Cells expressing chimeric antigen receptors and a glutamine transporter, uses and methods thereof

Abstract

The present invention relates to cells expressing chimeric antigen receptors against BCMA and a glutamine transporter, and uses thereof in the treatment of cancer or autoimmune diseases. The invention also relates to a method for the prognosis of multiple myeloma in a subject.

Description

[0001] CELLS EXPRESSING CHIMERIC ANTIGEN RECEPTORS AND A GLUTAMINE TRANSPORTER, USES AND METHODS THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention is comprised within the field of biotechnology and biomedicine. It specifically relates to cells expressing specific chimeric antigen receptors against BCMA and an exogenous glutamine transporter and uses thereof in the treatment of cancer or autoimmune diseases. The invention also relates to a method for the prognosis of multiple myeloma in a subject.

[0004] BACKGROUND OF THE INVENTION

[0005] Redirection of immune cell activity towards cancer cells by targeting tumor- associated or tumor specific antigens is a successful therapeutic approach against certain types of cancer. Several cancer immunotherapies based on the recognition of tumor associated or tumor-specific antigens by T cells have emerged over the past few decades, such as therapies based on T cells manipulated to express chimeric antigen receptors (CARs). CARs are engineered proteins that contain the antigen-binding domain of an antibody and signaling domains of the T cell receptor (TOR), and coactivator receptors.

[0006] B cell maturation antigen (BCMA) is a type III transmembrane protein expressed on mature B lymphocytes. Following binding of BCMA to its ligands, B cell activating factor (BAFF) or a proliferation inducing ligand (APRIL), a pro-survival cell signal is delivered to the B cell which has been found to be required for plasma cell survival. The expression of BCMA has been linked to several types of cancer, being expressed at significantly greater levels in cancer cells.

[0007] Due to the role of BCMA in cancer, BCMA is a promising therapeutic target for T- cell redirecting strategies. Various BCMA-binding chimeric antigen receptors (CARs), and cells expressing such CARs, are available. However, their efficacy is limited as most patients relapse within a few months after immunotherapy due to (i) the short-term persistence of the BCMA-CAR T cells in cancer patients, and (ii) the hostile immunosuppressive tumor microenvironment.

[0008] There still remains a need for improved BCMA-binding CARs and engineered BCMA-CAR expressing targeting cells, such as for use in adoptive cell therapy. Provided herein are embodiments that meet such needs. SUMMARY OF THE INVENTION

[0009] The authors of the present invention have generated cells that express a chimeric antigen receptor (CAR) that specifically target BCMA and that are also modified with an exogenous nucleic acid that results in the expression of a glutamine transporter. This results in an unexpected increase in their therapeutic antitumor effect against tumor cells expressing BCMA. The authors of the present invention have found that the BCMA CAR- T cells expressing a glutamine transporter- exert an enhanced antitumor response, with longer-term persistence of said CAR-T cells, increasing overall cancer survival with respect to BCMA CAR-T cells which do not express such a transporter.

[0010] Therefore, a first aspect of the present invention refers to a cell which expresses

[0011] (i) a chimeric antigen receptor (CAR) comprising: o an antigen binding domain specific for BCMA, o a transmembrane domain, and o at least one intracellular signaling domain and / or a costimulatory domain; and

[0012] (ii) a glutamine transporter, wherein the glutamine transporter is expressed from an exogenous nucleic acid present in the cell and wherein the glutamine transported is selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2.

[0013] A second aspect of the present invention relates to a nucleic acid construct comprising

[0014] (i) a first region encoding a chimeric antigen receptor (CAR), said chimeric antigen receptor comprising: o an antigen binding domain specific for BCMA, o a transmembrane domain, and o at least one intracellular signaling domain and / or a costimulatory domain; and

[0015] (ii) a second region encoding a glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2; or a nucleic acid composition comprising

[0016] (i) a first polynucleotide encoding a chimeric antigen receptor (CAR), said chimeric antigen receptor comprising: o an antigen binding domain specific for BCMA, o a transmembrane domain, and o at least one intracellular signaling domain and / or a costimulatory domain; and

[0017] (ii) a second polynucleotide encoding a glutamine transporter selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2.

[0018] A third aspect of the present invention relates to a vector comprising the nucleic acid construct according to the second aspect of the invention or a vector composition comprising a pair of vectors in which the first and second nucleic acids of the composition are respectively comprised within the first and second vectors of said pair of vectors.

[0019] A fourth aspect of the present invention relates to an ex vivo method for obtaining a cell expressing

[0020] (i) a chimeric antigen receptor comprising: o an antigen binding domain specific for BCMA, o a transmembrane domain, and o at least one intracellular signaling domain and / or a costimulatory domain; and

[0021] (ii) a glutamine transporter selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2 which comprises inserting into the cell the nucleic acid construct or the nucleic acid composition according to the second aspect of the invention, or the vector or vector composition according to the third aspect of the invention and selecting those cells which express the CAR on their surface and which express the glutamine transporter.

[0022] A fifth aspect of the present invention relates to a cell obtainable by the method according to the fourth aspect of the invention.

[0023] A sixth aspect of the present invention relates to a pharmaceutical composition comprising the cell according to the first aspect of the invention or the fifth aspect of the invention, the nucleic acid construct or the nucleic acid composition according to the second aspect of the invention, or the vector or vector composition according the third aspect of the invention, and at least one pharmaceutically acceptable excipient and / or vehicle.

[0024] A seventh aspect of the present invention relates to the cell according to the first aspect of the invention or the fifth aspect of the invention, the nucleic acid construct or the nucleic acid composition according to the second aspect of the invention, the vector or vector composition according the third aspect of the invention, or the pharmaceutical composition according the sixth aspect of the invention, for use in medicine.

[0025] An eighth aspect of the present invention relates to the cell according to the first aspect of the invention or the fifth aspect of the invention, the nucleic acid construct or the nucleic acid composition according to the second aspect of the invention, the vector or vector composition according the third aspect of the invention, or the pharmaceutical composition according the sixth aspect of the invention, for use in the prevention and / or treatment of cancer or an autoimmune disease.

[0026] A ninth aspect of the present invention relates to an in vitro method for the prognosis of multiple myeloma in a subject, the method comprising:

[0027] (i) determining the expression level of a glutamine transporter in a tumor sample obtained from said subject, and

[0028] (ii) comparing said expression level obtained in (i) to a reference value, wherein if a deviation in the expression level of the glutamine transporter with respect to the reference value is an increase, it is indicative of poor prognosis of multiple myeloma in said subject, and wherein if a deviation of the expression level of the glutamine transporter with respect to the reference value is a decrease or if there is no deviation between the expression level of the glutamine transporter and the reference value, it is indicative of good prognosis of multiple myeloma in said subject.

[0029] BRIEF DESCRIPTION OF THE FIGURES

[0030] Figure 1. ASCT2 expression associates with a poor prognosis in multiple myeloma. (A, B) Correlation between ASCT2 expression and clinical outcomes in multiple myeloma (MM) in progression-free survival (A) and overall survival (B). (C) ASCT2 mRNA expression levels in Naive B cells (NB), centroblasts (CB), centrocytes (CC), memory (MEM), tonsillar (TPC) and bone marrow plasma cells (BMPC) as well as in MM aspirates and MM cell lines.

[0031] Figure 2. ASCT2 overexpression enhances BCMA-CAR T cell activity. (A) Schematic representation of second-generation BCMA CAR and PSMA CAR constructs. (B) Proliferation and IFN-y production of BCMA and PSMA CAR T cells in response to BCMA, TACI or ovalbumin-coated plates. (C) IFN-y production and percentage of lysis induced by BCMA and PSMA CAR-T cells in response to MM cell lines (5080 and 9275) or to B16-OVA melanoma cell line. (D) Chromatogram showing BCMA expression of he cell lines used in (C). (E) Schematic representation of second-generation of ASCT2- BCMA CAR construct. (F) CAR expression in T lymphocytes transduced with retroviruses (RV) expressing BCMA or ASCT2+-BCMA CAR constructs. Flow cytometry histograms represent the mean fluorescence intensity (MFI) in RV-transduced CD4+and CD8+T cells. (G) ASCT2 expression measured by flow cytometry. (H)3H-labeled Gin uptake by CD4+and CD8+BCMA CAR and ASCT2+BCMA CAR T cells (purified by Flow cytometric cell sorting). (I) IFN-y secretion of CD4+or CD8+BCMA CAR and ASCT2+BCMA CAR T cells in response to BCMA-coated plates under different Gin concentrations. (J) Specific lysis of 5080 MM cells by BCMA CAR and ASCT2+BCMA CAR T cells under different Gin concentrations. Data represent mean ± SEM and were analyzed via 2-way ANOVA, and 1-way ANOVA (****p < 0.0001 , ***p < 0.001 , **p < 0.01 , *p<0.05.

[0032] Figure 3. ASCT2 overexpression enhances the cellular metabolism of CD8+and CD4+BCMA CAR-T cells. SeaHorse metabolic assay was used to measure the Oxygen Consumption Rate (OCR) and the Extracellular Acidification Rate (ECAR) of control and ASCT2-overexpressing CD4+(A), and CD8+(B) BCMA CAR-T cells. Data represent mean ± SEM and were analyzed via 2-way ANOVA, and 1-way ANOVA (****p < 0.0001 , ***p < 0.001 , **p < 0.01 , *p<0.05). (C-E) Seahorse experiments following pre-incubation of CART cells in the presence / absence of GLUT 1 inhibitor WZB117 or the glutaminase 1 inhibitor BPTES.

[0033] Figure 4 Transcriptomic analysis of Asct2 Bcma-CART cells and Bcma-CART cells before and after BCMA antigen stimulation. (A) Expression of Slc1a5 (Asct2). (B) Number of differentially expressed genes between Asct2 Bcma CART cells and Bcma- CART cells before and after BCMA antigen stimulation. (C) Principal component analysis (PCA) of gene expression profiles. (D) Gene signatures upregulated in Asct2 Bcma- CART cells compared to Bcma-CART cells, before and after BCMA antigen stimulation. (E) Heatmap showing differential expression of SLC family members in Asct2 Bcma- CART cells compared to Bcma-CART cells after antigen stimulation. Data represent mean ± SEM and were analyzed via Student's t-test. (**p < 0.01).

[0034] Figure 5. The antitumor efficacy of ACT immunotherapy with BCMA-CAR-T cells was improved by ASCT2 overexpression. (A) Gin concentration measured in the bone marrow of healthy mice or mice challenged with 5080 MM cell line (25 days after tumor challenge). (B) Experimental design to evaluate antitumor activity of CAR-T cells in a MM murine model based on the intravenous injection of 5080 MM cells. (C) Percent of survival of 5080 MM-challenged mice treated with 1 x 106 CAR-T cells (1 :1 ratio of CD4 and CD8 CAR-T cells). (D) Total number of tumor cells (B220+GFP+) found in the bone marrow of mice treated with ASCT2+BCMA-CAR-T and BCMA-CAR-T cells. (E-G) Total numbers of CAR-T cells measured in the bone marrow (E and G), and the spleen (F) of mice treated with ASCT2+BCMA CAR-T or BCMA CAR-T cells at day 14 (E and F), and at day 21 (G) after tumor challenge.

[0035] Figure 6. ASCT2 BCMA CAR-T cells exert antitumor activity in a genetic model of Multiple myeloma. (A) Gin concentration in the bone marrow (BM) aspirates and the sera of 170-day-old M lcyimice compared to the Gin levels in healthy mice BM. Glutamine concentration in bone marrow aspirates of 170-day-old MICYgimice compared to levels in healthy animals’ BM or serum. (B) Experimental design for testing CAR-T cell immunotherapy against the MlcyiMM genetic model. (C, D) Fc gamma fraction levels (C) and survival curves (D) after CAR-T treatment. (E) Number of tumor cells in both the spleen and the BM of mice treated with BCMA CAR-T and ASCT2+BCMA CAR-T cells compared to untreated mice. (F-H) CAR-T cells in the spleen (F, H) and in the BM (G, H) of Mlcyimice treated with BCMA or ASCT2+BCMA CAR-T cells analyzed in the CD3+(F, G) or in the CD4+and CD8+compartments (H).

[0036] Figure 7. ASCT2 overexpression enhances anti-human BCMA-CART cell activity. (A) Schematic diagrams of lentiviral vectors used to overexpress ASCT2 in human BCMA- CART (BCMA-CAR T) cells. (B) Expression levels of the CAR construct, measured by BFP fluorescence. (C) Human ASCT2 expression detected on CAR T cells by flow cytometry. (D) Proliferation of CAR T cells in response to BCMA-coated plate stimulation at different glutamine concentrations, measured by [3H]-thymidine incorporation. (E) Phenotypic analysis of CD4+and CD8+CAR T cells at the time of manufacture. (F) Asct2 silencing in H929 MM cells using three different sgRNAs TadCBEd. (G) Measurement of [3H]-glutamine uptake in wild-type and ASCT2-silenced H929 MM cells. (H) Cytotoxic activity of BCMA-CART cells against wild-type and ASCT2-silenced H929 MM cells. Data represent mean ± SEM and were analyzed via two-way ANOVA with Bonferroni multiple comparisons test (***p < 0.001). DETAILED DESCRIPTION OF THE INVENTION

[0037] Given the limitations of the current cell immunotherapies directed to BCMA- expressing cells for the treatment of tumors, the present invention relates to CAR cells expressing BCMA, in order to take advantage of the specificity of tumor-specific antigens and combine it with the potency of targeting tumor-associated antigens homogeneously expressed at higher levels in tumor cells. The present work discloses cells expressing a chimeric antigen receptor specific for BCMA and expressing a glutamine transporter, both expressed from exogenous nucleic acid(s) introduced into the cell.

[0038] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0039] All the embodiments and definitions disclosed in the context of one aspect of the invention are also applicable to the other aspects of the invention.

[0040] Cell of the invention

[0041] In a first aspect, the present invention refers to a cell, hereinafter referred to as “the cell of the invention”, which expresses

[0042] (i) a chimeric antigen receptor (CAR) comprising: o an antigen binding domain specific for BCMA, o a transmembrane domain, and o at least one intracellular signaling domain and / or a costimulatory domain; and

[0043] (ii) a glutamine transporter selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, wherein the glutamine transporter is expressed from an exogenous nucleic acid present in the cell.

[0044] In an alternative first aspect, the invention relates to a cell which expresses

[0045] (i) a chimeric antigen receptor (CAR) comprising: o an antigen binding domain specific for BCMA, o a transmembrane domain, and o at least one intracellular signaling domain and / or a costimulatory domain; and

[0046] (ii) a glutamine transporter In an alternative first aspect, the invention relates to a cell which expresses

[0047] (i) a chimeric antigen receptor (CAR) comprising: o an antigen binding domain specific for BCMA, o a transmembrane domain, and o at least one intracellular signaling domain and / or a costimulatory domain; and

[0048] (ii) a glutamine transporter wherein the glutamine transporter is not SLC7A5.

[0049] As used herein, the term “cell” or “engineered cell” refers to any cell of any organism that is modified, transformed, or manipulated by addition or modification of a gene, a DNA or RNA sequence, or protein or polypeptide. It also refers to the progeny of such cells. Cells or genetically engineered cells of the present invention include immune cells (such as T, NK, or NKT cells), somatic cells and progenitor cells. Isolated immune cells, somatic cells or progenitor cells contain the DNA or RNA sequences encoding a chimeric antigen receptor or a chimeric antigen receptor complex specific for BCMA and express the chimeric receptor on the cell surface and a glutamine transporter. Isolated cells and engineered cells may be used, for example, for enhancing an NK or NKT cell activity or a T lymphocyte activity, treatment of cancer, and treatment of infectious diseases.

[0050] In an embodiment, the cell of the invention is selected from a group consisting of an immune cell, a somatic cell or a progenitor cell.

[0051] As used herein, the term “immune cell” refers to a cell that plays a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, neutrophils, mast cells, basophils, dendritic cells and granulocytes. In some embodiments, the cell is a T cell; a NK cell; a NKT cell; lymphocytes, such as B cells and T cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0052] As used herein, the term “somatic cell” refers to any cell other than germ cells, such as an egg, a sperm, or the like, which does not directly transfer its DNA to the next generation. Typically, somatic cells have limited or no pluripotency. Somatic cells used herein may be naturally-occurring or genetically-modified. “Somatic cells” include, but are not limited to, skeletal cells, muscle cells, fibroblasts, cardiac cells, enterocytes, fat cells, blood cells, epithelial cells, neurons, chondrocytes, tissue cells, organ cells, etc., their respective precursor and progenies.

[0053] As used herein, the term "progenitor cell" refers to an undifferentiated cell which is capable of proliferation and giving rise to more progenitor cells having the ability to generate a large number of mother cells that can in turn give rise to differentiated, or differentiable daughter cells. As used herein, the term "progenitor cell" is also intended to encompass a cell which is sometimes referred to in the art as a "stem cell". In a preferred embodiment, the term "progenitor cell" refers to a generalized mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues. “Progenitor cell” include, but are not limited to, hematopoietic stem cells, mesenchymal stem cells, etc., their respective precursor and progenies.

[0054] In a particular embodiment, the cell of the invention is an immune cell selected from a group consisting of a T cell, a B cell, a NK cell, a neutrophil, an eosinophil, a basophil, a monocyte, a macrophage, a dendritic cell and a mast cell, or a precursor of any of these cells.

[0055] As used herein, the term “T cell” refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes such as B lymphocytes by the presence of T cell receptors on the cell surface. T cells can also be isolated or obtained from commercially available sources. T cells are of any type expressing CD3, including helper T cells (CD4+cells), cytotoxic T cells (CD8+cells), natural killer T cells, regulatory T cells (Tregs) and gammadelta T cells. “Cytotoxic cells” include CD8+T cells, natural-killer (NK) cells, and neutrophils capable of mediating a cytotoxic response. The terms "T cell" and "T lymphocyte" are interchangeable and are used interchangeably herein.

[0056] “Natural killer cells” or “NK cells” are well known in the art. In one embodiment, natural killer cells include cell lines, such as NK- 92 cells. Further examples of NK cell lines include NKG, YT, NK-YS, HANK-1 , YTS cells, and NKL cells. NK cells can be detected by specific surface markers, such as CD16, CD56, and CD8 in humans. NK cells do not express T-cell antigen receptors, the pan T marker CD3, or surface immunoglobulin B cell receptors.

[0057] Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties of both T cells and natural killer cells. Thus, NKT cells are a subset of T cells that coexpress an op T-cell receptor, but also express a variety of molecular markers that are typically associated with NK cells, such as NK1 . Many of these cells recognize the non-polymorphic CD1d molecule, an antigen-presenting molecule that binds self and foreign lipids and glycolipids. They constitute only approximately 0.1 % of all peripheral blood T cells. Natural killer T cells should not be confused with natural killer cells.

[0058] As used herein, the term “B cell” or “B lymphocyte” refer to immune cells which express a cell surface immunoglobulin molecule and which, upon activation, terminally differentiate into cells, which secrete antibodies.

[0059] As used herein, the term “neutrophil” refers to neutrophil granulocytes which are the most abundant (up to 75 percent) type of white blood cells in mammals and are a central part of the innate immune system. They derive from stem cells in the bone marrow. Together with basophils and eosinophils, they form part of the polymorphonuclear cell family (PMNs).

[0060] As used herein, the term “eosinophil” refers to an eosinophil granulocyte.

[0061] As used herein, the term “basophil” refers to a basophil granulocyte.

[0062] As used herein, the term “monocyte” refers to white blood cells that circulate in the blood stream. Monocytes differentiate into macrophages upon migration into the tissues.

[0063] As used herein, the term "macrophage" refers to a subgroup of phagocytic cells produced by the differentiation of monocytes. Macrophages which are activated by inflammation, immune cytokines or microbial products nonspecifically engulf and kill foreign pathogens within the macrophage by hydrolytic and oxidative attack resulting in degradation of the pathogen. Peptides from degraded proteins are displayed on the macrophage cell surface where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, resulting in T and B cell activation and further stimulation of the immune response. Macrophages belong to the class of antigen presenting cells. In one embodiment, the macrophages are splenic macrophages.

[0064] As used herein, the term “mast cell” or “mastocyte” or “labrocyte” refer to a granulocyte that contains granules with in histamine and heparin.

[0065] The term “dendritic cell”, as used herein, refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. Dendritic cells are a class of “professional” antigen presenting cells, and have a high capacity for sensitizing H LA-restricted T cells. Specifically, the dendritic cells include, for example, lymphocytic dendritic cells (including cells which induce Th2 or immune tolerance), bone marrow dendritic cells (generally used dendritic cells, including immature and mature dendritic cells), Langerhans cells (dendritic cells important as antigen-presenting cells in the skin), interdigitating cells (distributed in the lymph nodes and spleen T cell region, and believed to function in antigen presentation to T cells), and follicular dendritic cells (important as antigen-presenting cells for B cells). Dendritic cells may be recognized by function, or by phenotype, particularly by cell surface phenotype. These cells are characterized by their distinctive morphology (having veil-like projections on the cell surface), intermediate to high levels of surface H LA-class II expression and ability to present antigen to T cells, particularly to naive T cells. See Steinman R, et al., Ann. Rev. Immunol. 1991 ; 9:271-196. The cell surface of dendritic cells is characterized by the expression of the cell surface markers CD1a+, CD4+, CD86+, or HLA-DR+.

[0066] The term “dendritic cell precursor”, as used herein, refers to any cell capable of differentiating into an immature dendritic cell in the presence of an appropriate cytokine (i.e. G-CSF, GM-CSF, TNFa, IL-4, IL-13, SCF (c-kit ligand), Flt-3 ligand, ora combination thereof). Examples of dendritic precursor cells include, but are not limited to, myeloid dendritic precursor cells, lymphoid dendritic precursor cells and plasmacytoid dendritic precursor cells. Phenotypic surface markers expressed by various subsets of dendritic precursor cells are well known in the art and may be used for the purpose of identification, for example, by flow cytometry or using immunohistochemical techniques.

