T cells expressing Anti-BCMA / Anti-CD3 bispecific antibodies and uses thereof

Abstract

The present invention relates to T cells expressing bispecific antibodies for BCMA and CD3 and uses thereof in the treatment of cancer, in particular cancers which overexpress BCMA antigen.

Description

[0001] T CELLS EXPRESSING ANTI-BCMA / ANTI-CD3 BISPECIFIC ANTIBODIES AND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention is comprised within the field of biotechnology and biomedicine. It specifically relates to T cells expressing a bispecific antibody for BCMA and CD3 and uses thereof in the treatment of cancer.

[0004] BACKGROUND ART

[0005] Redirection of T cell activity towards cancer cells by targeting tumor-associated or tumorspecific antigens is a successful therapeutic approach against certain hematologic malignancies, aiming at stimulating and augmenting the immune response towards cancer cells. 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 chimeric antigen receptors (CARs) or bispecific antibodies.

[0006] CARs are synthetic receptors which bind specifically to targeted tumor antigens and kill these targeted tumor cells. CARs are composed of an extracellular antigen-binding domain of an antibody that recognizes targeted tumor-associated antigens (TAAs), and an intracellular signaling domain derived from the T cell receptor (TCR), composed of combinations of signaling domains such as the T cell activation complex transducer CD3 (the zeta chain associated with the T cell receptor complex). CARs can also contain co-stimulatory molecules, commonly CD28, ICOS or 4-1 BB, that enhance T cell function. Bispecific antibodies, commonly known as bispecific T cell engagers (TCEs or BiTEs) or T cell bispecific antibodies (TCBs), are engineered antibodies designed to simultaneously recognize two binding domains, one recognizing tumor-expressed antigens, and another recognizing an invariant subunit of the T cell receptor (TCR), typically CD3. The binding domains are two single chain fragment variable (scFv) regions from monoclonal antibodies, joined by a flexible peptide linker. When a BiTE molecule engages both a cytotoxic T cell and a tumor cell, the T cells proliferate, increasing overall numbers of effector cells and strengthening the potency of BiTE therapy. Malignant cell lysis in then triggered. Blinatumomab is an anti-CD19 / anti-CD3 tandem scFv BiTE therapy, approved by the US Food and Drug Administration for the treatment of relapsed / refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and for the treatment of BCP-ALL with minimal residual disease (MRD).

[0007] B cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor (TNFR) superfamily which regulates B cell proliferation, survival, and differentiation to plasma cells (PCs). BCMA is an attractive target for T cell-redirecting strategies since it is almost exclusively expressed on plasmablasts and PCs and is overexpressed on malignant PCs, while undetectable in naive B lymphocytes, hematopoietic stem cells or non-hematological normal tissue.

[0008] T cell-redirecting immunotherapies targeting BCMA have been extensively evaluated, by means of CAR-T cell therapies or bispecific antibodies, giving rise to the US Food and Drug Administration and the European Medicines Agency approval of the BCMA-specific CAR-T cell products idecabtagene vicleucel and ciltacabtagene autoleucel, and BCMA- specific TCE teclistamab, for the treatment of relapsed / refractory multiple myeloma. However, despite the encouraging results obtained with BCMA-specific immunotherapies, most patients eventually become refractoty and relapse, or show disease progression.

[0009] A T cell-redirecting strategy that combines both adoptive cell therapy (ACT) and bispecific antibodies, referred as secretion of TCE antibodies (STAb), relies on the in vivo production of TCEs by engineered T cells. This emerging approach leads to a sustained in vivo TCE secretion resulting in effective serum concentrations, expansion and persistence of adoptively transferred STAb-T cells, and polyclonal recruitment of both gene-modified STAb-T cells and unmodified bystander T cells present at the tumor microenvironment (TME), in contrast to CAR-T cells, leading to significant increased antitumor responses. Therapeutic potential of STAb-T cells has been demonstrated in B cells and T cell hematological malignancies, secreting a CD19 / CD3-, or a CD1a / CD3- targeting bispecific TCE antibody, respectively.

