / or the expression level of CD38 in tumor infiltrating lymphocytes (TILs) obtained from the subject; ii. comparing the NAD level determined at step i) with a predetermined reference value of NAD and / or comparing the expression level of CD38 determined at step i) with a predetermined reference value of CD38; wherein when the NAD level determined at step i) is equal or higher than its predetermined reference value and / or the expression level of CD38 determined at step i) is equal or lower than its predetermined reference value is indicative for good efficacy of the immune checkpoint blockade (ICB) therapy when applied to the subject, and when the NAD level determined at step i) is lower than its predetermined reference value and / or the expression level of CD38determined at step i) is higher than its predetermined reference value is indicative for poor efficacy of the immune checkpoint blockade (ICB) therapy when applied to the subject.

12. The method of any one of claims 6 to 11, wherein the predetermined reference value of NAD is the NAD level in T cells derived from population of young healthy subjects being less than 30 years old, and the predetermined reference value of CD38 is the expression level of CD38 in T cells derived from population of young healthy subjects being less than 30 years old.

13. An ex vivo method of producing rejuvenated CAR-T cells from unresponsive CAR-T cells, the method comprising i. providing a population of unresponsive CAR-T cells; ii. expanding the population of unresponsive CAR-T cells in the presence of an NAD booster and one or more compounds selected from a group consisting of a CD38 inhibitor, a PARP inhibitor and NAMPT activator, and, thereby obtaining rejuvenated CAR-T cells wherein CD38 is inhibited or deficient and wherein the NAD booster is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide (NAM), nicotinic acid (NA), and nicotinamide adenine dinucleotide (NAD), preferably the NAD booster is nicotinamide mononucleotide (NMN), preferably the NAD booster is nicotinamide mononucleotide (NMN).

14. The method of claim 13, wherein the unresponsive CAR-T cells are CD4+ and / or CD8+ CAR-T cells.

15. The method of any one of claims 13 to 14, wherein the CD38 inhibitor is selected from the group consisting of• flavonoids selected from quercetin, apigenin, luteolin, luteolinidin, kuromanin, or Rehin;• chemical compounds selected from CD38 inhibitor 78c, CD38 inhibitor lah, or CD38 inhibitor lai;• antibodies selected from Isatuximab, Daratumumab, TAK-079, or MOR-202;• NAD analogs selected from Ara-F-NAD, Ara-F-NFM, Ara-F-NMN, or Carba-NAD; and• Compound of Formula II preferably the CD38 inhibitor is CD38 inhibitor 78c.

16. The method of any one of claims 13 to 15, wherein the PARP inhibitor is selected from the group consisting of Olaparib, Rucaparib and Niraparib, preferably the PARP inhibitor is Olaparib.

17. The method of any one of claims 13 to 16, wherein the NAMPT activator is selected from the group comprisingSBI-136892SBI-797812Nampt activator-4Nampt activator-2 Nampt activator-3Nampt activator-5Nampt activator-6Nampt activator-7 Nampt activator-8

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