[0067] The term “precursor” or “T cell precursor” as described herein refers to an immature progenitor that have recently immigrated from the bone marrow to the thymus and which retain a multilineage differentiation potential (T-lymphoid, natural killer, dendritic and myeloid cell differentiation potential).

[0068] In a more particular embodiment, the cell of the invention is a T cell or a natural killer (NK) cell, or a precursor thereof. In a more particular embodiment, the cell of the invention is a primary T cell.

[0069] The term “primary T cell” as used herein refers to any T-cell which can be obtained from an organism in possession of T-cells. Typically, said organism is an animal, preferably, a mammal, more preferably a laboratory animal such as a mouse, a rat, a monkey or a rabbit or a pet or farming animal such as a dog, a cat, a horse, a cow, a sheep or a goat and, most preferably, a human. It will be understood that the said primary T-cell has not been genetically modified in order to be immortalized. Primary T-cells may be cytotoxic T-cells, T helper cells, regulatory T-cells, natural killer T-cells, T memory cells, or gammadelta T-cells. Typically, the primary T-cell may be selected from the group consisting of: CD8 positive primary T-cells, CD4 positive primary T-cells, CD3 positive primary T-cells. More preferably, it is a CD3-positive primary T-cell.

[0070] Chimeric antigen receptor The cells of the invention are characterized in that they express a chimeric antigen receptor (CAR) comprising: o an antigen binding domain specific for BCMA, o a transmembrane domain, and o at least one intracellular signaling domain and / or a costimulatory domain.

[0071] As used herein, a "chimeric antigen receptor” or “CAR" also known as chimeric T cell receptors, a T-body, artificial T cell receptors and chimeric immune receptors (CIR), are engineered receptors, which graft an arbitrary specificity onto an immune effector cell. In a classical CAR, the specificity of a monoclonal antibody is grafted on to a T cell. CARs are therefore fusion proteins which comprise at least, an extracellular domain or antigen binding domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from a polypeptide from which the extracellular domain is derived, and at least one intracellular costimulatory domain.

[0072] The antigen binding domain of the CAR expressed by the cell of the invention

[0073] According to the present invention, the expressions "extracellular domain”, “antigenbinding domain”, “antigen-binding region”, “antigen-binding fragment” or “antibody fragment” are used interchangeably and refer to any oligopeptide or polypeptide that can bind to a certain antigen. In the present invention said antigen is BCMA, i.e., the antigen-binding domain or antigen-binding region is specific for BCMA.

[0074] The antigen-binding domain may comprise an antibody fragment, which refers to at least one portion of an intact antibody, or recombinant variants thereof, for example an antigen variable region of an intact antibody that is sufficient to allow recognition and specific binding of an antibody fragment to a target. The antigen-binding domain of the invention comprises at least a VH region and a VL region. Examples of antibody fragments include, but are not limited to Fab, Fab'-, F(ab')2 and Fv fragments, ScFv antibody fragments and linear antibodies. Within the context of the present invention, the antigen-binding domain or antibody fragment comprise at least one VH and one VL regions, but it may comprise two VL regions and two VH regions. Thus, for example, in a particular embodiment, the antigen-binding domain specific for BCMA of the CAR expressed by the cell of the invention is a ScFv, and therefore, it will comprise only one VL and one VH region. In another embodiment, the antigen-binding domain specific for BCMA of the CAR expressed by the cell of the invention is a Fab fragment, in which case it will comprise one VL and VH (Fab or Fab’) or two VH and two VL regions (Fab2, or F(ab’)2). In another embodiment, the antigen-binding domain specific for BCMA of the CAR expressed by the cell of the invention is a single domain antibody (sdAb) or a biparatopic sdAb.

[0075] As used herein the term “single domain antibody”, or its acronym “sdAb”, has its general meaning in the art and refers to the single heavy chain variable domain (VH) of antibodies of the type that can be found in Camelid mammals, which are naturally devoid of light chains. Such single domain antibody is also known as “nanobody®”. SdAb are characterized by a molecular weight of about 12-15 kDa, with a peptide chain of about 100-120 amino acids long, comprising three “complementarity-determining regions” (CDR), CDR1 , CDR2 and CDR3, which determine the antigen specificity and the binding properties of the sdAb. sdAbs can be obtain from several sources, natural and synthetic. Thus, in the meaning of the present invention, the term "single domain antibody" comprises polypeptides, which are derived from a non-human source, preferably a camelid, preferably a camel heavy chain antibody. They may be humanized. In a preferred embodiment the single domain antibody (sdAb) that specifically binds to human B-cell maturation antigen (hBCMA) are humanized. Moreover, the term comprises polypeptides derived from non-camelid sources, e.g. mouse or human, which have been "camelized", as previously described, e.g. in WO 08 / 101985 and WO 08 / 142164. The term "single domain antibody" encompasses immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. It also includes fully human, humanized or chimeric immunoglobulin sequences. For example, it comprises camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized single variable domains. In a particular embodiment, the sdAb comprises a sequence of any one of SEQ ID NO: 104 or SEQ ID NO: 105.

[0076] The term “biparatopic sdAb” refers to an antigen-binding domain comprising a first and a second sdAb, wherein these two sdAbs are capable of binding to two nonoverlapping epitopes of an antigen. In a particular embodiment, the biparatopic sdAb comprises both sdAbs comprising the sequences of SEQ ID NO: 104 and 105; preferably said biparatopic sdAb comprises the sequence of SEQ ID NO: 142.

[0077] In a particular embodiment, the antigen-binding domain is humanized.

[0078] As used herein, “humanized” forms of non-human (e.g., murine) antibodies or antigen-binding domains are chimeric antibodies or antigen-binding domains that contain minimal sequence, or no sequence, derived from non-human immunoglobulin. For the most part, humanized antibodies or antigen-binding domains are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies or antigen-binding domains can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are generally made to further refine antibody or antigen-binding domain performance. In general, the humanized antibody or antigen-binding domain will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a nonhuman immunoglobulin and all or substantially all of the FR residues are those of a human immunoglobulin sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0079] The term “BCMA”, as used herein, refers to the B cell maturation antigen, which is a cell surface receptor of the tumor necrosis factor receptor family that is preferentially expressed in mature B-lymphocytes. BCMA is also highly expressed in multiple myeloma. BCMA can also be referred to as BCM, CD269 or TNFRSF17 (tumor necrosis factor receptor superfamily member 17). The term "BCMA" includes any BCMA variant, isoform, and species homolog, which is naturally expressed by cells (including T cells) or can be expressed on cells transfected with genes or cDNA encoding those polypeptides. BCMA is characterized by the sequence according to the UniProt database with accession number Q02223 (version 2, release of 24 July 2024) (SEQ ID NO: 106).

[0080] In a particular embodiment, the antigen-binding domain specific for BCMA of the CAR expressed by the cell of the invention is a ScFv.

[0081] As used herein, a "single chain variable fragment (ScFv)" means a single chain polypeptide derived from an antibody which retains the ability to bind to an antigen. An example of the ScFv includes an antibody polypeptide which is formed by a recombinant DNA technique and in which variable (Fv) regions of immunoglobulin heavy chain (VH chain) and light chain (VL chain) fragments are linked via a spacer sequence. Various methods for preparing a ScFv are known, and include methods described in US Patent No. 4694778, Nature, vol. 334, p. 54454 (1989), and Science, vol. 242, pp. 1038-1041 (1988). In another particular embodiment the antigen-binding domain specific for BCMA of the CAR expressed by the cell of the invention is a ScFv with the VL region at the N- terminus and the VH region at the C-terminus. In a particular embodiment, the antigen- binding domain specific for BCMA of the CAR expressed by the cell of the invention is a ScFv with the VH region at the N-terminus and the VL region at the C-terminus.

[0082] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) or complementary determining regions (CDRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0083] The term “CDRs”, “hypervariable region”, “HVR”, “complementarity determining regions” or as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops (“hypervariable loops”). Generally, native four-chain antibodies comprise six CDRs; three in the VH (H1 , H2, H3), and three in the VL (L1 , L2, L3). Thus, CDRs determine the protein's affinity (roughly, bonding strength) and specificity for specific antigens. The CDRs of the two chains of each pair are aligned by the framework regions, acquiring the function of binding a specific epitope. Consequently, both the heavy variable chain and the light variable chain are characterized by three CDRs, respectively VH-CDR1 , VH- CDR2, VH-CDR3 and VL-CDR1 , VL-CDR2, VL-CDR3.

[0084] The term “Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1 , FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1 (L1)-FR2-H2(L2)-FR3-H3 (L3)-FR4.

[0085] In a particular embodiment, the ScFv of the CAR expressed by the cell of the invention comprises a sequence of any one of SEQ ID NO: 4 to SEQ ID NO: 92, or a functionally equivalent variant thereof.

[0086] As it is used herein, the term "functionally equivalent variant of a ScFv sequence" refers to a sequence variant of a particular ScFv sequence having substantially similar sequence identity with it and substantially maintaining its capacity to bind to its cognate antigen when being part of an antibody, antibody fragment or antigen-binding domain. For example, a functionally equivalent variant of a ScFv sequence may be a polypeptide sequence derivative of said sequence comprising the addition, deletion or substitution of one or more amino acids. In one embodiment, the substitution of one amino acid by other in the functionally equivalent variant is a conservative substitution. As used herein, the term “conservative substitution” refers to the replacement of an amino acid by another amino acid having similar chemical properties. Conservative substitution tables providing functionally similar amino acids are well known in the art.

[0087] Functionally equivalent variants of a ScFv sequence according to the invention include ScFv sequences having at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the corresponding amino acid sequence of any one of SEQ ID NO: 4 to SEQ ID NO: 92. It is also contemplated that functionally equivalent variants of a ScFv sequence comprise additions consisting of at least 1 amino acid, or at least 2 amino acids, or at least 4 amino acids, or at least 6 amino acids, or at least 8 amino acids, or at least 10 amino acids or more amino acids at the N-terminus, or at the C-terminus, or both at the N- and C-terminus of the corresponding amino acid sequence of any one of SEQ ID NO: 4 to SEQ ID NO: 92. Likewise, it is also contemplated that variants comprise deletions consisting of at least 1 amino acid, or at least 2 amino acids, or at least 4 amino acids, or at least 6 amino acids, or at least 8 amino acids, or at least 10 amino acids or more amino acids at the N-terminus, or at the C-terminus, or both at the N- and C-terminus of the corresponding amino acid sequence of any one of SEQ ID NO: 4 SEQ ID NO: to 92.

[0088] Functionally equivalent variants of a ScFv sequence according to the invention will preferably maintain at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 200% or more of the capacity of the corresponding amino acid sequence of any one of SEQ ID NO: 4 to SEQ ID NO: 92 to bind to its cognate antigen when being part of an antibody, antibody fragment or antigen-binding domain of the CAR expressed by the cell of the invention. This capacity to bind to its cognate antigen may be determined as a value of affinity, avidity, specificity and / or selectivity of the antibody or antibody fragment to its cognate antigen.

[0089] The terms “identity”, “identical” or “percent identity” in the context of two or more amino acid or nucleotide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotide or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Publicly available software programs can be used to align sequences. Appropriate parameters for maximal alignment by particular alignment software can be determined by one skilled in the art.

[0090] The CDR sequences can be determined according to conventional criteria, for example by means of the criteria of IgBLAST: http: / / www.ncbi.nlm.nih.gov / igblast / (Ye et al., 2013, Nucleic Acids Res 41 (Web Server issue: W34-40), by following the numbering provided by Kabat et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), or by following the numbering provided by Chothia et al. (1989, Nature 342:877-83). This particular region has been described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared against each other. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0091] In an embodiment, the ScFv of the CAR expressed by the cell of the invention may be defined by its variable domains of the heavy chain and light chain (VH and VL, respectively. The term “variable region” or “variable domain” has been described above.

[0092] In a particular embodiment of the CAR expressed by the cell of the invention, the VH and VL regions of the ScFv of the CAR expressed by the cell of the invention are connected by a linker region.

[0093] The term “flexible polypeptide linker” or “linker” refers to a peptide linker that consists of amino acids such as glycine and / or serine residues used alone or in combination, to link variable heavy and variable light chain regions together; or to link any or the regions of the CAR expressed by the cell of the invention.

[0094] The linker peptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded. A linker can be a peptide of between about 6 and about 40 amino acids in length, or between about 6 and about 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acid sequence, bearing in mind that suitable linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art.

[0095] Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1 , 2, 3, 4, 5, 6, or 7 amino acids.

[0096] Exemplary flexible linkers include the linker having the sequence TGSTSGSGKPGSGEGS (SEQ ID NO : 107). Suitable linkers include as glycine polymers (G) n, glycine-serine polymers (including, for example, (GS)nj (GSGGS)n(SEQ ID NO: 108) and (GGGS)n(SEQ ID NO: 109), where n is an integer of at least one), glycinealanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In a particular embodiment the linker comprises a glycine polymer of formula (G4S)3. Glycine and glycine-serine polymers are of interest since both of these amino acids are relatively unstructured, and therefore may serve as a neutral tether between components. Glycine polymers are of particular interest since glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains. Exemplary flexible linkers include, but are not limited to GGSG (SEQ ID NO : 110) , GGSGG (SEQ ID NO : 111) , GSGSG (SEQ ID NO: 112) , GSGGG (SEQ ID NO: 113) , GGGSG (SEQ ID NO: 114) , GSSSG (SEQ ID NO: 115), and the like. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any elements described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure.

[0097] In a particular embodiment, the linker is located between the VH and the VL regions of the ScFv of the CAR expressed by the cell of the invention. In an embodiment, the ScFv of the CAR expressed by the cell of the invention comprises the structure VL-linker- VH. In another embodiment, the ScFv of the CAR expressed by the cell of the invention may have the structure VH-linker-VL or VL-linker-VH. In a particular embodiment, the linker is located C-terminally with respect to the VL region and N-terminally with respect to the VH region, that is, VL-linker-VH.

[0098] In an embodiment, the ScFv of the CAR expressed by the cell of the invention is characterized by showing a high affinity towards BCMA and will be referred to hereinafter as the “High Aff.”. In a particular embodiment, the ScFv of the CAR expressed by the cell of the invention has a dissociation constant (KD) of between about 0.1 to about 1.0 nM, preferably of about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM or about 0.9 nM.

[0099] In another embodiment, the ScFv of the CAR expressed by the cell of the invention is characterized by showing a medium affinity towards BCMA and will be referred to hereinafter as the “Med Aff.”. In a particular embodiment, the ScFv of the CAR expressed by the cell of the invention has a dissociation constant (KD) of between 500 and 2000 nM, preferably of about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1000 nM, about 1100 nM, about 1200 nM, about 1300 nM, about 1400 nM, about 1500 nM, about 1600 nM, about 1700 nM, about 1800 nM, about 1900 nM, about 2000 nM, more preferably of about 1020 nM, about 1040 nM, about 1060 nM, about 1080 nM, about 1120 nM, about 1140 nM, about 1160 nM, about 1180 nM.

[0100] As used herein, the term “binding” in the context of the binding of an antibody / ScFv / FV / CDR to a predetermined antigen or epitope typically is a binding with an affinity, or “binding affinity”, corresponding to a KD of about 10"7M or less, such as about 10"8M or less, such as about 10"9M or less, about 10"1° M or less, or about 10"11M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1 ,000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is lower is dependent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly specific), then the amount with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold.

[0101] The aforementioned method to determine the binding affinity of an antibody / ScFv / FV / CDR can also be used to compare the binding affinity between antibodies. In another particular embodiment of the antigen-binding domain of the CAR expressed by the cell of the invention, the antigen-binding domain Med Aff of the cell of the invention has a binding affinity which at least 2 fold lower, at least 3 fold lower, at least 4 fold lower, at least 5 fold lower than the antigen-binding domain High Aff of the cell of the invention.

[0102] The transmembrane domain of the CAR expressed by the cell of the invention The second element of the CAR expressed by the cell of the invention is a transmembrane domain that is attached to the extracellular domain of the CAR.

[0103] As used herein, “transmembrane domain” (TMD) refers to the area of CAR that crosses the cell membrane. The transmembrane domain of the CAR expressed by the cell of the invention is the transmembrane domain of a transmembrane protein (e.g., a type I transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g. , 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is one that is associated with one of the other domains of the CAR is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In a particular embodiment, the transmembrane domain of the CAR expressed by the cell of the invention is capable of homodimerization with another CAR on the CAR T cell surface. In a different particular embodiment of the CAR expressed by the cell of the invention, the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same CAR or CAR T cell.

[0104] The transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR has bound to a target. Non limiting examples or transmembrane domains of particular use in this invention may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD3 zeta, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD 278), 4-1 BB (CD137), GITR, CD40, CTLA4, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1 , VLA1 , CD49a, ITGA4, IA4 CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDGA, CDGA, CD103, ITGAL, CDLa, LFA-1 , ITGAM, CDIIb, ITGAX, CDIc, ITGB1 , CD29, ITGB2, CD18, LFA-1 , LGA ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD100 (SEMA4D), SLAMF6 (NTB-A) , LylOS), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, Kp30, NKp46, including NKG2D, and / or a transmembrane domain selected from the transmembrane domain of NKG2C.

[0105] In a particular embodiment of the CAR expressed by the cell of the invention, the transmembrane domain is selected from the group consisting of the CD4 transmembrane domain, the CD8 transmembrane domain, the CD28 transmembrane domain, the 4-1 BB transmembrane domain, the CTLA4 transmembrane domain, the CD27 transmembrane domain and the CD3 zeta transmembrane domain.

[0106] In a particular embodiment, the transmembrane domain is the CD8 transmembrane domain. In a particular embodiment, the CD8 transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 93).

[0107] In another particular embodiment of the CAR expressed by the cell of the invention, the transmembrane domain comprises sequences having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the sequence of SEQ ID NO: 93.

[0108] In another particular embodiment of the CAR expressed by the cell of the invention, the transmembrane domain comprises the sequence of SEQ ID NO: 93.

[0109] In another particular embodiment of the CAR expressed by the immune cell of the invention, the transmembrane domain consists of or essentially consists of the sequence SEQ ID NO: 93.

[0110] The intracellular signaling domain and / or costimulatory domain of the CAR expressed by the cell of the invention

[0111] The CARs forming part of the cells according to the present invention comprise at least one intracellular signaling domain and / or costimulatory domain.

[0112] “Intracellular signaling domain”, as the term is used herein, refers to the intracellular portion of a molecule and more specifically to any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell. The intracellular signaling domain generates a signal that stimulates the immune effector function of CAR-containing cells, for example, CAR-T cells. The effector function of a T cell, for example, may be cytolytic function or helper activity including the secretion of cytokines. Thus, the intracellular signaling domain may be a portion of a protein which transduces the effector function signal and directs the cell (e.g. T cell) to perform a specialized function.

[0113] Generally, the whole intracellular signaling domain can be used; however, it is appreciated that it is not necessary to use the entire domain, provided that whatever part of the signaling domain that is used is still capable of transducing the effector function signal. It will also be appreciated that variants of such intracellular signaling domains with substantially the same or greater functional capability may also be used. By this we include the meaning that the variants should have substantially the same or greater transduction of the effector functional signal. Typically, substantially the same or greater signal transduction includes at least 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, or 120%, or more of the signal transduction of the unmodified intracellular signaling domain, wherein signal transduction of the unmodified intracellular signaling domain corresponds to 100%. Methods for assessing transduction of effector function signal are well known to those skilled in the art and include, for example, assessing the amounts and / or activity of molecules (e.g. proteins such as cytokines) that are indicative of the transduced signal. Thus, when the signal is the cytolytic function of a T-cell, the methods may involve measurement of one or more cytokines secreted by the T-cell, which cytokines are known to have a cytolytic activity (e.g. IFN gamma). Another means of assessing the cytolytic function is by CFSE staining and counting positive cells by Flow cytometry or by a chromium release assay as is well known in the art.

[0114] Examples of intracellular signaling domains for use in the CAR expressed by the cell of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and coreceptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0115] It is known that signals generated through the TCR (T cell receptor) alone are generally insufficient for full activation of a T cell and that a secondary and / or costimulatory signal may also be required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen- independent manner to provide a secondary or costimulatory signal (secondary intracellular signaling domain, such as a costimulatory domain). Costimulatory domains promote activation of effector functions and may also promote persistence of the effector function and / or survival of the cell. In a particular embodiment of the CAR expressed by the cell of the invention, the at least one intracellular signaling domain of the CAR comprises a costimulatory domain, a primary signaling domain, or any combination thereof.

[0116] A primary intracellular signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs (e.g. 2, 3, 4, 5 or more ITAMs). Thus, the intracellular signaling domain may comprise one or more ITAMs. It will be appreciated that one or more ITAMs of the intracellular signaling domain may be modified, for example by mutation. The modification may be used to increase or decrease the signaling function of the ITAM as compared to the native ITAM domain.

[0117] Examples of ITAM containing primary intracellular signaling domains that are of particular use in the invention include those of CD3 zeta, Fc receptor gamma, Fc receptor beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0118] In a particular embodiment, the at least one intracellular signaling domain of the CAR expressed by the cell of the invention is selected from a group consisting of CD3 zeta, Fc receptor gamma, Fc receptor beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In a more particular embodiment, the at least one intracellular signaling domain of the CAR expressed by the cell of the invention is the CD3-zeta intracellular domain.

[0119] The term “CD3” refers to the human CD3 protein complex, which is composed of six distinct chains: a CD3y chain (UniProt database accession number P09693, version 1 , release of 24 July 2024), a CD35 chain (UniProt database accession number P04234, version 1 , release of 24 July 2024), two CD3E chains (UniProt database accession number P07766, version 2, release of 24 July 2024), and one CD3 zeta chain homodimer (UniProt database accession number P20963, version 2, release of 24 July 2024) (E y: E 5:^0, and which is associated with the T cell receptor a and chain. The term includes any CD3 variants, isoforms and species homologs which are naturally expressed by cells, including T cells, or are expressed on cells transfected with genes or cDNA encoding the aforementioned chains.

[0120] The term “zeta” or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta” is defined as the protein represented by GenBank entry No. BAG36664.1 (version of 24 May 2008), or variants thereof from a non-human species, such as a mouse, rodent, monkey, primate, etc., and a “zeta stimulating domain” or alternatively a “CD3 zeta stimulating domain” or “TCR zeta stimulating domain” is defined as amino acid residues of the cytoplasmic domain of the zeta chain that are sufficient for functional transmission of the primary signal required to activate T cells etc. In one aspect, the zeta cytoplasmic domain comprises residues 52 through 164 inclusive of a protein represented by GenBank entry No. BAG36664.1 (version of 24 May 2008), or equivalent residues from a non-human species, for example, a mouse, rodent, monkey, primate, and the like, which are their functional orthologists.

[0121] As mentioned above, the intracellular signaling domain may comprise a primary intracellular signaling domain by itself, or it may comprise a primary intracellular signaling domain in combination with one or more secondary intracellular signaling domains, such as one or more costimulatory signaling domains. Thus, the intracellular signaling domain of the CAR may comprise the CD3 zeta signaling domain by itself or in combination with one or more other intracellular signaling domains such as one or more costimulatory signaling domains.

[0122] The costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.

[0123] The term “co-stimulating molecule” refers to a recognizable T-cell binding partner that specifically binds to a co-stimulating ligand, thereby mediating the co-stimulatory response exerted by the T-cell, such as, but not limited to, proliferation. Co-stimulating molecules are cell surface molecules other than antigen-specific receptors or their ligands, which are necessary for an effective immune response. A costimulatory molecule may be a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of immune cells (eg lymphocytes) to an antigen. A costimulatory molecule can be represented in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins) and NK cell activation receptors. Examples of such molecules include, but are not limited to 0X40, ICOS, DAP10, CD27, CD28, CDS, CD30, CD137 (4-1 BB), CD40, ICOS, lymphocyte function- associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, GITR, NKG2C, SLAMF7, NKp80, BAFFR, HVEM, BTLA, ICAM-1 , LFA-1 (CD11a / CD18), B7- H3, and a ligand that specifically binds with CD83, and the like. For example, CD27 co- stimulation has been demonstrated to enhance expansion, effector function, and survival of human CAR T cells in vitro and augments human T cell persistence and anti-tumor activity in vivo (Song et al. Blood. 2012; 1 19(3):696-706).

[0124] In another embodiment, the at least one intracellular signaling domain of the cell of the invention comprises the intracellular domain of a costimulatory molecule selected from the group consisting of: 0X40, CD70, CD27, CD28, CD5, ICAM-1 , LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP 12, and 4-1 BB (CD137), or any combination thereof.

[0125] In a particular embodiment, the CAR expressed by the cell of the invention comprises the intracellular domain of the costimulatory molecule 4-1 BB. In a more particular embodiment, the intracellular domain of a costimulatory molecule comprises the sequence of SEQ ID NO: 95.

[0126] SEQ ID NO : 95

[0127] KRGRKKLLYI FKQPFMRPVQTTQE EDGCSCRF PE E E EGGCE L

[0128] In a particular embodiment, the at least one intracellular signaling domain of the CAR expressed by the cell of the invention further comprises the CD3-zeta intracellular domain. In one more particular embodiment the at least one intracellular signaling domain of the CAR expressed by the cell of the invention further comprises the sequence of SEQ ID NO: 96.

[0129] SEQ ID NO : 96

[0130] RVKFSRSADAPAYQQGQNQLYNE LNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE LQKDKMAEA YSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0131] In one particular embodiment, the at least one intracellular signaling domain of the CAR expressed by the cell of the invention is arranged on an N-terminal side relative to the CD3-zeta intracellular domain. In another particular embodiment, the at least one intracellular signaling domain of the CAR expressed by the cell of the invention is arranged on a C-terminal side relative to the CD3-zeta intracellular domain.

[0132] The intracellular signaling sequences within the intracellular portion of the CAR expressed by the cell of the invention may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequences. In one embodiment, a glycine- serine doublet can be used as a suitable linker. In another embodiment, a single amino acid, such as an alanine or a glycine, can be used as a suitable linker.

[0133] In one embodiment, the intracellular signaling domain is designed to comprise two or more, for example 3, 4, 5, or more, costimulatory signaling domains. In an embodiment, the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains, are separated by a linker molecule, such as one described herein. In one embodiment, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0134] In preferred embodiments, the intracellular portion of the CAR expressed by the cell of the invention comprises: the signaling domain of CD3-zeta and the signaling domain of CD28, the signaling domain of CD3-zeta and the signaling domain of 4-1 BB, the signaling domain of CD3-zeta and the signaling domain of 0X40, the signaling domain of CD3-zeta and the signaling domain of ICOS, the signaling domain of CD3-zeta and the signaling domain of DAP10 the signaling domain of CD3-zeta, the signaling domain of 4-1 BB and the signaling domain of 0X40. the signaling domain of 4-1 BB and the signaling domain of CD28.

[0135] In another embodiment, the intracellular portion of the CAR expressed by the cell of the invention comprises the signaling domain of CD3-zeta and the signaling domain of 4-1 BB.

[0136] The intracellular signaling domain may include the entire intracellular portion, or the entire natural intracellular signaling domain, the molecule from which it originates, or a functional fragment thereof.

[0137] In another embodiment, the CAR expressed by the cell of the invention further comprises a hinge domain between the antigen binding domain and the transmembrane domain.

[0138] As used herein, “hinge domain”, “hinge region” or “spacer” refers to an amino acid region that allows for separation and flexibility of the binding moiety and the T cell membrane. The length of the flexible hinges also allows for better binding to relatively inaccessible epitopes, e.g., longer hinge domains are allowed for optimal binding. One skilled in the art will be able to determine the appropriate hinge for the given CAR target.

[0139] In some cases, the first polypeptide of the CAR expressed by the cell of the invention comprises a hinge domain, where the hinge domain is interposed between the antigenbinding domain and the transmembrane domain. In some cases, the hinge domain is an immunoglobulin heavy chain hinge domain. In some cases, the hinge domain is a domain region polypeptide derived from a receptor (e.g., a CD8-derived hinge domain).

[0140] The hinge domain can have a length of from about 10 amino acids to about 200 amino acids, preferably, between 50 and 150 amino acids, more preferably between 75 and 125 amino acids. Exemplary hinge domains include glycine polymers (G) n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 108) and (GGGS)n (SEQ ID NO: 109), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains. Exemplary spacers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 110) , GGSGG (SEQ ID NO: 111) , GSGSG (SEQ ID NO: 112) , GSGGG (SEQ ID NO: 113) , GGGSG (SEQ ID NO: 114) , GSSSG (SEQ ID NO: 115) and the like.

[0141] In some cases, the hinge domain of the CAR expressed by the cell of the invention includes at least one cysteine. For example, in some cases, the hinge domain can include the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 116). If present, a cysteine in the hinge domain of a first CAR can be available to form a disulfide bond with a hinge domain in a second CAR.

[0142] Immunoglobulin hinge domain amino acid sequences are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res. 14:1779. As non-limiting examples, an immunoglobulin hinge domain can include one of the following amino acid sequences: DKTHT (SEQ ID NO: 117) ; CPPC (SEQ ID NO: 116) ; CPEPKSCDTPPPCPR (SEQ ID NO: 118) (see, e.g., Glaser et al. (2005) J. Biol. Chern. 280:41494); ELKTPLGDTTHT (SEQ ID NO : 119) ; KSCDKTHTCP (SEQ ID NO: 120) ; KCCVDCP (SEQ ID NO : 121) ; KYGPPCP (SEQ ID NO : 122) ; EPKSCDKTHTCPPCP (SEQ ID NO : 123) (human lgG1 hinge); ERKCCVECPPCP (SEQ ID NO: 124) (human lgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 125) (human lgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 126) (human lgG4 hinge); and the like.

[0143] The hinge domain can comprise an amino acid sequence of a human lgG1 , lgG2, lgG3, or lgG4, hinge domain. The hinge domain can include one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally- occurring) hinge domain. For example, His 229 of human IgG 1 hinge can be substituted with Tyr, so that the hinge domain comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 127); see, e.g., Yan et al. (2012) J. Biol. Chem. 287:5891).

[0144] In an embodiment, the hinge domain of the CAR expressed by the cell of the invention is selected from the group consisting of CD8, CD28, and lgG4 hinge domain, or a variant thereof. In a particular embodiment, the hinge domain of the CAR expressed by the cell of the invention is the CD8 hinge domain.

[0145] The hinge domain can comprise an amino acid sequence derived from human CD8 or a variant thereof.

[0146] In a particular embodiment, the hinge domain comprises, consists or essentially consists of the CD8 hinge domain.

[0147] In another particular embodiment the hinge domain comprises the sequence of SEQ ID NO: 94.

[0148] SEQ ID NO : 94

[0149] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0150] In a particular embodiment of the CAR expressed by the cell of the invention, the hinge domain is the CD8 hinge domain, the transmembrane domain is the CD8 transmembrane domain and the intracellular signaling domain is the 4-1 BB costimulatory domain.

[0151] In a particular embodiment, the CAR expressed by the cell of the invention comprises the CD8 hinge domain, the CD8 transmembrane domain and the CD3 zeta intracellular signaling domain and the 4-1 BB intracellular costimulatory signaling domain.

[0152] In another embodiment, the CAR expressed by the cell of the invention includes from the N-terminus to the C-terminus an anti-BCMA light chain variable domain, a linker domain, an anti-BCMA heavy chain variable domain, a CD8 hinge domain, a CD8 transmembrane domain, a 4-1 BB intracellular co-stimulatory signaling domain followed by a CD3 zeta intracellular signaling domain. In another embodiment, the CAR expressed by the cell of the invention includes from the N-terminus to the C-terminus an anti-BCMA heavy chain variable domain, a linker domain, an anti-BCMA light chain variable domain, a CD8 hinge domain, a CD8 transmembrane domain, a 4-1 BB intracellular co-stimulatory signaling domain followed by a CD3 zeta intracellular signaling domain.

[0153] In a particular embodiment, the CAR expressed by the cell of the invention comprises the sequence of any one of SEQ ID NO: 97 to SEQ ID NO: 102 or SEQ ID NO: 143.

[0154] Glutamine transporter

[0155] In addition to the CAR, the cell of the invention further expresses an exogenous glutamine transporter selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, which is expressed from an exogenous nucleic acid introduced into the cell.

[0156] The term “glutamine transporter” or “Gin transporter” in the context of the present invention refers to a membrane transporter that transports glutamine across the cell membrane.

[0157] The term “ASCT2” or “SLC1A5” or “ATBO”, in the context of the present invention, refers to the solute carrier family 1 , member 5 (neutral amino acid transporter B(0)), encoded by the SLC1A5 gene. ASCT2 is characterized by the sequence according to the UniProt database with accession number Q15758 (version 2, release of 24 July 2024). ASCT2 has three human isoforms: Q15758-1 (SEQ ID NO: 1), Q15758-2 (SEQ ID NO: 2) and Q15758-3 (SEQ ID NO: 3).

[0158] The term “SLC7A5” or “LAT1”, in the context of the present invention, refers to the solute carrier family 7, member 5 (large neutral amino acids transporter small subunit 1), encoded by the SLC7A5 gene. SLC7A5 is characterized by the sequence according to the UniProt database with accession numbers Q01650 (version 2, release of 24 July 2024).

[0159] The term “SLC7A8” or “LAT2”, in the context of the present invention, refers to the solute carrier family 7, member 8 (large neutral amino acids transporter small subunit 2), encoded by the SLC7A8 gene. SLC7A8 is characterized by the sequence according to the UniProt database with accession numbers Q9UHI5 (version 1 , release of 24 July 2024).

[0160] The term “SLC6A14” or “ATB0,+”, in the context of the present invention, refers to the solute carrier family 6, member 14 (sodium- and chloride-dependent neutral and basic amino acid transporter B(0+)), encoded by the SLC6A14 gene. SLC6A14 is characterized by the sequence according to the UniProt database with accession numbers Q9UN76 (version 1 , release of 24 July 2024).

[0161] The term “SLC38A1” or “SNAT1” or “SAT1” or “ATA1” or “NAT2”, in the context of the present invention, refers to the solute carrier family 38, member 1 (sodium-coupled neutral amino acid symporter 1), encoded by the SLC38A1 gene. SLC38A1 is characterized by the sequence according to the UniProt database with accession numbers Q9H2H9 (version 1 , release of 24 July 2024).

[0162] The term “SLC38A2” or “SNAT2” or “SAT2” or “ATA2” or “KIAA1382”, in the context of the present invention, refers to the solute carrier family 38, member 2 (sodium-coupled neutral amino acid symporter 2), encoded by the SLC38A2 gene. SLC38A2 is characterized by the sequence according to the UniProt database with accession numbers Q96QD8 (version 2, release of 24 July 2024).

[0163] In a particular embodiment, the glutamine transporter expressed by the cell of the invention is ASCT2. In a more particular embodiment, the glutamine transporter ASCT2 expressed by the cell of the invention comprises a sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 3, or a functionally equivalent variant thereof.

[0164] A variant of a protein, e.g., the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, as used herein, may be (i) a protein in which one or more of the amino acid residues is / are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) a protein having one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) a modified protein said protein being the result of an alternative splicing of the mRNA encoding the glutamine transporter protein, and / or (iv) a fragment of the protein. The term "fragment" includes also a peptide or protein generated via proteolytic cleavage (including multisite proteolysis) of an original protein. Variants are deemed to be within the scope of those skilled in the art from the teaching herein.

[0165] The term “protein” or “peptide”, as used herein, refers to a molecular chain of amino acids, linked by covalent or non-covalent bonds. The term includes all the forms of post- translational modifications, for example, glycosylation, phosphorylation or acetylation.

[0166] As known in the art, the "similarity" between two proteins is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one protein to a sequence of a second protein. Variants according to the present invention include peptides or protein having amino acid sequences that are at least 60%, at least 65%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 95%, or even more, similar or identical to the original amino acid sequence.

[0167] The terms “identity” and “identical” have been defined in the context of the CAR expressed by the cell of the invention and apply equally to the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2 expressed by the cell of the invention.

[0168] The degree of identity between two proteins can be determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)).

[0169] In one more particular embodiment, the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2 expressed by the cell of the invention comprises a sequence having at least 74%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 3.

[0170] In another embodiment, the functionally equivalent variant of the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, is one which substantially maintains the ability to transport glutamine across the cell membrane as that of a sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 3 when tested by a method as defined in the Examples, Materials and Methods, Glutamine uptake assay of the present document and as illustrated in Figure 2G. In some embodiments, the functionally equivalent variant of the glutamine transporter maintains at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the activity of one or more of the glutamine transporters as defined above including the ASCT2, SLC7A5, SLC7A8, SLC6A14, SLC38A1 and SLC38A2 glutamine transporters.

[0171] In an embodiment, the cell of the invention does not comprise an exogenous nucleic acid encoding for tryptophanyl-tRNA-synthetase. The term “tryptophanyl-tRNA- synthetase” or “tryptophan-tRNA ligase”, as disclosed herein, refers to an enzyme which mediates the aminoacylation reaction between the amino acid tryptophan and a tRNA, encoded by the WARS gene. In an embodiment, the tryptophanyl-tRNA-synthetase comprises a sequence of any one of SEQ ID NO: 144 to SEQ ID NO: 155.

[0172] Nucleic acids, compositions of nucleic acids and vectors of the invention

[0173] The cell of the invention can be obtained by the use of nucleic acid constructs and / or compositions which encode the CAR and the glutamine transporter which is to be expressed by the cells of the invention.

[0174] Accordingly, a second aspect of the present invention relates to a nucleic acid construct, hereinafter referred to as “the nucleic acid construct of the invention”, comprising (i) a first region encoding a chimeric antigen receptor (CAR), said chimeric antigen receptor comprising: o an antigen binding domain specific for BCMA, o a transmembrane domain, and o at least one intracellular signaling domain and / or a costimulatory domain; and

[0175] (ii) a second region encoding a glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2; or a nucleic acid composition, hereinafter referred to as “the nucleic acid composition of the invention”, comprising

[0176] (i) a first polynucleotide encoding a chimeric antigen receptor (CAR), said chimeric antigen receptor comprising: o an antigen binding domain specific for BCMA, o a transmembrane domain, and o at least one intracellular signaling domain and / or a costimulatory domain; and

[0177] (ii) a second polynucleotide encoding a glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2.

[0178] The terms and expressions “CAR”, antigen binding domain”, “transmembrane domain”, “intracellular signaling domain”, costimulatory domain” and “glutamine transporter” and their particulars have been defined in the context of the cell of the invention and apply equally to the nucleic acid construct of the invention and nucleic acid composition of the invention.

[0179] In particular embodiment of the nucleic acid construct of the invention or of the nucleic acid composition of the invention, the CAR antigen binding domain specific for BCMA, the CAR transmembrane domain, the at least one CAR intracellular signaling domain and / or a CAR costimulatory domain, and the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, are as defined in any of the previous aspects and / or embodiments in the context of the cell of the invention.

[0180] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and their polymers in either single or double stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding capabilities similar to those of a reference nucleic acid and which are metabolized similarly to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implies conservatively modified variants (e.g., substitutions with degenerate codons), alleles, orthologs, SNPs and complementary sequences, as well as sequences indicated in direct form. In particular, substitutions with degenerate codons can be obtained by creating sequences in which the third position of one or more selected (or all) codons is replaced by residues with mixed bases and / or deoxyinosine residues.

[0181] The term “nucleic acid construct” as used herein refers to one single nucleotide sequence which comprises all the elements required to encode the chimeric antigen receptor and the glutamine transporter, i.e., the first and second regions of the polynucleotide. The term “nucleic acid composition” as used herein refers to a composition comprising at least two nucleotide sequences or polynucleotides, one encoding for the CAR expressed by the cell of the invention and the other encoding for the glutamine transporter expressed by the cell of the invention.

[0182] In a particular embodiment of the nucleic acid construct of the invention or of the nucleic acid composition of the invention, the chimeric antigen receptor encoded by the first region of the nucleic acid construct of the invention or by the first polynucleotide of the nucleic acid composition of the invention, and / or the glutamine transporter encoded by the second region of the nucleic acid construct of the invention or by the second polynucleotide of the nucleic acid composition of the invention further comprise a sequence encoding signal sequence which is located within the same open reading frame with respect to the sequence encoding the chimeric antigen receptor and / or with respect to the sequence encoding the glutamine transporter.

[0183] The term “signal sequence”, also known as “signal peptide” or “leader peptide”, is used herein according to its ordinary meaning in the art and refers to a peptide having a length of about 5-30 amino acids. A leader peptide is present at the N-terminus of newly synthesized proteins that form part of the secretory pathway. Proteins of the secretory pathway include, but are not limited to proteins that reside either inside certain organelles (the endoplasmic reticulum, Golgi or endosomes), are secreted from the cell, or are inserted into a cellular membrane. In some embodiments, the leader peptide forms part of the transmembrane domain of a protein.

[0184] In some embodiments of the nucleic acid construct of the invention, the construct contains a region encoding a signal sequence preceding the sequence encoding the chimeric antigen receptor but does not contain a region encoding a signal sequence preceding the sequence encoding the glutamine transporter.

[0185] In some embodiments of the nucleic acid construct of the invention, the construct contains regions encoding signal sequences preceding both the sequence encoding the chimeric antigen receptor and the sequence encoding the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, which may be the same or different.

[0186] In some embodiments of the nucleic acid composition of the invention, the nucleic acid encoding the chimeric antigen receptor contains a region encoding a signal sequence whereas the nucleic acid encoding the glutamine transporter does not contain a region encoding a signal sequence.

[0187] In some embodiments of the nucleic acid composition of the invention, both the nucleic acid encoding the chimeric antigen receptor and the nucleic acid encoding the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, contain regions encoding signal sequences, which may the same or different.

[0188] In a particular embodiment of the nucleic acid construct of the invention or of the nucleic acid composition of the invention, whenever the sequence encoding the chimeric antigen receptor and / or the sequence encoding the glutamine transporter selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, is preceded by a sequence encoding a signal sequence, the sequence of the signal sequence is that of SEQ ID NO: 103.

[0189] SEQ ID NO : 103 MALPVTALLLPLALLLHAAR

[0190] It will be obvious to the expert in the field that the nucleic acid construct of the invention, which comprises two regions, one encoding for the CAR expressed by the cell of the invention and the other encoding for the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, expressed by the cell of the invention, allows two separate polypeptides to be obtained, i.e., the CAR and the glutamine transporter selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2. Likewise, it will be obvious to the expert in the field that the nucleic acid composition of the invention, which comprises two polynucleotides, one encoding for the CAR expressed by the cell of the invention and the other encoding for the glutamine transporter expressed by the cell of the invention, allows two separate polypeptides to be obtained, i.e., the CAR and the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2. Several mechanisms are available to produce two or more polypeptides from the same nucleic acid construct or nucleic acid composition. One such way is the existence in said nucleic acid of different transcription promoting elements, e.g., promoters and enhancers, for each of the different coding regions (see aspects and embodiments further below). Another way is the existence of single promoting elements which originate a unique messenger RNA (mRNA) which encodes for both the CAR and the glutamine transporter. In said case the mRNA can be monocistronic, i.e., both regions or polynucleotides are contained in the same open reading frame of the mRNA, or bicistronic wherein the mRNA contains two different opening reading frames, one encoding for the first region or first polynucleotide which corresponds to the CAR expressed by the cell of the invention, and the other encoding for the second region or second polynucleotide which corresponds to the glutamine transporter selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, expressed by the cell of the invention.

[0191] In a particular embodiment, the nucleic acid construct of the invention or the nucleic acid composition of the invention is a monocistronic construct or monocistronic composition, respectively, wherein the chimeric antigen receptor encoded by the first region or first polynucleotide and the glutamine transporter selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, encoded by the second region or second polynucleotide are found in the same open-reading frame and are separated by a self-cleaving peptide region or polynucleotide.

[0192] As used herein, the term "self-cleaving peptide region or polynucleotide" means a peptide sequence having a cleavage activity that occurs between two amino acid residues in the peptide sequence itself. Examples of the self-cleaving peptide include a 2A peptide and a 2A-like peptide. For example, in 2A or 2A-like peptides, cleavage occurs between glycine and proline residues on these peptides. This is caused by the "ribosome skipping mechanism", which prevents the formation of normal peptide bonds between glycine residues and proline residues during translation, and does not affect downstream translation. The ribosome skip mechanism is known in the art and is used for the expression of multiple proteins encoded by a single molecule of mRNA. The selfcleaving peptide used in the present invention can be obtained from a viral 2A peptide or a 2A-like peptide having an equivalent function. Examples of 2A self-cleaving peptides include 2A peptides (F2A) derived from foot-and-mouth disease virus (FMDV) (VKQTLNFDL LKLAGDVESNPGP (SEQ ID NO: 128)), 2A peptides (E2A) derived from horse rhinitis A virus (ERA V) (QCTNYALLKLAGDVESNPGP (SEQ ID NO: 129)), 2A peptide (P2A) derived from Porcine teschovirus (PTV-1) (ATNFSLLKQAGDVEENPGP (SEQ ID NO: 130)), and 2A peptides (T2A) derived from Thosea signa virus (TaV) (EGRGSLLTCGDVEENPGP (SEQ ID NO: 131) ). Mutations may be appropriately introduced into the self-cleaving peptide domain as long as its activity is not significantly impaired.

[0193] In another particular embodiment, the nucleic acid construct of the invention or the nucleic acid composition of the invention, which is a monocistronic construct or monocistronic composition, the chimeric antigen receptor encoded by the first region or first polynucleotide and the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, encoded by the second region or second polynucleotide are found in the same open-reading frame and are separated by a cis-acting hydrolase element.

[0194] A "cis-acting hydrolase element" or "CHYSEL" refers to a peptide sequence that causes a ribosome to release the growing polypeptide chain that it is being synthesized without dissociation from the mRNA. In this respect, the ribosome continues translating and therefore produces a second polypeptide. Peptides such as the FMDV 2A sequence (GSGSRVTELLYRMKRAETYC PRPLLAIHPTEARHKQKIVAPVKQLLNFDLLKLAGDVESNPGP, SEQ ID NO : 132), Sponge (Amphimedon queenslandica) 2A sequence (LLCFLLLLLSGDVELNPGPj SEQ ID NO: 133; or HHFMFLLLLLAGDIELNPGP, SEQ ID NO:

[0195] 134); acorn worm (Saccoglossus kowalevskii) (WFLVLLSFILSGDIEVNPGP SEQ ID NO :

[0196] 135) 2A sequence; amphioxus (Branchiostoma floridae) (KNCAIWILLLSGDVETNPGP SEQ ID NO : 136; or MVISQLMLKLAGDVEENPGP SEQ ID NO : 137) 2A sequence Porcine teschovirus-1 (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO: 138) 2A sequence; Thoseaa signa virus (GSGEGRGSLLTCGDVEENPGP, SEQ ID NO: 139) 2A sequence; and Equine rhinitis A virus (GSGQCTNYALLKLAGDVESNPGP, SEQ ID NO: 140) 2A sequence are CHYSELs of use in this invention. In some embodiments, the 2A sequence is a naturally occurring or synthetic sequence that includes the 2A consensus sequence D-X- E-X- NPGP (SEQ ID NO : 141), in which X is any amino acid residue.

[0197] In a particular embodiment, the nucleic acid construct of the invention or the nucleic acid composition of the invention, which is a multicistronic construct or monocistronic composition, the chimeric antigen receptor encoded by the first region or first polynucleotide and the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, encoded by the second region or second polynucleotide are found in different open-reading frames and are separated by an internal ribosome entry site or a cis-acting hydrolase element. The term "internal ribosome entry site" or "IRES" defines a sequence motif that promotes attachment of ribosomes to that motif on internal mRNA sequences. Consequently, an mRNA containing an IRES sequence motif results in two translational products, one initiating from the 5'-end of the mRNA and the other by an internal translation mechanism mediated by the IRES. A number of IRES have been described and can be used in the nucleic acid construct of this invention. See, e.g., US 8,192,984; WO 2010 / 119257; and US 2005 / 0112095.

[0198] A third aspect of the present invention relates to a vector comprising the nucleic acid construct of the invention, hereinafter referred to as “the vector of the invention”, or a vector composition comprising a pair of vectors in which the first and second nucleic acids of the composition are respectively comprised within the first and second vectors of said pair of vectors, hereinafter referred to as “the vector composition of the invention”.

[0199] As used herein, “vector,” “cloning vector,” and “expression vector” are vehicles by which the host is transformed and expression of introduced sequences (e.g., transcription and translation) may be accomplished. The term “vector” is also meant to include plasmids, phages, viruses and the like.

[0200] A nucleotide sequence encoding any of the CARs expressed by the cell of the invention and / or the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, expressed by the cell of the invention can be present in an expression vector and / or a cloning vector. An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, e.g., plasmids, viral vectors, and the like.

[0201] Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating suitable recombinant constructs. The following vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1 , pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0202] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors, in particular lentiviral (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus; adeno-associated virus; SV40; herpes simplex virus; human immunodeficiency virus; a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0203] In a particular embodiment of the vector of the invention or of the vector composition of the invention, the vector is a lentiviral vector.

[0204] The term "promoter" or “regulatory sequence” as used herein refers to a DNA sequence recognized by the transcription machinery of the cell / host required to initiate the specific transcription of a polynucleotide sequence and / or required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0205] Examples of promoters / regulatory sequences suitable for the present invention are, without limitation human cytomegalovirus promoter (CMV) (Hosseini Rad et al, PLoS One, 2020, 15(7): e0232915), Chinese Hamster Elongation Factor-1a (CHEF-1) (Ebadat, et al, PLoS One, 2017, 12(10):e018596), Human elongation factor-1 alpha (EF- 1) (Hosseini Rad et al, PLoS One, 2020, 15(7): e0232915), Human Phosphoglycerate Kinase (hPGK) (Hosseini Rad et al, PLoS One, 2020, 15(7): e0232915), RPBSA (Hosseini Rad et al, PLoS One, 2020, 15(7): e0232915), MND promoter (WO2018085690), murine stem cell virus (MSCV) long terminal repeat (LTR) (Hughes, M. et al., Hum Gene Ther. 2005 Apr; 16(4): 457-472) and the NFAT-minimal IL2 promoter (Hoojberg et al., Blood, 2000 Jul; 96(2): 459-466.

[0206] In a particular embodiment of the vector of the invention or of the vector composition of the invention, the nucleic acid construct, the first polynucleotide and / or the second polynucleotide are under the operative control of one or more promoters, wherein the promoters can be identical or distinct. In a particular embodiment of the vector of the invention or of the vector composition of the invention, the nucleic acid construct, the first polynucleotide is under the operative control of the LTR promoter and the second polynucleotide is under the operative control of the NFAT-minimal IL2 promoter.

[0207] In another particular embodiment of the vector of the invention or of the vector composition of the invention, the nucleic acid construct, the first polynucleotide and / or the second polynucleotide are under operative control of the murine stem cell virus (MSCV) long terminal repeat.

[0208] The term "operably linked" or alternatively "transcriptional control" refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.

[0209] As noted above, in some embodiments, a nucleic acid comprising any of the CARs of the invention will in some embodiments be RNA, e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known in the art; any known method can be used to synthesize RNA comprising a nucleotide sequence encoding the first and / or the second polypeptide of a heterodimeric, conditionally active CAR of the present disclosure. Methods for introducing RNA into a host cell are known in the art. Introducing RNA comprising a nucleotide sequence encoding the first and / or the second polypeptide of a heterodimeric, conditionally active CAR of the present disclosure into a host cell can be carried out in vitro or ex vivo or in vivo. For example, a host cell (e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding the first and / or the second polypeptide of a heterodimeric, conditionally active CAR of the present disclosure.

[0210] In order to assess the expression of a CAR polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors; in other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic -resistance genes, such as neomycin and the like. Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene. Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.

[0211] All the terms and embodiments described in any of the previous aspects of the invention are equally applicable to the present aspects of the invention.

[0212] Methods for obtaining the cells of the invention

[0213] The present invention further provides methods that allow obtaining the cells of the invention which express a CAR and a glutamine transporter. Therefore, a fourth aspect of the present invention relates to an ex vivo method, hereinafter referred to as “the first method of the invention”, for obtaining a cell expressing

[0214] (i) a chimeric antigen receptor comprising: o an antigen binding domain specific for BCMA, o a transmembrane domain, and o at least one intracellular signaling domain and / or a costimulatory domain; and

[0215] (ii) a glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2 which comprises inserting into the cell the nucleic acid construct of the invention or the nucleic acid composition of the invention, or the vector of the invention or vector composition of the invention and selecting those cells which express the CAR on their surface and which express the glutamine transporter.

[0216] The terms and expressions “cell”, “CAR”, antigen binding domain”, “transmembrane domain”, “intracellular signaling domain”, costimulatory domain”, “glutamine transporter”, “nucleic acid” and “vector” and their particulars have been defined in the context of the previous aspects of the invention and apply equally to the first method of the invention. In particular embodiment of the first method of the invention, the CAR antigen binding domain specific for BCMA, the CAR transmembrane domain, the at least one CAR intracellular signaling domain and / or a CAR costimulatory domain, and the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, are as defined in any of the previous aspects and / or embodiments in the context of the cell of the invention.

[0217] In a particular embodiment of the first method of the invention, the cell is a mammalian cell.

[0218] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61 , CRL9096), 293 cells (e.g., ATCC No. CRL- 1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0219] In some instances, the cell is not an immortalized cell line, but is instead a cell (e.g., a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cell obtained from an individual.

[0220] The engineered cells may be obtained from peripheral blood, cord blood, bone marrow, tumor infiltrating lymphocytes, lymph node tissue, or thymus tissue. The cells may include placental cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells. The cells may be obtained from humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. The cells may be obtained from established cell lines.

[0221] The above cells may be obtained by any known means. The cells may be autologous, syngeneic, allogeneic, or xenogeneic to the recipient of the engineered cells. The term "autologous" refer to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0222] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenic ally.

[0223] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0224] The term "syngeneic" refers to an extremely close genetic similarity or identity especially with respect to antigens or immunological reactions. Syngeneic systems include for example, models in which organs and cells (e.g. cancer cells and their non- cancerous counterparts) come from the same individual, and / or models in which the organs and cells come from different individual animals that are of the same inbred strain.

[0225] In a particular embodiment of the first method of the invention, the cell is selected from a group consisting of an immune cell, a somatic cell and a progenitor cell. In a more particular embodiment of the first method of the invention, the cell is a cell from the immune system.

[0226] The immune cell can be obtained from a subject having or diagnosed as having cancer, a plasma cell disorder, or an autoimmune disease or disorder. For example, the immune cell can be obtained from a subject having a cancer, e.g., multiple myeloma, smoldering myeloma, or Waldenstrom’s macroglobulenemia. In some embodiments, the immune cell is obtained from a subject resistant to anti-BCMA therapy. Immune cells can also be obtained from allogeneic donors, which are non-genetically identical individuals of the same species as the intended recipients of the cells.

[0227] Immune cells (e.g., human immune cells) that can be used in the invention include autologous cells, obtained from the subject to whom the cells are later to be administered, after ex vivo modification and expansion. For example, the immune cells can be obtained from an individual having or diagnosed as having cancer, a plasma cell disorder, or autoimmune disease or disorder. Immune cells can also be obtained from allogeneic donors, which are non-genetically identical individuals of the same species as the intended recipients of the cells. Immune cells useful for the invention include T cells and NK cells.

[0228] In a particular embodiment of the method of the invention, the cell is an immune cell selected from a group consisting of a T cell, a B cell, a NK cell, a neutrophil, an eosinophil, a basophil, a monocyte, a macrophage, a dendritic cell and a mast cell, or a precursor of any of these cells.

[0229] In another particular embodiment of the method of the invention the immune cell is a T cell, a natural killer (NK) cell or a macrophage. In a more particular embodiment of the first method of the invention, the cell is a T cell or a natural killer (NK) cell, or a precursor thereof. In a more particular embodiment of the first method of the invention, the cell is a primary T cell.

[0230] In an embodiment, immune cells (e.g., human immune cells) that can be used in the invention include autologous cells, obtained from the subject to whom the cells are later to be administered, after ex vivo modification and expansion. For example, the immune cells can be obtained from an individual having or diagnosed as having cancer. Immune cells can also be obtained from allogeneic donors, which are non-genetically identical individuals of the same species as the intended recipients of the cells. Immune cells useful for the invention include T, NK and NKT cells.

[0231] Methods for obtaining T, NK and NKT cells are known in the art and can be useful for the engineered immune cells described herein. T, NK and NKT cells are typically obtained from peripheral blood that is collected from a subject by, e.g., venipuncture or withdrawal through an implanted port or catheter. Optionally, the blood can be obtained by a process including leukapheresis, in which white cells are obtained from the blood of a subject, while other blood components are returned to the subject. Blood or leukapheresis product (fresh or cryopreserved) is processed to enrich for T, NK or NKT cells using methods known in the art. For example, density gradient centrifugation (using, e.g., Ficoll) and / or counter-flow centrifugal elutriation can be carried out to enrich for mononuclear cells (including T, NK or NKT cells). In one example, for T cells, a T cell stimulation step employing, e.g., CD3 / CD28 antibodies coated on magnetic beads or artificial antigen presenting cells (aAPCs) expressing, e.g., cell surface-bound anti-CD3 and anti-CD28 antibody fragments (see below), can further be carried out in order to stimulate T cells and to deplete other cells, e.g., B cells. The T cells of enriched T cell preparations can then be subject to genetic modification.

[0232] As an alternative to peripheral blood, tissues including bone marrow, lymph nodes, spleen, and tumors can be used as a source for T cells and NK cells. The T cells and NK cells can be of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog, or cat origin, but any other mammalian cell may be used. In certain embodiments of any aspect, the T or NK cell is human.

[0233] Immune cells such as T, NK or NKT cells can be obtained from a number of sources peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Any number of cell lines (e.g. immune cell lines such as T cell lines) available in the art, may also be used. In an embodiment, immune cells (e.g. T, NK or NKT cells) are obtained from a unit of blood collected from a subject using any suitable techniques known in the art such as Ficoll™ separation. In another embodiment, cells from the circulating blood of a subject are obtained by apheresis. The apheresis product typically contains lymphocytes, including T, NK or NKT cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. It will be appreciated that the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. For example, the cells may be washed with phosphate buffered saline (PBS). Alternatively, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation. A washing step may be accomplished by methods known to those in the art, such as by using a semi- automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[0234] In an embodiment, immune cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient or by counter-flow centrifugal elutriation. Specific subpopulations of immune cells, like T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+T cells, may be further isolated by positive or negative selection techniques known in the art. For example, T cells may be isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a time period sufficient for positive selection of the desired T cells. Additionally or alternatively, a population of T cells may be enriched by negative selection, for instance by a combination of antibodies directed to surface markers unique to the negatively selected cells. Cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry may be used.

[0235] It will be understood that cells derived from subjects that are to be modified to express the CAR and the glutamine transporter expressed by the cell of the invention may be stored for a period of time prior to their use (see, for example, therapeutic methods below). For example, the cells may be frozen, optionally after they have been washed, or they may be incubated under suitable conditions for them to remain viable until needed (e.g. on a rotator at 2-10°C or at room temperature). In this way, the cells can be stored until such time as they might be needed. They may be stored in an unmodified state (i.e. wherein they do not express the CAR and / or the glutamine transporter) or in a modified state (i.e. wherein they have been modified to express the CAR and / or the glutamine transporter). Prior to use in the therapeutic applications described further below, the cells may be activated and expanded generally using methods known in the art. For example, T cells may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (eg bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art.

[0236] T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apherised peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.

[0237] In a particular embodiment, the immune cell is a T cell or an NK cell. In a more particular embodiment the T cell is a CD8+T cell.

[0238] Particularly, the host cells of the invention may be expanded prior to transduction with the vector of the invention.

[0239] It is worth noting that after some cancer treatments, in particular, treatments with drugs that damage the immune system, shortly after treatment during the period of time when patients should normally recover from treatment, the quality of the obtained immune cells may be optimal or improved in relation to their ability to reproduce ex vivo. Also, after ex vivo manipulation using the methods described herein, these cells may be in a preferred condition for enhanced engraftment and in vivo propagation. Thus, in connection with the present invention provides for the production of blood cells, including T cells, dendritic cells or other cells of the hematopoietic line, during this phase of recovery. In addition, in some aspects, mobilization modes (e.g., mobilization using GM- CSF) and the establishment of a specific condition can be used to create a condition in a subject in which repopulation, recirculation, regeneration and I or reproduction of specific cell types is advantageous, especially in time of a certain time window after therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0240] The engineered cells of the present disclosure may also include a suicide system. Suicide systems provide a mechanism whereby the engineered cell, as described above, may be deactivated or destroyed. Such a feature allows precise therapeutic control of any treatments wherein the engineered cells are used. As used herein, a suicide system provides a mechanism by which the cell having the suicide system can be deactivated or destroyed. Suicide systems are well known in the art.

[0241] The expression “inserting into the cell a nucleic acid construct of the invention or a nucleic acid composition of the invention or a vector of the invention or vector composition of the invention” as used herein refers the introduction of nucleic acids or vectors into the cells of interest in a way that said nucleic acids or vectors will produce or express the nucleic acids of interest.

[0242] Methods of introducing and expressing genes into a cell are known in the art. In the context of one or more expression vectors, the vectors can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0243] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0244] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like.

[0245] Chemical means for introducing a polynucleotide into a host cell include transposons (such as nanoparticle-based transposons) and colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0246] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyi phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyi phosphatidylglycerol ("DM PG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. "Liposome" is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self - rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as non-uniform aggregates of lipid molecules. Also contemplated are lipofectamine- nucleic acid complexes.

[0247] In a particular embodiment, the first method of the invention further comprises a step of selecting the cells which express the CAR on their surface and which express the glutamine transporter.

[0248] Regardless of the method used to introduce exogenous polynucleotides into a host cell or otherwise expose a cell to the polynucleotide of the present disclosure, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed, which will allow for the selection of those cells expressing the CAR on their surface and expressing the glutamine transporter. Such assays include, for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure. It will be obvious to the expert in the art that in order to select the cells expressing both the CAR and the glutamine transporter, two or more techniques of the exemplified above may be used in order to obtain confirmation of the expression of both polypeptides of interest.

[0249] In another aspect, the invention relates to a cell obtained by the methods according to the invention. Therefore, a fifth aspect of the present invention relates to a cell obtainable by the first method of the invention, hereinafter referred to as “the cell obtainable by the first method of the invention”.

[0250] All the terms and embodiments described in any of the previous aspects of the invention are equally applicable to the present aspects of the invention.

[0251] Pharmaceutical compositions of the invention A sixth aspect of the present invention relates to a pharmaceutical composition, hereinafter referred to as “the pharmaceutical composition of the invention”, comprising the cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, or the vector of the invention or the vector composition of the invention, and at least one pharmaceutically acceptable excipient and / or vehicle.

[0252] The terms and expressions “cell”, “nucleic acid” and “vector” and their particulars have been defined in the context of the previous aspects of the invention and apply equally to the cell obtainable by the first method of the invention and to the pharmaceutical composition of the invention.

[0253] The term “pharmaceutical composition” is such a form that allows the biological activity of the active ingredient contained therein to be effective and has unacceptable toxicity for the subject to which the composition is administered. Refers to a preparation that does not contain additional ingredients.

[0254] The term “pharmaceutically acceptable excipient and / or vehicle”, as used herein, refers to an ingredient of a pharmaceutical composition other than an active ingredient that is non-toxic to a subject. Pharmaceutically acceptable excipients and / or vehicles include but are not limited to buffers, excipients, stabilizers or preservatives.

[0255] In a particular embodiment, the pharmaceutical composition of the invention comprises the cells of the invention, more particularly, the immune cells (e.g. T, NK or NKT cells) that have been genetically engineered to express the CAR and the glutamine transporter of the cell invention. In another embodiment, the pharmaceutical composition of the invention comprises the cell obtainable by the first method of the invention. In another embodiment, the pharmaceutical composition of the invention comprises the nucleic acid construct of the invention or the nucleic acid composition of the invention. In another embodiment, the pharmaceutical composition of the invention comprises the vector of the invention or the vector composition of the invention.

[0256] Pharmaceutical compositions and formulations as described herein can be prepared by mixing the cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, or the vector of the invention or the vector composition of the invention having the desired degree of purity with one or more optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 22nd edition, 2012), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers, excipients or stabilizers are generally nontoxic to recipients at the dosages and concentrations employed. The choice of the carrier, excipient or stabilizer is determined in part by the particular cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, or the vector of the invention or the vector composition of the invention. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention.

[0257] Pharmaceutical compositions comprising the cells of the invention

[0258] Pharmaceutical compositions and formulations as described herein comprising the cell of the invention or the cell obtainable by the first method of the invention can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

[0259] Sterile injectable solutions can be prepared by incorporating the pharmaceutical compositions of the invention of the presently disclosed subject matter, e.g., a pharmaceutical composition comprising the cells of the invention or the cells obtainable by the first method of the invention, in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The pharmaceutical compositions can also be lyophilized. The pharmaceutical compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “Remington's Pharmaceutical Sciences 22nd edition, 2012”, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation. Various additives which enhance the stability and sterility of the pharmaceutical compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the presently disclosed subject matter, however, any vehicle, diluent, or additive used would have to be compatible with the cells of the invention or the cells obtainable by the first method of the invention.

[0260] The pharmaceutical compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of the presently disclosed subject matter may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is suitable particularly for buffers containing sodium ions.

[0261] The viscosity of the pharmaceutical compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose can be used because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The concentration of the thickener can depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. The choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid- filled form).

[0262] Those skilled in the art will recognize that the components of the pharmaceutical compositions should be selected to be chemically inert and will not affect the viability or efficacy of the cells of the invention or the cells obtainable by the first method of the invention. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

[0263] The skilled artisan can readily determine the amount of cells and optional additives, vehicles, and / or carriers in the pharmaceutical compositions to be administered to the subject in need. Typically, any additives (in addition to the active cell(s)) are present in an amount of from about 0.001 % to about 50 % by weight solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as from about 0.0001 wt % to about 5 wt %, from about 0.0001 wt % to about 1 wt %, from about 0.0001 wt % to about 0.05 wt %, from about 0.001 wt % to about 20 wt %, from about 0.01 wt % to about 10 wt %, or from about 0.05 wt % to about 5 wt %.

[0264] Pharmaceutical compositions comprising the nucleic acids, compositions of nucleic acids and vectors of the invention

[0265] Pharmaceutical compositions and formulations as described herein comprising the nucleic acid construct of the invention or the nucleic acid composition of the invention, or the vector of the invention or the vector composition of the invention may include, for example, pharmaceutically acceptable excipients include, but are not limited to, dimethylsulfoxide or dextran-40. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. A mixture of two or more preservatives optionally may be used. The preservative or mixtures thereof are typically present in an amount of about 0.0001 % to about 2% by weight of the total composition.

[0266] Pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0267] In addition, buffering agents may be used in the composition. Suitable buffering agents include, for example, but are not limited to, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. A mixture of two or more buffering agents optionally may be used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001 % to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1 , 2005).

[0268] The pharmaceutical composition of the invention may be formulated in, but not limited to, macromolecule complexes, microspheres, microcapsules, beads, in colloidal drug delivery systems (for example, albumin microspheres, microemulsions, nanoparticles and nanocapsules), in macroemulsions, in lipid-based systems including, but not limited to, oil-in-water emulsions, micelles, mixed micelles, and liposomes, or transposons (for example nanoparticle-based transposons). In some embodiments, the pharmaceutical composition of the invention can be formulated as an inclusion complex, such as cyclodextrin inclusion complex, or as a liposome. Liposomes can serve to target the cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, or the vector of the invention or the vector composition of the invention to a particular tissue. Liposomes also can be used to increase the half-life of the cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, or the vector of the invention or the vector composition of the invention. Many methods are available for preparing liposomes, such as those described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and U.S. Patents 4,235,871 ; 4,501 ,728; 4,837,028; and 5,019,369.

[0269] The pharmaceutical composition of the invention can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the pharmaceutical composition of the invention occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Many types of release delivery systems are available and known to those of ordinary skill in the art. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.

[0270] All the terms and embodiments described in any of the previous aspects of the invention are equally applicable to the present aspects of the invention.

[0271] Medical uses of the invention The cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, the vector of the invention or the vector composition of the invention, or the pharmaceutical composition of the invention finds uses in medicine, specifically in the treatment of cancer.

[0272] Therefore, a seventh aspect of the present invention, hereinafter referred to as “the first medical use of the invention”, relates to the cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, the vector of the invention or the vector composition of the invention, or the pharmaceutical composition of the invention, for use in medicine.

[0273] Alternatively, the seventh aspect of the invention relates to the use of the cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, the vector of the invention or the vector composition of the invention, or the pharmaceutical composition of the invention, in the manufacture of a medicament.

[0274] The term “medicament” as used herein refers to a composition comprising a therapeutically effective amount of an agent, preferably an anti-cancer agent. The medicament also comprises at least one pharmaceutically acceptable excipient or carrier.

[0275] An eighth aspect of the present invention, hereinafter referred to as “the second medical use of the invention”, relates to the cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, the vector of the invention or the vector composition of the invention, or the pharmaceutical composition of the invention, for use in the prevention and / or treatment of cancer or an autoimmune disease.

[0276] Alternatively, the eighth aspect of the invention relates to the use of the cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, the vector of the invention or the vector composition of the invention, or the pharmaceutical composition of the invention, in the manufacture of a medicament for the prevention and / or treatment of cancer or an autoimmune disease. Alternatively, this aspect may be reformulated as a method of prevention and / or treatment of cancer or an autoimmune disease, comprising administering the cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, the vector of the invention or the vector composition of the invention, or the pharmaceutical composition of the invention, to a subject in need thereof.

[0277] The term "subject" or "patient", as used herein, refers to all animals classified as mammals and includes but is not limited to domestic and farm animals, primates and humans, for example, human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, the subject is a human man or woman of any age or race.

[0278] As used herein, the terms "prevent", "prevention" and "preventing" refer to the reduction in the risk of acquiring or developing a given disease or disorder, e.g., cancer, or the reduction or inhibition of the recurrence or a disease or disorder, e.g., cancer.

[0279] As used herein, the terms "treat", "treatment", or "amelioration" refer to therapeutic treatment, the purpose of which is to reverse, reduce, suppress, delay or stop the progression or severity of the condition associated with the disease or disorder. The term "treatment" includes reducing or alleviating at least one adverse effect or condition of a condition, such as cancer, a disease or disorder. Treatment is usually "effective" when one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if disease progression is delayed or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the interruption of at least a condition that indicates the progression or worsening of symptoms that would be expected in the absence of treatment. The beneficial or desirable clinical outcome, whether detectable or not, is a reduction in one or more symptoms, a reduction in the extent of the disease, a stable (i.e., not aggravated) condition of the disease, a disease These include, but are not limited to, delayed or slowed progression, amelioration or alleviation of the disease state, and remission (partial or total). The term "treatment" of a disease also includes providing relief from symptoms or side effects of the disease (including symptomatic treatment). In some embodiments, treating cancer includes reducing tumor volume, reducing the number of cancer cells, suppressing cancer metastasis, prolonging life, reducing cancer cell growth, reducing cell survival, or reducing cancerous status. It involves amelioration of the various physiological symptoms involved.

[0280] In certain embodiments of the present disclosure, the cells, i.e., immune cells, are delivered to an individual in need thereof, such as an individual that has cancer. The cells then enhance the individual's immune system to attack the respective cancer cells. In some cases, the individual is provided with one or more doses of the cells. In cases where the individual is provided with two or more doses of the cells, the duration between the administrations should be sufficient to allow time for propagation in the individual, and in specific embodiments the duration between doses is 1 , 2, 3, 4, 5, 6, 7, or more days.

[0281] In certain embodiments, a growth factor that promotes the growth and activation of the cells is administered to the subject either concomitantly with the cells or subsequently to the cells. The cell growth factor can be any suitable growth factor that promotes the growth and activation of the cells. Examples of suitable cell growth factors include interleukin (IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL-2.

[0282] Therapeutically effective amounts of the cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, the vector of the invention or the vector composition of the invention, or the pharmaceutical composition of the invention can be administered by a number of routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal, or intraarticular injection, or infusion.

[0283] The cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, the vector of the invention or the vector composition of the invention, or the pharmaceutical composition of the invention can be administered in treatment regimens consistent with the disease, for example a single or a few doses over one to several days to ameliorate a disease state or periodic doses over an extended time to inhibit disease progression and prevent disease recurrence. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. The therapeutically effective number of cells will be dependent on the subject being treated, the severity and type of the affliction, and the manner of administration. In some embodiments, a therapeutically effective number of the cell of the invention or the cell obtainable by the first method of the invention administered can vary from about 5 x 106cells per kg body weight to about 7.5 x 108cells per kg body weight, such as about 2 x 107cells to about 5 x 108cells per kg body weight, or about 5 x 107cells to about 2 x 108cells per kg body weight. In certain embodiments, a therapeutically effective amount of the nucleic acid construct of the invention, the nucleic acid composition of the invention, the vector of the invention, the vector composition of the invention, or the pharmaceutical composition of the invention are administered at a dose resulting in a number of cells that can vary from about 5 x 106cells per kg body weight to about 7.5 x 108cells per kg body weight, such as about 2 x 107cells to about 5 x 108cells per kg body weight, or about 5 x 107cells to about 2 x 108cells per kg body weight.

[0284] The exact number of cells is readily determined by one of skill in the art based on the age, weight, sex, and physiological condition of the subject. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0285] The term "cancer" or “carcinoma” or "tumor" or "tumor disease", as used herein, refers to a broad group of diseases involving unregulated cell growth and which are also referred to as malignant neoplasms. The term is usually applied to a disease characterized by uncontrolled cell division (or by an increase of survival or apoptosis resistance) and by the ability of said cells to invade other neighboring tissues (invasion) and spread to other areas of the body where the cells are not normally located (metastasis) through the lymphatic and blood vessels, circulate through the bloodstream, and then invade normal tissues elsewhere in the body. Depending on whether or not they can spread by invasion and metastasis, tumors are classified as being either benign or malignant: benign tumors are tumors that cannot spread by invasion or metastasis, i.e., they only grow locally; whereas malignant tumors are tumors that are capable of spreading by invasion and metastasis. Biological processes known to be related to cancer include angiogenesis, immune cell infiltration, cell migration and metastasis. Cancers usually share some of the following characteristics: sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and eventually metastasis. Cancers invade nearby parts of the body and may also spread to more distant parts of the body through the lymphatic system or bloodstream. Cancers are classified by the type of cell that the tumor cells resemble, which is therefore presumed to be the origin of the tumor.

[0286] Examples of cancer or tumor include without limitation, breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovarian, prostate, brain, rectum, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicles, hepatobiliary and liver tumors. In particular, the tumor / cancer can be selected from the group of adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, hepatoblastoma, leukaemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, hepatobiliary cancer, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, teratoma, acrallentiginous melanoma, actinic keratosis adenocarcinoma, adenoid cystic carcinoma, adenosarcoma, adenosquamous carcinoma, astrocytictumors, bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, carcinosarcoma, cholangiocarcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Swing's sarcoma, focal nodular hyperplasia, germ cell tumors, glucagonoma, hemangioblastoma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, insulinoma, intraepithelial neoplasia, interepithelial squamous cell neoplasia, invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma, malignant melanoma, malignant mesothelialtumor, medulloepithelioma, mucoepidermoid carcinoma, neuroepithelial adenocarcinoma, nodular melanoma, papillary serous adenocarcinoma, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma, serous carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vipoma, Wilm's tumor.

[0287] In a particular embodiment, the cancer is a BCMA positive cancer.

[0288] The term “BCMA” has already been defined within the context of the CARs expressed by the cell of the invention and said definition applies equally to the present uses of the invention.

[0289] A “cancer that is BCMA positive” refers to a cancer in which at least a portion of the cancer cells contain BCMA, as determined by immunohistochemistry (IHC), Western blot, VeraTag® assay (Monogram Biosciences), or flow cytometry.

[0290] In a more particular embodiment, the cancer is selected from the group consisting of multiple myeloma (MM), plasma cell leukemia (PCL), Waldenstrom's macroglobulinemia (WM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), Hodgkin’s lymphoma (HL), primary central nervous system lymphoma (PCNSL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), breast cancer, lung cancer and melanoma.

[0291] As used herein, the term “multiple myeloma (MM)” also known as plasma cell myeloma, is a cancer of plasma cells, a type of white blood cell which normally produces antibodies. Often, no symptoms are noticed initially. When advanced, bone pain, bleeding, frequent infections, and anemia may occur. Complications may include amyloidosis.

[0292] The term “plasma cell leukemia (PCL)”, as used herein, refers to an aggressive hematological malignancy characterized by an uncontrolled clonal proliferation of plasma cells (PCs) in the bone marrow and peripheral blood.

[0293] The term “Waldenstrom's macroglobulinemia (WM)” or “Waldenstrom macroglobulinemia”, as used herein, refers to also known as lymphoplasmacytic lymphoma, is cancer involving a subtype of white blood cells called lymphocytes. It is characterized by an uncontrolled clonal proliferation of terminally differentiated B lymphocytes. It is also characterized by the lymphoma cells making an antibody called immunoglobulin M (IgM).

[0294] The term “chronic lymphocytic leukemia (CLL)”, as used herein, refers to a chronic lymphoproliferative disorder characterized by monoclonal B cell proliferation.

[0295] The term “diffuse large B-cell lymphoma (DLBCL)”, as used herein, refers to a neoplasm of the germinal center B lymphocytes with a diffuse growth pattern and a high intermediate proliferation index. DLBCL represents approximately 30 percent of all lymphomas.

[0296] The term “mantle cell lymphoma (MCL)”, as used herein, refers to a lymphoma characterized as aggressive, usually diffuse non-Hodgkin lymphoma composed of small to medium sized B-lymphocytes (centrocytes). Most patients present with advanced stage disease with lymphadenopathy, hepatosplenomegaly, and bone marrow involvement. The diagnosis and determination of mantle cell lymphoma is readily determined by one of skill in the art, e.g., in accordance with the current accepted guidelines. For example, guidelines set forth by the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) are widely accepted. Markers for mantle cell lymphoma include surface markers of B cells (e.g. CD20); overexpression of cyclin DI; and (11 ;14) translocation.

[0297] The term “follicular lymphoma (FL)”, as used herein, refers to any of several types of non-Hodgkin's lymphoma in which the lymphomatous cells are clustered into nodules or follicles. The term "follicular" is used because the cells tend to grow in a circular, or nodular, pattern in lymph nodes.

[0298] The term “marginal zone lymphoma (MZL)” or “marginal zone B-cell lymphomas”, as used herein, refers to a group of indolent type of B-cell non-Hodgkin lymphoma that begins forming in certain areas (the marginal zones) of lymph tissue. There are three types based on whether it forms in the spleen, lymph nodes, or other lymphoid tissue that contains a lot of B cells (a type of white blood cell).

[0299] The term “Hodgkin lymphoma (HL)” or “Hodgkin’s lymphoma” or “Hodgkin disease”, as used herein, refers to a type of blood cancer that affects the lymphatic system.

[0300] The term “primary central nervous system lymphoma (PCNSL)”, as used herein, refers to a diffuse large B cell lymphoma in which the brain, spinal cord, leptomeninges and / or eyes are exclusive sites of disease.

[0301] The term “acute myeloid leukemia (AML)” or “acute myelogenous leukemia”, as used herein, refers to a cancer of the myeloid line of blood cells, characterized by the rapid growth of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells.

[0302] The term “myelodysplastic syndrome (MDS)”, as used herein, refers to a group of blood disorders in which the bone marrow stops functioning normally, resulting in a deficiency in the number of healthy blood cells.

[0303] The term “breast cancer”, as used herein, refers to a malignant neoplasm of the breast or pectoral area characterized by the abnormal proliferation of cells, the growth of which cells exceeds and is uncoordinated with that of the normal tissues around it.

[0304] The term “lung cancer” relates to the cancer arising from the cells of the respiratory epithelium, and can be divided into two broad categories. The term lung cancer, as used herein, refers to small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC), including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. As used herein, the term lung cancer refers to stage IA, stage IB, stage HA, stage I IB, stage I HA stage 11 IB or stage IV lung cancer. Moreover, as used herein, lung cancer refers both to primary lung tumors as well as to secondary lung cancer, i.e. lung cancer which results from the metastasis from a primary cancer elsewhere in the body.

[0305] The term “melanoma”, as used herein, refers to a malignant skin tumor of melanocytes and includes, but is not limited to, melanomas, metastatic melanomas, melanomas derived from either melanocytes or melanocyte related nevus cells, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma in situ, superficial spreading melanoma, modular melanoma, lentigo malignant melanoma, acral lentiginous melanoma, invasive melanoma and familial atypical mole and melanoma (FAM-M) syndrome. Moreover, the term “melanoma” refers not only to primary melanomas but also to "melanoma metastasis" which, as used herein, refers to the spread of melanoma cells to regional lymph nodes and / or distant organs. This event is frequent, given that melanomas contain multiple cell populations characterized by diverse growth rates, karyotypes, cell-surface properties, antigenicity, immunogenicity, invasion, metastasis, and sensitivity to cytotoxic drugs or biologic agents. Melanoma shows frequent metastasis to brain, lungs, lymph nodes, and skin.

[0306] In a more preferred embodiment, the cancer is multiple myeloma.

[0307] The term "autoimmune disease" or “autoimmune disorder”, as used herein, refers to a condition in a subject characterized by cellular, tissue and / or organ injury caused by an immunological reaction of the subject to its own cells, tissues and / or organs. Illustrative, non-limiting examples of autoimmune diseases include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia- fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, sarcoidosis, scleroderma, progressive systemic sclerosis, Sjogren's syndrome, Good pasture's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteristis / giant cell arteritis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, Wegener's granulomatosis, Anti-Glomerular Basement Membrane Disease, Antiphospholipid Syndrome, Autoimmune Diseases of the Nervous System, Familial Mediterranean Fever, Lambert-Eaton Myasthenic Syndrome, Sympathetic Ophthalmia, polyendocrinopathies, psoriasis, etc. In a particular embodiment, the autoimmune disease is as lupus, sarcoidosis, chronic obstructive pulmonary disease, asthma, primary biliary cholangitis, sclerosing cholangitis, autoimmune hepatitis, inflammatory bowel disease, Crohn’s disease, multiple sclerosis, ulcerative colitis, encephalomyelitis, rheumatoid arthritis, atopic dermatitis, multiple sclerosis, type 1 diabetes, Behcet's disease, Sjogren's syndrome, myasthenia gravis, scleroderma, polyarteritis nodosa, Kikuchi disease, collagen disease, Hashimoko thyroiditis, psoriasis, vitiligo, hyperthyroidism, uveitis, fibromyalgia, alopecia areata, or allergy.

[0308] In a particular embodiment, the autoimmune disease responds to plasma cell depletion.

[0309] An “autoimmune disease that responds to plasma cell depletion” refers to a disease which responds to the depletion or elimination of plasma cells producing antibodies, wherein the plasma cells depleted express BCMA, as determined by immunohistochemistry (IHC), Western blot, VeraTag® assay (Monogram Biosciences), or flow cytometry.

[0310] In an embodiment, the autoimmune disease is selected from the group consisting of lupus erythematosus, an autoimmune disease of the nervous system, neuromyelitis optica spectrum disorder, myasthenia gravis, chronic inflammatory demyelinating polyradiculoneuropathy, immune-mediated necrotizing myopathy, idiopathic inflammatory myopathy, multiple sclerosis, autoimmune encephalitis, myelin oligodendrocyte glycoprotein antibody-associated disease and POEMS syndrome.

[0311] The term “lupus erythematosus”, as used herein, refers to a name given to a collection of autoimmune diseases that have common symptoms that affect joints, skin, kidneys, blood cells, heart and lungs. Lupus erythematosus may manifest as systemic disease or in a purely cutaneous form also known as incomplete lupus erythematosus. Lupus has four main types: systemic, discoid, drug-induced and neonatal. The term "lupus erythematosus" in the context of the present invention encompasses, without limitation, acute cutaneous lupus erythematosus, subacute cutaneous lupus erythematosus, discoid lupus erythematosus (chronic cutaneous), childhood discoid lupus erythematosus, generalized discoid lupus erythematosus, localized discoid lupus erythematosus, chilblain lupus erythematosus (Hutchinson), lupus erythematosus-lichen planus overlap syndrome, lupus erythematosus panniculitis (lupus erythematosus profundus), tumid lupus erythematosus, verrucous lupus erythematosus (hypertrophic lupus erythematosus), cutaneous lupus mucinosis, complement deficiency syndromes, drug-induced lupus erythematosus, neonatal lupus erythematosus and systemic lupus erythematosus. The most common severe form is systemic lupus erythematosus.

[0312] The term "autoimmune disease of the nervous system", as used herein, includes any disease in which nervous tissue or a component thereof comes under autoimmune attack. The term includes, for example central nervous system diseases having an autoimmune etiology such as multiple sclerosis (MS), perivenous encephalomyelitis, autoimmune myelopathies, paraneoplastic cerebellar degeneration, paraneoplastic limbic (cortical) degeneration, stiff man syndrome, choreas (such as Sydenham's chorea), stroke, focal epilepsy and migraine; and peripheral nervous system diseases having an autoimmune etiology such as Guillain-Barre syndrome, Miller Fisher syndrome, chronic inflammatory demyelinating neuropathy, multifocal motor neuropathy with conduction block, demyelinating neuropathy associated with anti-myelin-associated glycoprotein antibodies, paraneoplastyic sensory neuropathy, POEMS, dorsal root ganglion neuronitis, acute panautonomic neuropathy and brachial neutritis.

[0313] The term “neuromyelitis optica spectrum disorder (NMOSD)”, also known as “Devic disease”, as used herein, refers to an autoinmune disease characterized by a chronic disorder of the brain and spinal cord dominated by inflammation of the optic nerve (optic neuritis) and inflammation of the spinal cord (myelitis).

[0314] The term "myasthenia gravis (MG)", as used herein, refers to an autoimmune disease characterized by autoantibodies directed against epitopes of the post-synpatic muscle membrane, including the nicotinic acetylcholine receptor (AChR) and the musclespecific tyrosine kinase receptor (MuSK), and complement-mediated destruction of the post junctional membrane. Around 10-20 percent of MG patients do not have AChR antibodies (seronegative), of whom some have antibodies to a membrane-linked MuSK. Clinical manifestations include fluctuating weakness of ocular, bulbar, respiratory and limb muscles.

[0315] The term “chronic inflammatory demyelinating polyneuropathy (Cl DP)”, as used herein, refers to a peripheral nerve disease which features chronic progressive, chronic stepped or recurrent bilateral muscle weakness and sensation disturbance over 2 months or more in distal or proximal muscles of extremities as cardinal symptoms. The pathogenesis of Cl DP is thought to be an autoimmune disease caused by immune abnormality to the constituent components of peripheral nerve myelin, details of which are not yet known.

[0316] The term “immune-mediated necrotizing myopathy (IMNM)”, as used herein, refers to a type of autoimmune myopathy characterized by relatively severe proximal weakness, myofiber necrosis with minimal inflammatory cell infiltrate on muscle biopsy, and infrequent extra-muscular involvement.

[0317] The term “idiopathic inflammatory myopathy (I IM)”, as used herein, refers to a group of diseases, more particular a group of myopathies that have an unknown cause and are hence termed idiopathic diseases. Inflammatory myopathies are characterized by weakness and inflammation of muscles. Muscle pain has also been reported as disease feature in certain types. Inflammatory myopathies are typically diagnosed based on a combination of symptoms, parameters, electromyography, magnetic resonance imaging (MRI), and laboratory tests. A large number of myopathies have known causes, and the diagnosis of idiopathic myopathy fora subject can therefore only established once known causes have been ruled out.

[0318] The term “multiple sclerosis (MS)” or “encephalomyelitis disseminata”, as used herein, refers to an inflammatory demyelinating disease of the central nervous system (CNS). MS is characterized by destruction of myelin, associated with death of oligodendrocytes and axonal loss. The main pathologic finding in MS is the presence of infiltrating mononuclear cells, predominantly T lymphocytes and macrophages, which surpass the blood brain barrier (BBB) and induce an active inflammation within the brain and spinal cord. The neurological symptoms that characterize MS include, but are not limited to, complete or partial vision loss, diplopia, sensory symptoms, motor weakness that can worsen to complete paralysis, bladder dysfunction and cognitive deficits, which eventually may lead to a significant disability. The associated multiple inflammatory foci can lead to myelin destruction, plaques of demyelination, gliosis and axonal loss within the brain and spinal cord. These are the reasons which contribute to the clinical manifestations of neurological disability. There are four main varieties of multiple sclerosis: relapsing-remitting multiple sclerosis (RR-MS), secondary progressive multiple sclerosis (SP-MS), progressive relapsing multiple sclerosis (PR-MS), and primary progressive multiple sclerosis (PP-MS).

[0319] The term “autoimmune encephalitis (AE)”, as used herein, refers to acute to subacute, progressive inflammation of the brain associated with antibodies against neuronal cell surface and synaptic protein, most commonly being anti-N-methyl-D- aspartate receptor encephalitis.

[0320] The term “myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD)”, as used herein, refers to an inflammatory disorder of the central nervous system characterized by attacks of immune-mediated demyelination predominantly targeting the optic nerves, brain, and spinal cord. The disease has a predilection for children.

[0321] The term “POEMS syndrome”, as used herein, refers to a rare, multisystem disorder. POEMS stands for the disorder's features, which may include Polyneuropathy, Organomegaly, Endocrinopathy, Monoclonal gammopathy, and Skin changes. Signs and symptoms may include progressive sensorimotor polyneuropathy; enlarged liver, spleen, and / or lymph nodes; a disorder of the endocrine glands (often with multiple abnormalities); a monoclonal plasma cell proliferative disorder; and darkening of the skin (hyperpigmentation). Various other symptoms may also occur and may vary among affected people. The underlying cause of the disorder is not well understood.

[0322] In the context of the first and second medical uses of the invention, when referring to the use in medicine or in the prevention and / or treatment of cancer of the nucleic acid construct of the invention or the nucleic acid composition of the invention, or the vector of the invention or the vector composition of the invention, it will be understood that the nucleic acid construct of the invention or the nucleic acid composition of the invention act indirectly after being administered to a patient in need thereof through the in vivo generation of T cells that express the proteins encoded by the nucleic acid construct of the invention or the nucleic acid composition of the invention. This results in the generation in the patient of the cells expressing the anti-BCMA CAR and the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, which are then exerting its function.

[0323] In an embodiment of the second medical use of the invention, the cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, the vector of the invention or the vector composition of the invention, or the pharmaceutical composition of the invention is for use in a personalized therapy for a subject suffering from cancer, wherein said subject has increased glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, expression levels in a tumor sample obtained from said subject with respect to a reference value. In a preferred embodiment, the glutamine transporter is ASCT2.

[0324] The term “personalized therapy”, as used herein, refers to appropriate and optimal therapies based on a subject’s specific medical characteristics, resulting from its personal background. In the context of the present invention, said characteristic consists of the level of the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2 in a tumor sample isolated from the subject. In a preferred embodiment, the glutamine transporter is ASCT2.

[0325] The terms “subject”, “cancer”, “glutamine transporter” and “ASCT2” have already been defined above, within the context of the cell of the invention and the second medical use of the invention, and apply equally to the present embodiment.

[0326] The term “sample”, as used herein, relates to any sample which can be obtained from the patient and which contains any biological material suitable for detecting DNA, RNA or protein levels. In a particular embodiment, the sample contains genetic material, e.g., DNA, genomic DNA (gDNA), complementary DNA (cDNA), RNA, heterogeneous nuclear RNA (hnRNA), mRNA, etc., from the patient under study. In another particular embodiment the sample contains proteins. The sample can comprise cell and / or noncell material of the patient. The present method can be applied to any kind of biological sample from a patient, such as a biopsy sample, tissue, cell or biological fluid or biofluid (blood, plasma, serum, saliva, urine, semen, sputum, cerebral spinal fluid (CSF), tears, mucus, sweat, milk), feces, brain extracts, bone marrow, nipple aspirate, samples obtained by bronchial lavage, bronchoscopy, fine needle aspiration biopsy (FNAB), solid tumor biopsy sample, a buccal or buccal pharyngeal swab and the like. Said sample can be obtained by conventional methods, e.g., biopsy, surgical excision or aspiration, by using methods well known to those of ordinary skill in the related medical arts. Methods for obtaining the sample from the biopsy include gross apportioning of a mass, or microdissection or other art-known cell-separation methods. Tumor cells can additionally be obtained from fine needle aspiration cytology. In order to simplify conservation and handling of the samples, these can be formalin-fixed and paraffin-embedded or first frozen and then embedded in a cryosolidifiable medium, such as OCT-Compound, through immersion in a highly cryogenic medium that allows rapid freeze. The samples can also be a cell suspension, cell pellet, cell slide, frozen solid tumor biopsy.

[0327] In a particular embodiment, the sample is a tissue sample, preferably a biopsy of a tumor tissue sample, either fresh or frozen.

[0328] In a particular embodiment, said sample comprises cancer cells, preferably breast cancer, ovarian cancer, prostate cancer, gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, stomach / gastric cancer, endometrial / uterine / cervical cancer, bladder cancer, head and neck cancer, leukemia, sarcoma, cholangiocarcinoma, glioblastoma, multiple myeloma, lymphoma cells. In a preferred embodiment, the sample comprises multiple myeloma cancer cells. In a preferred embodiment it is a tumor tissue sample or portion thereof. Preferably, said tumor tissue sample is a breast tumor, ovarian tumor, prostate tumor, gastric tumor, pancreatic tumor, lung tumor, colorectal tumor, stomach / gastric tumor, endometrial / uterine / cervical tumor, bladder tumor, head and neck tumor, sarcoma tumor, cholangiocarcinoma tumor, glioblastoma tumor, multiple myeloma tumor, lymphoma tumor tissue, or portion thereof, sample. In a preferred embodiment, the sample is a multiple myeloma tumor sample.

[0329] In another particular embodiment, the sample from the subject according to the second medical use of the invention is a biofluid, preferably a biofluid from affected organs. A “biofluid”, “biological fluid sample” or “bodily fluid sample”, as used herewith, refers to any biological secretion or fluid, whether physiological or pathological, which is produced in the body of a subject. Such biofluids include, without limitation, blood, plasma, serum, bronchoalveolar washing fluid, urine, nasal secretion, ear secretion, urethral secretion, cerebrospinal fluid, pleural fluid, synovial fluid, peritoneal fluid, ascites fluid, pericardial liquid, amniotic fluid, gastric juice, lymphatic fluid, interstitial fluid, saliva, sputum, liquid deposition, tears, mucus, sweat, milk, semen, vaginal secretions, fluid coming from ulcer, blisters, abscesses and other surface eruptions. Said samples can be obtained by conventional methods, using processes known in the state of art by the person skilled in the art, such as blood extraction, instillation and aspiration of liquid during bronchofibroscopy, cisternal, ventricular or lumbar puncture, pleural puncture or thoracocentesis, joint or synovial percutaneous puncture, abdominal puncture, amniocentesis, expectoration, peritoneal percutaneous puncture, pericardial percutaneous puncture, etc., or by simple harvesting.

[0330] In a preferred embodiment, the biofluid is selected from the group consisting of bone marrow aspirate, blood, plasma and serum.

[0331] In some embodiments, the sample wherein the expression level of the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, e.g. ASCT2, is determined can be any sample containing cells from the potential tumor. In a particular embodiment, the sample containing cells from the potential tumor is a potential tumor tissue or a portion thereof. Said sample can be obtained by conventional methods, e.g., biopsy, surgical excision, or aspiration, by using methods well known to those of ordinary skill in the related medical arts. Methods for obtaining the sample from the biopsy include gross apportioning of a mass, or microdissection or other art-known cell-separation methods including partial tumorectomy. Tumor cells can additionally be obtained from fine needle aspiration cytology.

[0332] In order to simplify conservation and handling of the samples, these can be formalin- fixed and paraffin-embedded or first frozen and then embedded in a cryosolidifiable medium, such as OCT-compound, through immersion in a highly cryogenic medium that allows for rapid freeze.

[0333] As it is used herein, the term “expression level” refers to the expression level of a gene product, more specifically to a measurable quantity of a gene product produced by a specific gene in a specific sample of the patient. The term “gene product”, as used herein, refers to a transcriptional product ora translational product, and thus corresponds to the mRNA transcribed from said gene or to the protein encoded by a specific gene.

[0334] As the person skilled in the art understands, the expression levels of the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, e.g. ASCT2, can be measured by determining the mRNA expression levels of the gene or by determining the protein levels encoded by said gene. In a particular embodiment, said value can be determined by measuring the mRNA level of the gene of interest or a fragment thereof or by measuring the amount of protein encoded by said gene of interest or a variant thereof.

[0335] In a particular embodiment, the expression level of the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, is determined by measuring the glutamine transporter mRNA levels or of a fragment thereof. In a preferred embodiment, the glutamine transporter is ASCT2.

[0336] In order to measure the levels of the mRNA encoded by a given gene, the biological sample may be treated to physically, mechanically or chemically disrupt tissue or cell structure, to release intracellular components into an aqueous or organic solution to prepare nucleic acids for further analysis. The nucleic acids are extracted from the sample by procedures known to the skilled person and commercially available. RNA is then extracted from frozen or fresh samples by any of the methods typical in the art, for example, Sambrook, J., et al., 2001. Molecular cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Laboratory Press, N.Y., Vol. 1-3. In some embodiments, the RNA is extracted from formalin-fixed, paraffin embedded tissues. An exemplary deparaffinization method involves washing the paraffinized sample with an organic solvent, such as xylene, for example. Deparaffinized samples can be rehydrated with an aqueous solution of a lower alcohol. Suitable lower alcohols, for example include, methanol, ethanol, propanols, and butanols. Deparaffinized samples may be rehydrated with successive washes with lower alcoholic solutions of decreasing concentration, for example. Alternatively, the sample is simultaneously deparaffinised and rehydrated. The sample is then lysed and RNA is extracted from the sample. Commercially available kits may be used for RNA extraction from paraffin samples, such as PureLink™ FFPE Total RNA Isolation Kit (Thermofisher Scientific Inc., US). Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker (1987) Lab Invest. 56: A67, and De Andres et al., BioTechniques 18:42044 (1995). Preferably, care is taken to avoid degradation of the RNA during the extraction process. The expression level can be determined using mRNA obtained from a formalin-fixed, paraffin-embedded tissue sample. mRNA may be isolated from an archival pathological sample or biopsy sample which is first deparaffinized. An exemplary deparaffinization method involves washing the paraffinized sample with an organic solvent, such as xylene. Deparaffinized samples can be rehydrated with an aqueous solution of a lower alcohol. Suitable lower alcohols, for example, include methanol, ethanol, propanols and butanols. Deparaffinized samples may be rehydrated with successive washes with lower alcoholic solutions of decreasing concentration, for example. Alternatively, the sample is simultaneously deparaffinized and rehydrated. The sample is then lysed and RNA is extracted from the sample. Samples can be also obtained from fresh tumor tissue such as a resected tumor. In a particular embodiment, samples can be obtained from fresh tumor tissue or from OCT embedded frozen tissue.

[0337] Suitable methods to determine gene expression levels at the mRNA level include, without limitation, standard assays for determining mRNA expression levels such as qPCR, RT-PCR, RNA protection analysis, Northern blot, RNA dot blot, TaqMan®, tag based methods such as serial analysis of gene expression (SAGE) including variants such as LongSAGE and SuperSAGE, microarrays, nucleic acid sequence based amplification (NASBA).

[0338] In a particular embodiment, the determination of the expression level of the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, e.g. ASCT2, is carried out by RT-qPCR or nCounter.

[0339] In another particular embodiment, the expression level of the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, e.g. ASCT2, is determined by measuring the expression level of the protein encoded by this gene, because the increased expression of a gene is usually accompanied by an increase in the amount of corresponding protein. The determination of the amount of a protein corresponding to the expression of a specific gene can be performed using any conventional method for protein detection and quantification, for example by means of an immunoassay, etc. By way of non-limiting illustration, said determination can be performed using antibodies with the capability to bind specifically to the protein to be determined (or fragments thereof with the antigenic determinants) and subsequent quantification of the antigen-antibody complex derivatives. The antibodies can be, for example, polyclonal sera, hybridoma supernatants or monoclonal antibodies, fragments of antibodies, Fv, Fab, Fab' and F(ab')2, scFv, diabodies, triabodies, tetrabodies, humanized antibodies, etc. Said antibodies may (or may not) be labeled with a marker. Illustrative, non-limiting examples of markers that can be used in the present invention include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme cofactors, enzyme substrates, enzyme inhibitors, etc. There is a wide range of well-known assays that can be used in the present invention, such as, for example, assays based on Western-blot or immunoblot techniques, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), EIA (enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical or immunohistochemical techniques, etc. Other ways of detecting and quantifying the protein include affinity chromatography, ligand binding assay techniques, particle-enhanced turbidimetric immunoassay (PETIA), etc.

[0340] In a particular embodiment, the determination of the expression level of the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, e.g. ASCT2, is carried out by immunohistochemical techniques.

[0341] The term “reference value”, as used herein, refers to a laboratory value used as a reference for values / data obtained by means of samples collected from subjects. The reference value or reference level can be an absolute value, a relative value, a value that has an upper or a lower limit, a range of values, an average value, a median value, a mean value, or a value as compared to a particular control or baseline value. A reference value can be based on an individual sample value, such as, for example, a value obtained from a sample from the subject being tested, but at an earlier point in time. The reference value can be based on a large number of samples, for example on a group of patients considered to be representative, such as the values obtained from a population of subjects of matched group coinciding with that of the patient object of the study, or based on a pool of samples including or excluding the sample to be tested.

[0342] In the context of the second medical use of the invention for use in a personalized therapy for a subject suffering from cancer, a preferred reference value can be the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, e.g. ASCT2, expression level determined in a tumor sample from a subject having cancer or having had cancer which has shown a good response to treatment with the cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, the vector of the invention or the vector composition of the invention, or the pharmaceutical composition of the invention, said expression levels having been determined at the time that the patient was being treated. In another embodiment, the reference value can be the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, e.g. ASCT2, expression level determined in a tumor sample from a patient having cancer or having had cancer which has shown a poor response to treatment with the cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, the vector of the invention or the vector composition of the invention, or the pharmaceutical composition of the invention, said expression levels having been determined at the time that the patient was being treated. In another embodiment, the reference value can be the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, e.g. ASCT2, expression level determined in a tumor sample from a pool of patients having cancer or having had cancer wherein several of them have shown good response and the others have shown bad response to treatment with the cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, the vector of the invention or the vector composition of the invention, or the pharmaceutical composition of the invention, said expression levels having been determined right before the patient was being treated. In another embodiment, the reference value can be the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, e.g. ASCT2, expression level determined in a tumor sample from a pool of patients having cancer or having had cancer wherein several of them have shown poor response and the others have shown bad response to treatment with the cell of the invention or the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, the vector of the invention or the vector composition of the invention, or the pharmaceutical composition of the invention, said expression levels having been determined right before the patient was being treated. In another embodiment, the reference value can be the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, e.g. ASCT2, expression level in a healthy patient, i.e., a patient which has not been diagnosed with the type of cancer for which the personalized therapy is desired.

[0343] Once this reference value is established, the level of the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, e.g. ASCT2, expressed in the sample can be compared with said reference value, and thus be assigned a level of “increased”, “decreased” or “equal” expression level. In the context of the present invention, it is considered that an expression level of the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, e.g. ASCT2, in the sample from the patient is “above”, “increased” or “greater than” the reference value for said gene when the expression level of this gene in the sample from the patient increases, for example, 5%, 10%, 25%, 50%, 100% or even more when compared with the reference value for said gene, or when it increases, for example, at least 1.1-fold, 1.5-fold, 5-fold, 10-fold, 20- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more compared with the reference value of this gene.

[0344] When a comparison between the level of the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, e.g. ASCT2, and the reference value has been made, the second medical use of the invention allows deciding the suitable treatment for the patient tested. Thus, patients having an increased level of the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, e.g. ASCT2, with respect to the reference value should select a therapy comprising the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, the vector of the invention or the vector composition of the invention, or the pharmaceutical composition of the invention.

[0345] On the other hand, patients having an equal or a decreased level of the glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, e.g. ASCT2, with respect to the reference value should select an alternative therapy, i.e. , a therapy that does not comprise the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, the vector of the invention or the vector composition of the invention, or the pharmaceutical composition of the invention.

[0346] The expression “alternative therapy”, in the context of the second medical use of the invention, refers to any therapy different from a therapy comprising the cell obtainable by the first method of the invention, the nucleic acid construct of the invention or the nucleic acid composition of the invention, the vector of the invention or the vector composition of the invention, or the pharmaceutical composition of the invention. Alternative therapy, includes, without limitation, surgery, chemotherapy, radiation therapy, hormonal therapy and targeted therapy, including immunotherapy, and combinations thereof. Illustrative, non-limitative examples of alternative therapies include surgery, treatments with anti-cancer agents such as chemotherapeutic agents which have been previously defined in the context of the second medical use of the invention.

[0347] All the terms and embodiments described in any of the previous aspects of the invention are equally applicable to the present aspects of the invention.

[0348] Prognostic method

[0349] The authors of the present invention have also found a correlation between the expression level of a glutamine transporter and the survival in subjects suffering from multiple myeloma.

[0350] Accordingly, in a ninth aspect, the invention relates to an in vitro method, hereinafter referred to as the “second method of the invention”, for the prognosis of multiple myeloma in a subject, the method comprising:

[0351] (i) determining the expression level of a glutamine transporter in a tumor sample obtained from said subject, and

[0352] (ii) comparing said expression level obtained in (i) to a reference value, wherein if a deviation in the expression level of the glutamine transporter with respect to the reference value is an increase, it is indicative of poor prognosis of multiple myeloma in said subject, and wherein if a deviation of the expression level of the glutamine transporter with respect to the reference value is a decrease or if there is no deviation between the expression level of the glutamine transporter and the reference value, it is indicative of good prognosis of multiple myeloma in said subject.

[0353] In a preferred embodiment of the second method of the invention, the glutamine transporter is ASCT2.

[0354] The term “in vitro”, as used herein, refers to the fact that an experimental protocol or a method is not carried out in the body of a human or animal subject or patient, but in samples isolated from said subject, and already present in a laboratory tool, such as a test tube, dish or plate.

[0355] As it is used herein, the term “prognosis” refers to the prediction in a subject having multiple myeloma of the likelihood of cancer-attributable death or progression, including recurrence, metastatic spread, and drug resistance, of multiple myeloma. Prognosis may also be referred to in terms of "aggressiveness" or "severity": an aggressive cancer is determined to have a high risk of negative outcome (i.e., negative or poor prognosis) and a non-aggressive cancer has a low risk of negative outcome (i.e., positive or favorable prognosis). An "aggressive" or "severe" tumor is a cell-proliferation disorder that has the biological capability to rapidly spread outside of its primary location or organ. Indicators of tumor aggressiveness that are standard in the art include, without limitation, tumor stage, tumor grade, Gleason grade, Gleason score, nodal status, and survival. In this context, the term "survival" is not limited to mean survival until mortality (wherein said mortality may be either irrespective of cause or related to a cell proliferation disorder), but may also be used in combination with other terms to define clinical outcomes (e.g., "recurrence-free survival", in which the term "recurrence" includes both localized and distant recurrence; "metastasis-free survival"; "disease-free survival", in which the term "disease" includes cancer and diseases associated therewith). The length of the survival may be calculated by reference to a defined starting point (e.g., time of diagnosis or start of treatment) and a defined end point. Accordingly, a negative or poor prognosis is defined by a lower post-treatment survival term or survival rate. Conversely, a positive or good prognosis is defined by an elevated post-treatment survival term or survival rate. Usually prognosis is provided as the time of progression free survival or overall survival. As one skilled in the art will understand, said determination is not usually correct for all (i.e., 100%) of the patients to be identified. However, the term requires being able to identify a significant part of the subjects. One skilled in the art can readily determine if a part is statistically significant using several well-known statistical evaluation tools, for example, the determination of confidence intervals, the determination of p-values, Student’s t-test, Mann-Whitney test, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley and Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p-values are preferably 0.1 , 0.05, 0.01 , 0.005, or 0.0001. More preferably, at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a population can be suitably identified by the method of the present invention.

[0356] In a first step, the second method of the invention comprises the determination of the expression level of a glutamine transporter in a tumor sample obtained from a subject. In a preferred embodiment, the glutamine transporter is ASCT2.

[0357] The terms “subject”, “sample”, “multiple myeloma”, “glutamine transporter”, “ASCT2” and “expression level” have been defined within the context of the cell of the invention and the second medical use of the invention, and apply equally to the second method of the invention.

[0358] In an embodiment of the second method of the invention, the sample is a tissue sample, preferably a biopsy of a tumor tissue sample, either fresh or frozen. In a preferred embodiment of the second method of the invention, the sample is a tumor sample containing bone marrow cells, cells derived from the bone marrow, RNA derived from bone marrow cells, or RNA derived from cells derived from the bone marrow. In a more preferred embodiment, the tumor sample is a bone marrow biopsy or bone marrow aspirate.

[0359] In another particular embodiment of the second method of the invention, the sample from the subject is a biofluid, preferably a biofluid from affected organs. In a preferred embodiment, the biofluid is selected from the group consisting of blood, plasma and serum. The term “biofluid” has been defined in the context of the second medical use of the invention and applies equally to the second method of the invention.

[0360] In some embodiments of the second method of the invention, the sample wherein the expression level of the glutamine transporter, e.g. ASCT2, is determined can be any sample containing cells from the potential tumor. In a particular embodiment, the sample containing cells from the potential tumor is a potential tumor tissue or a portion thereof. Methods to obtain the sample have been defined in the context of the second medical use of the invention and apply equally to the second method of the invention.

[0361] As the person skilled in the art understands, the expression levels of the glutamine transporter, e.g. ASCT2, can be measured by determining the mRNA expression levels of the gene or by determining the protein levels encoded by said gene. In a particular embodiment, said value can be determined by measuring the mRNA level of the gene of interest or a fragment thereof or by measuring the amount of protein encoded by said gene of interest or a variant thereof.

[0362] In a particular embodiment, the expression level of the glutamine transporter is determined by measuring the glutamine transporter mRNA levels or of a fragment thereof. In a preferred embodiment, the glutamine transporter is ASCT2.

[0363] Methods to determine the mRNA level of a gene have been defined in the context of the second medical use of the invention and apply equally to the second method of the invention.

[0364] In a particular embodiment, the determination of the expression level of the glutamine transporter, e.g. ASCT2, is carried out by RT-qPCR or nCounter.

[0365] Methods to determine the expression level of the protein encoded by a gene have been defined in the context of the second medical use of the invention and apply equally to the second method of the invention.

[0366] In a particular embodiment, the determination of the expression level of the glutamine transporter, e.g. ASCT2, is carried out by immunohistochemical techniques. In a second step, the second method of the invention comprises comparing said expression level obtained in the first step to a reference value.

[0367] The term “reference value” has been defined within the context of the second medical use of the invention, and applies equally to the second method of the invention.

[0368] In the context of the second method of the invention, a preferred reference value can be the glutamine transporter, e.g. ASCT2, expression level determined in a tumor sample from a subject having cancer and in which the cancer has not progressed or who has had a good progression. In another embodiment, the reference value can be the glutamine transporter, e.g. ASCT2, expression level determined in a tumor sample from a patient having cancer and in which the cancer has progressed or who has had a poor progression. In another embodiment, the reference value can be the glutamine transporter, e.g. ASCT2, expression level determined in a tumor sample from a pool of patients having cancer and in which the cancer has not progressed or who has had a good progression. In another embodiment, the reference value can be the glutamine transporter, e.g. ASCT2, expression level determined in a tumor sample from a pool of patients having cancer and in which the cancer has progressed or who has had a poor progression. In another embodiment, the reference value can be the glutamine transporter, e.g. ASCT2, expression level in a healthy patient, i.e., a patient which has not been diagnosed with the type of cancer for which the determination of the prognosis is desired.

[0369] Once this reference value is established, the level of the glutamine transporter, e.g. ASCT2, expressed in the tumor sample from subjects can be compared with the reference value, and thus be assigned a level of deviation with respect to a reference value. The “deviation” can be either an increase or a decrease in the expression level of the glutamine transporter, e.g. ASCT2, with respect to the reference value. For example, an increase in the expression level of a gene above the reference value of at least 1.1- fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80- fold, 90-fold, 100-fold or even more compared with the reference value is considered as “increased” expression level, or an upregulation in the expression level of a gene in a sample with respect to the reference value. Similarly, the expression level of a gene is considered increased in a sample of the subject under study when its expression levels increase with respect to the reference sample by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100%, by at least 110%, by at least 120%, by at least 130%, by at least 140%, by at least 150%, or more. On the other hand, a decrease in the expression level of a gene below the reference value of at least 0.9-fold, 0.75-fold, 0.2-fold, 0.1 -fold, 0.05-fold, 0.025-fold, 0.02-fold, 0.01 -fold, 0.005-fold or even less compared with reference value is considered as “decreased” expression level, or a downregulation in the expression level of a gene in a sample with respect to the reference value. Similarly, the expression level of a gene is considered decreased in a sample of the subject under study when its expression levels decrease with respect to the reference sample by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100% (i.e. , absent).

[0370] When a comparison between the level of the glutamine transporter, e.g. ASCT2, and the reference value has been made, the second method of the invention allows the determination of the prognosis of a subject suffering from multiple myeloma. Thus, if a deviation in the expression level of the glutamine transporter, e.g. ASCT2, with respect to the reference value is an increase, it is indicative of poor prognosis of multiple myeloma in said subject. On the other hand, if a deviation of the expression level of the glutamine transporter, e.g. ASCT2, with respect to the reference value is a decrease or if there is no deviation between the expression level of the glutamine transporter, e.g. ASCT2, and the reference value, it is indicative of good prognosis of multiple myeloma in said subject

[0371] All the terms and embodiments described in any of the previous aspects of the invention are equally applicable to the present aspects of the invention.

[0372] ***

[0373] The invention will be described by way of the following examples which are to be considered as merely illustrative and not limitative of the scope of the invention.

[0374] EXAMPLES

[0375] Materials and Methods

[0376] ASCT2 expression and survival analyses in MM patients

[0377] For survival analyses, the survival data in the IA18 release of the Multiple Myeloma Research Foundation (MMRF) CoMMpass Study dataset (Valcarcel L.V., et al., 2021 , Leukemia; 35:3012-3016) was used, accessed via the data portal https: / / research.themmrf.org. The progression-free survival (PFS) data, the overall survival (OS) data, and gene expression data in TPM of a total of 664 patients that possessed all information was used. The maxStat package (R) was used to select a threshold value for the expression of the ASCT2, which was discretized as lowly or highly expressed and univariate COX regressions for PFS and OS of MM patients was then performed. The corrected p-value was calculated using conditional Monte-Carlo.

[0378] Adoptive Cell Transfer experiments

[0379] 5080 MM model: C57BI / 6 mice were challenged with 6 x 1065080 MM cells intravenously. 5080 MM cells were first reported by M. Larrayoz et al. (Larrayoz M., et al., 2023, Nat Med; 29:632-645). Seven days later, mice received total body irradiation of 2,5 Gy and were treated intravenously with 5 x 105ASCT2-overexpressing or control CD8+and CD4+BCMA CAR-T cells (1 x 106CAR-T cells in total) and 20,000 lll / mouse of IL-2. IL-2 was administered for 4 consecutive days after ACT. Mice were sacrificed when they showed signs of disease, (hunching, ruffled fur, labored breathing, low mobility, leg paralysis, and / or >20% weight loss). For characterization experiments, 5080 MM tumor-bearing mice were treated with CD8+(5 x 105) and CD4+(5 x 105) ASCT2- overexpressing or control BCMA CAR-T cells. Seven and fourteen days later, mice were sacrificed for flow cytometry analysis to study the presence of tumor and transferred T cells within the tumors and the periphery.

[0380] Mlcyigenetic MM model was described previously (Larrayoz M., et al., 2023, Nat Med; 29:632-645). 170 days-old MICYI mice were analysed for disease levels by measuring gamma fraction in serum and were grouped homogenously according to this parameter. They received a total body irradiation dose of 2.5 Gy and ACT immunotherapy consisting of 3 x 106CD8+and 3 x 106CD4+control or ASCT2-overexpressing BCMA CAR-T cells (6 x 106total CAR-T cells), along with 20,000 lU / mouse of IL-2. Blood samples were collected weekly and gamma fractions were measured to monitor disease progression. Mice were sacrificed when they showed signs of discomfort, as mentioned above.

[0381] Purification and activation of CD4+ and CD8+ cells

[0382] CD4+and CD8+T cells were purified from mouse splenocytes using the Miltenyi microbead negative selection kits following manufacturer’s instructions. (CD4+: 130-104- 454 / CD8+: 130-104-075). Purified T cells were activated by plating them at 1 x 106 cells / mL in complete RPMI medium with anti-CD3 / CD28 dynabeads at 1 :2 beads: T cell ratio and 100 lll / mL IL-2. Activated T cells were incubated at 37°C for 24 hours.

[0383] Transduction of activated T cells with retroviral supernatants

[0384] To overexpress ASCT2, the mSCV-IRES-ASCT2-Thy1.1 vector that was synthesized by GeneScript was designed. The mSCV-IRES-Thy1.1 vector was used as control. ASCT2-overexpressing or control T cells were sorted based on Thy1.1 expression using an anti-CD90.1 (Thy1.1)-PeCy7 (BioLegend) antibody. The pRubiG vector was used to generate retroviruses expressing eGFP linked to F2A auto cleavable protease and to an anti-murine BCMA ScFv or an anti PSMA ScFv (to be used as a negative CAR-T control) and followed by a murine 4-1 BB-CD3 expression cassette to express the BCMA CAR and PSMA CAR respectively). Also, a pRubiG vector expressing ASCT2-F2A-BCMA-CAR including also the murine 4-1 BB-CD3 expression cassette was generated to prepare the ASCT2 BCMA CAR-T cells (ASCT2-BCMA CAR).

[0385] The Platinum-Ecotropic Retroviral Packaging Cell Line (PlatE) was used for retrovirus production. PlatE packaging cells were transfected with 5 pg of retroviral plasmids in combination with 2.5 pg pCL-Eco plasmid DNA using Lipofectamine2000 (ThermoFisher Scientific). Retroviral supernatants were collected at 48 and 72 hours after transfection.

[0386] T cells previously activated for 24 hours were infected with retroviral supernatants containing 100 IIJ / mL of IL-2 and 10 pg / mL protamine sulfate (Sigma) and incubated overnight in the presence of IL-2. The next day, T cells were re-infected using clarified 72 hours retroviral supernatants. T cells were then cultured at 0.1 x 106cells / mL in complete RPMI medium containing 100 UI / mL of IL-2 and left to expand for 3 days. ASCT2+-BCMA and BCMA CAR-T cells were transduced using the same procedure but using the retroviral vector ASCT2+-BCMA CAR.

[0387] Flow cytometry

[0388] The RV transduction efficiency of lymphocytes was measured using flow cytometry. Both the control and the ASCT2 vectors contained a Thy1.1 (CD90) transduction marker that enabled the detection of successfully infected cells. To measure the percentage of infection of the lymphocytes, an anti-Thy1.1-PeCy7 antibody was used at a 1 :1000 dilution. T cells were stained for 15 minutes at room temperature in the dark and then analyzed using a BD CANTO flow cytometer. ASCT2 overexpression was measured by staining T cells with an anti-ASCT2 primary Antibody (V501 , CellSignaling Technologies), and a goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) secondary antibody after fixation with 4% paraformaldehyde and permeabilization with 90% ice-cold methanol.

[0389] To identify CAR-T cells, transduced T cells were stained with a biotinylated goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch) and streptavidin-APC (BioLegend) secondary antibody. Subsequently, to assess T cell memory phenotype, CAR-T cells were stained with anti-CD44-Pecy7 and anti-CD62L-Alexa Fluor 405 antibodies (Abeam).

[0390] For immune characterization experiments after ACT immunotherapy in the 5080MM and Mlcyi models, antibodies described in Table 1 were used to stain TILs and circulating T cells. Samples were analysed in a Beckman Coulter CytoFLEX LX flow cytometer. iTAg™ MHC tetramer-SUNFEKL-APC (H-2Kb) (MBL) (1 :100 dilution) and anti-CD45.1- BUV496 (BD OptiBuild™) (1 :50 dilution) antibodies were used to characterize immune cells in the B16-OVA models.

[0391] Table 1 : Antibodies used for CART cell characterization by flow cytometry.

[0392] Proliferation and cytokine secretion assays

[0393] To evaluate the proliferation of control and ASCT2-overexpressing CD4+and CD8+T cells, 100.000 cells / well were plated in SILAC RPMI 1640 media (lacking Glucose, Phenol red, HEPES, L-arginine, L-glutamine, L-lysine) (Gibco), and supplemented with 250 ng / mL L-Arginine-HCI for SILAC, 100 mg / mL L-Lysine-2HCI for SILAC, 200 g / L D- glucose solution, and 10% OneShot dialyzed FBS (Gibco) when it was indicted. Glutamine was added to the culture medium at the indicated concentrations. BCMA CAR-T cells were activated with 0.5 .g / mL of recombinant BCMA (R&D Systems) coated plates. T cell cultures were incubated for 48 hours in SILAC medium supplemented with different concentrations of Gin. T cell culture supernatants were collected to analyse the production of IFN-y using BD OptEIA mouse IFN-y ELISA Set according to the manufacturer’s instructions (BD Biosciences) and by ELISPOT as previously described (Navarro F., et al., 2022, Oncoimmunology; 11 :2070337). The number of spots was automatically counted by an ELISPOT reader (CTL, Germany).

[0394] Cytotoxicity assay

[0395] The cytotoxic activity of control and BCMA CAR-T and ASCT2-overexpressing BCMA CAR-T cells against 5080 MM, was assessed by flow cytometry. T cells were cocultured for 18 hours with their respective target tumor cells at different effector-to-tumor (E: T) ratios in SILAC RPMI media containing decreasing Gin concentrations. T cells were subsequently stained with anti-CD8-APC or anti-CD4-PE antibodies. Then, 10,000 Cytognos counting beads / well were added. Using flow cytometry and Cytognos beads, the remaining number of tumor cells / well was determined and % lysis was calculated.

[0396] Tumor interstitial fluid (TIF) metabolic assay

[0397] Gin concentration present in bone marrow tissues was measured using the Glutamine Detection Assay Kit (Abeam, ab197011) following the manufacturer’s instructions, and measured using a spectrophotometer at OD 450 nm. Known glutamine standards were used to generate a standard curve and calculate concentrations. Bone marrow samples were obtained from healthy mice, mice challenged with 5080 MM (25 days after tumor challenge), and 170-days-old MIcyt mice. The femur and tibia from one leg of each mouse were collected, and the tissue surrounding the bones was carefully removed. The ends of both sides of the bone were cut, and the opened bones were placed in 0.6 mL Eppendorf tubes and centrifuged at 10,000rpm for 10 minutes until the contents of the bones were extracted. The bone marrow contents were carefully collected and diluted at 1 :10 in ddH2O.

[0398] The cleared serums and interstitial fluids were deproteinated using a 10kDa column (Vivaspin 500, Abeam), and the Gin concentration was then measured.

[0399] Glutamine uptake assay Control and ASCT2-overexpressing CAR-T cells were plated at 100.000 cells / well in the Gin-deficient SILAC RPMI medium. T cells were activated with anti-CD3 / CD28 Dynabeads at 1 :2 beads: T cell ratio. Following a 15-minute incubation at 37°C, 1 pCi of L-[2,3,4, H3]-glutamine (American Radiolabelled Chemicals, ART-0149) was added to all conditions, and cells were incubated for additional 15 minutes. T cells were removed and washed in the uptake buffer separately. Cells were lysed with 1 N NaOH, and radioactivity was measured using a Beckman LS 6500 Scintillation counter for 3-5 min.

[0400] SeaHorse

[0401] The Seahorse XFp microplates were coated with 22.4 pg / mL Cell-Tak in 0.1 M NaHCOs. Control or ASCT2-overexpressing T cells were resuspended in Seahorse XF DMEM (pH 7.4) medium containing 15 mM glucose, 1 mM pyruvate and 2mM glutamine. 100,000 T cells were added to each well of the microplates and centrifuged at 200xg for 1 minute to ensure adherence to the Cell-Tak coating. Once the cells were attached to the well, 130 pL of Seahorse medium was slowly added to each well, taking care not to disturb the cells. Microplates were incubated at 37°C and 0% CO2 and then analyzed using the Agilent Seahorse XF. ATP production rate was measured according to the manufacturer’s instructions, using 1 pM Oligomycin, 1.5 pM Carbonyl cyanide-p- trifluoromethoxy phenylhydrazone (FCCP), 1 pM antimycin / rotenone and 50mM 2- desoxi-D-glucose (2-DG)) all from Sigma. OCR and ECAR data were normalized to the protein content as assessed by Bradford assay from Bio-Rad.

[0402] Bulk RNAsec analysis of CART cell products.

[0403] Ilumina strand-specific mRNA: Roughly 100 ng of high-quality total RNA (RIN > 8) was used for the transcriptomic interrogation of control Bcma-CART and Asct2 Bcma- CART cells using Illumina's Stranded mRNA Prep ligation according to the manufacturer's instructions. Briefly, Oligo(dT) magnetic beads were used to isolate mRNA from total RNA samples. The purified mRNA was then fragmented and reverse transcribed into first-strand complementary DNA (cDNA) using random primers. During second-strand synthesis, the RNA template was removed and dllTP was incorporated in place of dTTP to maintain strand specificity. Next, double stranded cDNA was A-tailed, then ligated to Illumina anchors bearing T-overhangs. PCR-amplification of the library allowed the barcoding of the samples with 10 bp dual indexes and the completion of Illumina sequences for cluster generation. Libraries were quantified with Qubit dsDNA HS Assay Kit and their profile was examined using Agilent's HS D1000 ScreenTape Assay. Sequencing was carried out in an Illumina NextSeq2000 using paired end, dualindex sequencing (Rd1 : 59 cycles; i7: 10 cycles; i5: 10 cycles, Rd2 59 cycles) at a depth of 30 million reads per sample.

[0404] RNAseq reads are trimmed using Trim Galore vO.4.4 using default parameters to remove the Nextera adapter sequence. Mapping is performed using STAR (2.7) against the mouse GRCm39 genome. Quantification and generation of gene expression matrices were performed with the function featurecounts, implemented in the R package Rsubread. Aligned fragments are imported into RStudio and before statistical analysis, the function filterby Expr, implemented in the R package edgeR, was used to determine genes with enough counts for further analyses. Differential gene expression analysis is performed using the DESeq2 algorithm within R and RStudio. Gene set enrichment analysis was carried out using GSEA software (https: / / www.gsea-msigdb.org / ).

[0405] Asct2 silencing.

[0406] Human ASCT2. For base editing, sgRNAs targeting exon 1 of the human SLC1A5 (ASCT2) gene were designed using BE-Designer from CRISPR RGEN Tools (www.rgenome.net). The sequence of the sgRNAs can be found in Table S2. spCas9- TadCBEd mRNA was synthetized by IVT from SpCas9-TadCBEd-ivt-template (Addgene plasmid #193843) as described previously (Neugebauer ME, Hsu A, Arbab M, et al. Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity. Nat Biotechnol. 2023;41(5):673-685) . For ASCT2 knockout, 2x106cells were electroporated with 3pig of mRNA coding for the spCas9-TadCBEd editor and 4.5 .M of BE-sgRNA in a total volume of 20 J using the ExPERT ATx™ Electroporator (MaxCyte) according to manufacturer’s instructions. Genomic DNA was isolated with NucleoSpin Tissue for DNA extraction kit (Macherey-Nagel) 72h after electroporation and subjected to targeted PCR amplification using primer described in Table S1. 0 to T modifications were corroborated by Sanger sequencing of the amplified PCR product and Asct2KO was confirmed by flow cytometry.

[0407] Mouse Asct2 sgRNAs targeting exon 4 of the mouse Asct2 gene were designed and selected as described previously (Zabaleta N, Barberia M, Martin-Higueras C, et al. CRISPR / Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type I. Nat Commun. 2018;9(1):5454), using Benchling software (www.benchling.com). Sequences for Asct2 sgRNAs can be found in Table 2. Retroviral pRubiG-U6- expressing sgRNAs to target Asct2 were generated by using pRubiG-U6-BFP plasmid and used as previously described (Lozano T, Conde E, Martin-Otal C, et al. TCR-induced FOXP3 expression by CD8(+) T cells impairs their anti-tumor activity. Cancer Lett. 2022;528:45-58). T cells from Rosa26-Cas9 mice (kindly provided by Dr. Beatriz Pelacho, Universidad de Navarra) were used for CART cell preparation. In vitro cleavage efficiency was evaluated by TIDE 7.

[0408] Table S2. Sequences for mouse and human sgRNA for Asct2 silencing

[0409] Statistics

[0410] Data are represented as averages ± standard error of the mean (SEM). Student's t tests were used for statistical comparisons between two groups of normally distributed variables, and one-way or two-way ANOVA and subsequent Tukey’s post-hoc test were used for comparisons between more than two groups. For tumor growth, data were analysed using nonlinear third-order polynomial (cubic) regression curves. Kaplan-Meier survival curves were evaluated for statistical significance with the Log-rank Mantel-Cox test. For all tests a p value <0.05 was considered statistically significant. GraphPad PRISM software was used for statistical analysis.

[0411] Results

[0412] ASCT2 expression is associated with a poor prognosis in Multiple myeloma (MM)

[0413] Multiple myeloma (MM) cells exhibit a reliance on extracellular glutamine, indicating characteristics of glutamine addiction (Koppenol W.H., et al., 2011 , Nat Rev Cancer; 11 :325-337). It was studied if ASCT2 expression could serve as a prognostic indicator for MM. Leveraging RNA-seq data from the CoMMpass (IA18) study involving 664 MM patient samples, the progression-free survival (PFS) and overall survival (OS) of MM patients according to the level of expression of ASCT2 was analyzed, separating cases into two groups based on the distribution of expression levels (high or low). High ASCT2 expression was significantly associated with reduced PFS (Figure 1A) and OS in MM patients (Figure 1 B). Analysis of RNAseq samples from bone marrow aspirates of untreated MM patients compared ASCT2 expression levels in various B cell subtypes (Naive, centroblasts, centrocytes, memory, tonsillar and bone marrow plasma cells) obtained from healthy donors as previously described (van der Windt G.J., et al., 2012, Immunity; 36:68-78), showed a marked upregulation in MM cells compared to normal B cell subsets (Figure 1C). These findings suggested a potential prognostic relevance of ASCT2 expression in MM.

[0414] ASCT2-overexpression in murine BCMA CAR-T cells improves their functionality in vitro

[0415] Since MM expresses B cell maturation antigen (BCMA), it was sought to generate a murine anti-BCMA CAR-T that was effective against MM in vivo. Then, it was tested if ASCT2 overexpression could improve their efficacy in vivo. A RV construct encoding an anti-murine BCMA ScFv linked to the murine CD8 transmembrane, the 41 BB costimulatory, and the CD3^ domains in combination with the green fluorescent protein (GFP) was generated (BCMA CAR). As control, an anti-prostate specific membrane antigen (PSMA) CAR encoded in the same vector as the BCMA CAR-T was used (Figure 2A). To validate the specificity and functionality of BCMA CAR-T cells, they were cultured on plates coated with recombinant BCMA or irrelevant proteins (OVA and TACI). BCMA CAR-T cells exhibited robust proliferation and high IFN-y secretion when stimulated with BCMA, while no activation occurred in response to OVA or TACI, whereas PSMA CAR- T cells did not respond to any protein (Figure 2B). Subsequent co-culture experiments with BCMA-expressing MM cells confirmed that BCMA CAR-T cells selectively secreted IFN-y and displayed cytolytic activity against MM cells expressing BCMA (5080 and 9257 MM cell lines) (Figure 2C). ) The expression levels of BCMA in the cells used in Figure 2C are showed in Figure 2D.

[0416] To evaluate the potential enhancement of BCMA CAR-T cell function through ASCT2 overexpression, the GFP transduction marker in the CAR vector was replaced with the murine ASCT2 sequence to generate T cells co-expressing the BCMA CAR and ASCT2 (ASCT2-BCMA CAR-T cells) (Figure 2E). Flow cytometry analysis revealed similar expression levels of the CAR in both BCMA and ASCT2+-BCMA CAR-T cells (Figure 2F). Flow cytometry revealed also an overexpression of ASCT2 in the membrane of ASCT2+-BCMA CAR-T cells (Figure 2G).

[0417] It was then evaluated the glutamine transport activity of ASCT2 by utilizing3H- glutamine. Both CD4+and CD8+ASCT2+-BCMA CAR-T cells exhibited a significantly enhanced glutamine uptake compared to control BCMA CAR-T cells. Additionally, upon the addition of excess non-labeled glutamine, the uptake of radiolabeled glutamine decreased in both CD4+and CD8+CAR-T cells from both groups, although it remained significantly higher in the CD4+ASCT2+-BCMA CAR-T cells (Figure 2H).

[0418] To assess the functional activity of BCMA CAR-T and ASCT2+-BCMA CAR-T cells, CD4+and CD8+CAR-T cells were activated on plates coated with recombinant BCMA, with varying glutamine (Gin) concentrations. Both CD4+and CD8+ASCT2+-BCMA CAR- T cells exhibited significantly higher IFN-y secretion than BCMA CAR-T cells under conditions of low Gin availability. However, at higher Gin concentrations (2 mM), the IFN- y secretion was comparable between the two groups, suggesting that ASCT2 overexpression enhances T cell functionality under restricted Gin conditions (Figure 2I).

[0419] In assays evaluating the lytic activity of BCMA CAR constructs, co-culture of 5080 MM cells with CD4+or CD8+BCMA CAR-T or ASCT2+-BCMA CAR-T cells revealed a higher lytic capacity in ASCT2+-BCMA CAR-T cells, particularly at low Gin levels (0.5 mM) (Figure 2J). These findings indicated that ASCT2 overexpression provides BCMA CAR-T cells with a competitive edge in antigen-specific activation under Gin-limited conditions.

[0420] Consistent with observations in OT-I and OT-II T cells, both CD4+and CD8+ASCT2+-BCMA CAR-T cells exhibited enhanced oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) compared to BCMA CAR-T cells (Figure 3). This advantage is maintained when glycolysis in inhibited with WZB117 but is lost when glutaminolysis is inhibited with BPTES (Figure 3C-E). These results suggested improved metabolic fitness in activated T cells overexpressing ASCT2, akin to previous findings in OT-I T cells.

[0421] We compared the transcriptomic profiles of Asct2 Bcma-CART and Bcma-CART cells before and after antigen stimulation. CD8+ CART cells were incubated with or without Bcma protein for 12 hours, followed by RNA sequencing. Asct2 expression was higher in Asct2 Bcma-CART cells and increased further upon Bcma activation in both cell types, peaking in Asct2 Bcma-CART cells (Figure 4A). Initially, 352 genes were differentially expressed between the two cell types, rising to 2,271 after stimulation (Figure 4B). This highlights significant transcriptomic changes in Asct2 Bcma-CART cells (Figure 4C), with increased mTORCI activity and pathways related to cholesterol homeostasis, hypoxia, and glycolysis (Figure 4D). Several studies demonstrate that glutamine uptake via the Asct2 transporter is essential for mTORCI activation (Nakaya M, Xiao Y, Zhou X, et al. Inflammatory T cell responses rely on amino acid transporter ASCT2 facilitation of glutamine uptake and mTORCI kinase activation. Immunity. 2014;40(5):692-705), which is closely linked to the regulation of many SLC transporters (Chen R, Chen L. Solute carrier transporters: emerging central players in tumour immunotherapy. Trends Cell Biol. 2022;32(3): 186-201). Importantly, the repertoire of SLC transporters differed from conventional Bcma-CART cells (Figure 4E), indicating specific metabolic reprogramming due to Asct2 overexpression.

[0422] ASCT2 overexpression enhances the function and anti-tumor activity of BCMA CAR-T cells in different murine models of MM

[0423] To evaluate if ASCT2 overexpression could enhance ACT immunotherapy with BCMA CAR-T cells a murine model of MM based on the injection of the multiple myeloma-derived 5080 MM cell line was used. The 5080 MM cell line was previously established and genetically characterized and found to be able to induce syngeneic multiple myeloma when transplanted into immunocompetent C57BL / 6 mice (Larrayoz M., et al., 2023, Nat Med; 29:632-645). As occurred in the bone marrow aspirates of MM patients, Gin concentration in the interstitial fluid of bone marrow from mice with 5080 tumors was significantly lower than that found in bone marrows from control healthy mice (Figure 5A).

[0424] For comparative studies between BCMA CAR-T and ASCT2+-BCMA CAR-T, mice were challenged with 5080 MM cells and treated intravenously with 1x 106CD8+and CD4+BCMA CAR-T (1 :1 ratio) or ASCT2+-BCMA CAR-T cells (Figure 5B). ASCT2+- BCMA CAR-T cells significantly increased overall survival in mice challenged with 5080 tumors compared to BCMA CAR-T cells, with a notable cure rate observed (9 out of 10 mice cured by ASCT2-BCMA CAR-T cell treatment versus 0 cured by BCMA CAR-T cells) (Figure 5C).

[0425] An in vivo characterization of the 5080-challenged mice was conducted 14 or 21 days following the adoptive transfer of CAR-T cells to assess the remaining tumor cells, as well as the presence of tumor-infiltrating and circulating CAR-T cells in the spleen and bone marrow. After 14 days post-CAR-T transfer, there was a significant reduction in tumor cells in the bone marrow of both CAR-T-treated mice groups, being more significant in the bone marrow of mice treated with ASCT2+-BCMA CAR-T compared to those treated with BCMA CAR-T cells (Figure 5D). Moreover, there was a higher number of infiltrating CAR-T cells in both the bone marrow (Figure 5E) and spleen (Figure 5F) of mice receiving ASCT2+-BCMA CAR-T cells. By day 21 , the number of infiltrating and circulating CAR-T cells had decreased in both groups, but a notably higher number of CAR-T cells persisted in the bone marrow of mice treated with ASCT2+-BCMA CAR-T cells compared to BCMA CAR-T cells (Figure 5G).

[0426] It was also evaluated the antitumor effect of the BCMA CAR-T cells in a genetic MM preclinical model based on the generation of transgenic mice carrying the MM genetic drivers MYC and I KK2NF-KB. This model closely mimics common genetic alterations observed in human MM, presenting bone marrow tumors comprising over 10% GFP+CD138+B220"slgM" plasma cells that morphologically resemble human MM cells. The BM infiltration pattern, expression of typical MM markers (such as acid phosphatase, Bcma, Slamf7, and Taci), immunoglobulin secretion, clonal IghV gene rearrangements, and other hallmark MM features were also observed in this model (Larrayoz M., et al., 2023, Nat Med; 29:632-645).

[0427] In this Mlcyi strain mice, survival is significantly compromised, with a majority of mice succumbing around 200 days post-birth. Notably, a decrease in glutamine concentration was observed in bone marrow aspirates of 170-day-old Mlcyi mice compared to levels in healthy animals' BM or serum (Figure 6A).

[0428] To investigate if MM progression could be delayed by BCMA CAR-T cell immunotherapy in Mlcyi mice, 6 x 106BCMA and ASCT2+-BCMA CAR-T cells (3 x 106CD4+and 3 x 106CD8+CAR-T cells for each group) were intravenously administered at day 170 post-birth (Figure 6B). Analysis of the gamma-globulin (y) fraction in diseased mice at different time points revealed a transient reduction in mice receiving BCMA CAR- T, notably deeper and sustained following treatment with ASCT2+-BCMA CAR-T cells compared to conventional BCMA CAR-T cells (Figure 6C, p < 0.005). Importantly, treatment with ASCT2+-BCMA CAR-T significantly enhanced overall survival compared to conventional BCMA CAR-T, with median survival times of 208 days for untreated, 245 days for BCMA CAR-T, and 275 days for ASCT2+-BCMA CAR-T-treated mice (Figure 6D, Log-rank test p < 0.005).

[0429] Concurrently, an experiment to characterize the CAR-T product 14 days after their administration was conducted. Initially, a decrease in the total number of tumor cells in both the spleen and the bone marrow of mice treated with BCMA CAR-T compared to untreated mice was observed (Figure 6E). Notably, mice treated with ASCT2+-BCMA CAR-T exhibited a higher number of CAR-T cells in the spleen (Figure 6F). A similar pattern was observed in the CAR-T cells present in the bone marrow, indicating a longer persistence of ASCT2+-BCMA CAR-T cells (Figure 6G). These differences were observed in both CD4+and CD8+CAR-T cells (Figure 6H). Asct2-overexpression in human Bcma-CART cells improves their functionality in vitro.

[0430] To explore the clinical relevance of ASCT2 overexpression in human CART cells, we created two lentiviral constructs encoding a second generation anti-human BCMA- CAR using scFv from the C11 D5.3 clone (SEQ ID NO: 156), used in the FDA-approved Idecabtagene vicleucel (Ide-cel) for MM. We prepared a conventional BCMA-CAR fused to blue fluorescent protein (BFP, SEQ ID NO 163) and the ASCT2 BCMA-CAR construct co-expressing ASCT2 (Figure 7A). Both were expressed at similar levels in CART cells (Figure 7B). Flow cytometry confirmed ASCT2 overexpression in ASCT2 BCMA-CART cells (Figure 7C). Notably, these cells showed greater proliferation and higher IFN-y production upon BCMA stimulation at various glutamine concentrations (Figure 7D). Additionally, they had a higher proportion of naive stem cell memory and central memory subsets (Figure 7E).

[0431] To further demonstrate the importance of ASCT2, we performed cytotoxicity assays with BCMA-CART cells against tumor cells with different ASCT2 levels. We silenced ASCT2 in H929 MM cells using three sgRNAs, with guide 1 being more effective (Figure 7F). Tritiated glutamine uptake assays confirmed reduced uptake in ASCT2 knockout cells (Figure 7G). BCMA-CART cells more efficiently lysed ASCT2 KO H929 cells, likely due to increased amino acid availability (Figure 7H).

Claims

CLAIMS1 . A cell which expresses(i) a chimeric antigen receptor (CAR) comprising: o an antigen binding domain specific for BCMA, o a transmembrane domain, and o at least one intracellular signaling domain and / or a costimulatory domain; and(ii) a glutamine transporter, wherein the glutamine transporter is expressed from an exogenous nucleic acid present in the cell and wherein the glutamine transporter is selected from the group consisting of ASCT2, , SLC7A8, SLC6A14, SLC38A1 and SLC38A2.

2. The cell according to claim 1 , wherein the glutamine transporter is ASCT2, preferably comprising a sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 3, or a functionally equivalent variant thereof.

3. The cell according to any one of the preceding claims, wherein the antigen binding domain specific for BCMA is a ScFv.

4. The cell according to claim 3, wherein the ScFv comprises a sequence of any one of SEQ ID NO: 4 to SEQ ID NO: 92, or a functionally equivalent variant thereof.

5. The cell according to any one of the preceding claims, wherein transmembrane domain of the CAR is selected from the group consisting of the CD4 transmembrane domain, the CD8 transmembrane domain, the CD28 transmembrane domain, the 4- 1 BB transmembrane domain, the CTLA4 transmembrane domain, the CD27 transmembrane domain and the CD3 zeta transmembrane domain.

6. The cell according to claim 5, wherein the transmembrane domain is the CD8 transmembrane domain, preferably comprising the sequence of SEQ ID NO: 93.

7. The cell according to any one of the preceding claims, wherein the CAR further comprises a hinge domain between the antigen binding domain specific for BCMA and the transmembrane domain.

8. The cell according to claim 7, wherein the hinge domain is selected from the group consisting of CD8, CD28, and lgG4 hinge domain; preferably, the hinge domain is the CD8 hinge domain, more preferably the hinge domain comprises the sequence of SEQ ID NO: 94.

9. The cell according to any one of the preceding claims, wherein the at least one intracellular signaling domain of the CAR comprises a costimulatory domain, a primary signaling domain, or any combination thereof.

10. The cell according to claim 9, wherein the at least one intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of: 0X40, CD70, CD27, CD28, CD5, ICAM-1 , LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP 12, and 4-1 BB (CD137), or any combination thereof.

11. The cell according to claim 10, wherein the at least one intracellular signaling domain comprises the intracellular domain of the costimulatory is 4-1 BB, preferably comprising the sequence of SEQ ID NO: 95.

12. The cell according to any one of the preceding claims, wherein the at least one intracellular signaling domain of the CAR further comprises a CD3 zeta intracellular domain, preferably comprising the sequence of SEQ ID NO: 96.

13. The cell according to any one of the preceding claims, wherein the hinge domain is the CD8 hinge domain, the transmembrane domain is the CD8 transmembrane domain, the intracellular signaling domain comprises the 4-1 BB intracellular region and the CD3 zeta intracellular domain.

14. The cell according to any one of the preceding claims, wherein the CAR comprises a sequence of any one of SEQ ID NO: 97 to SEQ ID NO: 102 or SEQ ID NO: 143.

15. The cell according to any one of the preceding claims, wherein said cell is selected from a group consisting of an immune cell, a somatic cell and a progenitor cell.

16. The cell according to claim 15, wherein the immune cell is selected from a group consisting of a T cell, a B cell, a NK cell, a neutrophil, an eosinophil, a basophil, a monocyte, a macrophage, a dendritic cell and a mast cell, or a precursor of any of these cells.

17. The cell according to any one of the preceding claims, wherein said cell is selected from a T cell or a precursor thereof.

18. The cell according to claim 17, wherein said T cell is a primary T cell.

19. A nucleic acid construct comprising(i) a first region encoding a chimeric antigen receptor (CAR), said chimeric antigen receptor comprising: o an antigen binding domain specific for BCMA, o a transmembrane domain, ando at least one intracellular signaling domain and / or a costimulatory domain; and(ii) a second region encoding a glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2; or a nucleic acid composition comprising(i) a first polynucleotide encoding a chimeric antigen receptor (CAR), said chimeric antigen receptor comprising: o an antigen binding domain specific for BCMA, o a transmembrane domain, and o at least one intracellular signaling domain and / or a costimulatory domain and(ii) a second polynucleotide encoding a glutamine transporter selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2.

20. The nucleic acid construct or the nucleic acid composition according to claim 19, wherein the CAR antigen binding domain specific for BCMA, the CAR transmembrane domain, the at least one CAR intracellular signaling domain and / or the CAR costimulatory domain, and the glutamine transporter are as defined in any one of claims 1 to 14.

21. The nucleic acid construct or the nucleic acid composition according to claims 19 or 20, wherein the chimeric antigen receptor encoded by the first region of the nucleic acid construct or by the first polynucleotide of the nucleic acid composition, and / or the glutamine transporter encoded by the second region of the nucleic acid construct or by the second polynucleotide of the nucleic acid composition further comprise a signal sequence.

22. The nucleic acid construct or the nucleic acid composition according to claim 21 , wherein whenever the sequence encoding the chimeric antigen receptor and / or the sequence encoding the glutamine transporter is preceded by a sequence encoding a signal sequence, the sequence of the signal sequence is that of SEQ ID NO: 103.

23. A vector comprising the nucleic acid construct according to any one of claims 19 to 22 or a vector composition comprising a pair of vectors in which the first and second nucleic acids of the composition are respectively comprised within the first and second vectors of said pair of vectors.

24. The vector or the vector composition according to claim 23, wherein the vector is a lentiviral vector.

25. An ex vivo method for obtaining a cell expressing(i) a chimeric antigen receptor comprising: o an antigen binding domain specific for BCMA, o a transmembrane domain, and o at least one intracellular signaling domain and / or a costimulatory domain; and(ii) a glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, which comprises inserting into the cell the nucleic acid construct or the nucleic acid composition according to any one of claims 20 to 23, or the vector or vector composition according to claims 24 or 25 and selecting those cells which express the CAR on their surface and which express the glutamine transporter.

26. The method according to claim 25, wherein the CAR antigen binding domain specific for BCMA, the CAR transmembrane domain, the at least one CAR intracellular signaling domain and / or the CAR costimulatory domain, and the glutamine transporter are as defined in any one of claims 1 to 14.

27. The method according to claim 25 or 26, wherein said cell is selected from a group consisting of an immune cell, a somatic cell and a progenitor cell.

28. The method according to claim 27, wherein the immune cell is selected from a group consisting of a T cell, a B cell, a NK cell, a neutrophil, an eosinophil, a basophil, a monocyte, a macrophage, a dendritic cell and a mast cell, or a precursor of any of these cells.

29. The method according to any one of claims 25 to 28, wherein said cell is selected from a T cell or a precursor thereof.

30. The method according to claim 29, wherein said T cell is a primary T cell.31 . A cell obtainable by the method according to any one of claims 25 to 30.

32. A pharmaceutical composition comprising the cell according to any one of claims 1 to 18 or claim 31 , the nucleic acid construct or the nucleic acid composition according to any one of claims 19 to 22, or the vector or vector composition according to claims 23 or 24, and at least one pharmaceutically acceptable excipient and / or vehicle.

33. The cell according to any one of claims 1 to 18 or claim 31 , the nucleic acid construct or the nucleic acid composition according to any one of claims 19 to 22, the vector or vector composition according to claims 23 or 24, or the pharmaceutical composition according to claim 32, for use in medicine.

34. The cell according to any one of claims 1 to 19 or claim 32, the nucleic acid construct or the nucleic acid composition according to any one of claims 20 to 23, the vector or vector composition according to claims 24 or 25, or the pharmaceutical composition according to claim 33, for use in the prevention and / or treatment of cancer or an autoimmune disease.

35. The cell, the nucleic acid construct or nucleic acid composition, the vector or vector composition, or the pharmaceutical composition for use according to claim 34, wherein the cancer is BCMA positive or wherein the autoimmune disease responds to plasma cell depletion.

36. The cell, the nucleic acid construct or nucleic acid composition, the vector or vector composition, or the pharmaceutical composition for use according to claim 34 or 35, wherein:- the cancer is selected from the group consisting of multiple myeloma (MM), plasma cell leukemia (PCL), Waldenstrom's macroglobulinemia (WM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), Hodgkin lymphoma (HL), primary central nervous system lymphoma (PCNSL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), breast cancer, lung cancer and melanoma, and / or- the autoimmune disease is selected from the group consisting of lupus erythematosus, an autoimmune disease of the nervous system, neuromyelitis optica spectrum disorder, myasthenia gravis, chronic inflammatory demyelinating polyradiculoneuropathy, immune-mediated necrotizing myopathy, idiopathic inflammatory myopathyand, multiple sclerosis, autoimmune encephalitis, myelin oligodendrocyte glycoprotein antibody-associated disease and POEMS syndrome.

37. The cell, the nucleic acid construct or nucleic acid composition, the vector or vector composition, or the pharmaceutical composition for use according to any one of claims 34 to 36, wherein the cancer is multiple myeloma.

38. The cell, the nucleic acid construct or nucleic acid composition, the vector or vector composition, or the pharmaceutical composition for use according to any one ofclaims 34 to 37 in a personalized therapy for a subject suffering from cancer, wherein said subject has increased glutamine transporter, selected from the group consisting of ASCT2, SLC7A8, SLC6A14, SLC38A1 and SLC38A2, expression levels in a tumor sample obtained from said subject with respect to a reference value.

39. The cell, the nucleic acid construct or nucleic acid composition, the vector or vector composition, or the pharmaceutical composition for use according to claim 38, wherein the glutamine transporter is ASCT2.

40. An in vitro method for the prognosis of multiple myeloma in a subject, the method comprising:(i) determining the expression level of a glutamine transporter in a tumor sample obtained from said subject, and(ii) comparing said expression level obtained in (i) to a reference value, wherein if a deviation in the expression level of the glutamine transporter with respect to the reference value is an increase, it is indicative of poor prognosis of multiple myeloma in said subject, and wherein if a deviation of the expression level of the glutamine transporter with respect to the reference value is a decrease or if there is no deviation between the expression level of the glutamine transporter and the reference value, it is indicative of good prognosis of multiple myeloma in said subject.

41. The method according to claim 40, wherein the expression levels of the glutamine transporter is determined by the glutamine transporter mRNA levels or of a fragment thereof.

42. The method according to claim 40 or 41 , wherein the glutamine transporter is ASCT2.

43. The method according to any one of claims 40 to 42, wherein the tumor sample is a sample containing bone marrow cells, cells derived from the bone marrow, RNA derived from bone marrow cells, or RNA derived from cells derived from the bone marrow.

44. The method according to any one of claims 40 to 43, wherein the tumor sample is a bone marrow biopsy or bone marrow aspirate.

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