[0010] Therefore, given the limitations of the present T cell immunotherapies, more effective alternatives are required taking advantage of the therapeutic potential of STAb-T cells, to specifically target BCMA, allow a wider effect of BCMA targeting, generate long-term BCMA-specific responses and prevent tumor progression. SUMMARY OF THE INVENTION

[0011] Described herein is a cell expressing and secreting a bispecific antibody for BCMA and CD3. These T cell can be used for the treatment of cancer. The cells are characterized by a higher efficiency in recruiting bystander T cells and in preventing tumor escape, when compared with BCMA CAR-T cells, leading to a more effective cancer treatment. In a first aspect the invention relates to a cell, characterized in that it comprises a polynucleotide that encodes a bispecific antibody comprising:

[0012] - an anti-BCMA single chain fragment variable (scFv), and

[0013] - an anti-CD3 single chain fragment variable (scFv), wherein:

[0014] - the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 1 , 2 and 3, or a functionally equivalent variant thereof,

[0015] - the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 4, SAS and 6, or a functionally equivalent variant thereof,

[0016] - the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 7, 8 and 9, or a functionally equivalent variant thereof, and

[0017] - the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 10, DTS and 12, or a functionally equivalent variant thereof, wherein the cell is selected from a group consisting of an immune cell, a somatic cell and a progenitor cell.

[0018] In a second aspect, the invention provides an ex vivo method for obtaining a cell expressing a bispecific antibody, the method comprising transducing the cell or precursor thereof with a polynucleotide encoding said bispecific antibody, or a vector comprising the same, wherein said bispecific antibody comprises:

[0019] - an anti-BCMA single chain fragment variable (scFv), and

[0020] - an anti-CD3 single chain fragment variable (scFv), wherein: - the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 1 , 2 and 3, or a functionally equivalent variant thereof,

[0021] - the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 4, SAS and 6, or a functionally equivalent variant thereof,

[0022] - the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 7, 8 and 9, or a functionally equivalent variant thereof, and

[0023] - the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 10, DTS and 12, or a functionally equivalent variant thereof, wherein the cell is selected from a group consisting of an immune cell, a somatic cell and a progenitor cell.

[0024] In a third aspect, the present invention relates to a cell obtainable by the method of the second aspect of the invention.

[0025] In a fourth aspect, the invention relates to a pharmaceutical composition comprising the cell according to the first aspect of the invention and to the third aspect of the invention, and at least one pharmaceutically acceptable excipient.

[0026] In another aspect, the invention relates to a bispecific antibody comprising:

[0027] - an anti-BCMA single chain fragment variable (scFv), and

[0028] - an anti-CD3 single chain fragment variable (scFv), wherein:

[0029] - the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 1 , 2 and 3, or a functionally equivalent variant thereof,

[0030] - the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 4, SAS and 6, or a functionally equivalent variant thereof,

[0031] - the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 7, 8 and 9, or a functionally equivalent variant thereof, and the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 10, DTS and 12, or a functionally equivalent variant thereof.

[0032] In further aspects, the invention relates to a polynucleotide encoding the bispecific antibody according to the invention as well as to an expression vector comprising said polynucleotide

[0033] In another aspect, the invention relates to a pharmaceutically composition comprising the bispecific antibody according to the invention, the polynucleotide according to the invention or the expression vector according to the invention and at least one pharmaceutically acceptable excipient.

[0034] In another aspect, the invention relates to the bispecific antibody according to the invention, to the polynucleotide according to the invention or to the expression vector according to the invention for use as a medicament.

[0035] In another aspect, the invention relates to the bispecific antibody according to the invention, to the polynucleotide according to the invention or to the expression vector according to the invention for use in the treatment of cancer or for use in the treatment of an hematological malignancy.

[0036] BRIEF DESCRIPTION OF THE FIGURES

[0037] Figure 1. Comparative in vitro study of engineered BCMA-specific STAb-T and CAR-T cells. Schematic diagrams showing the genetic (A) and domain structure (B) of the bispecific anti-BCMA x anti-CD3 antibody (BCMA-TCE), bearing a signal peptide from the human k light chain signal peptide (white box), the anti-BCMA (J22.9) scFv gene (orange boxes), the anti-CD3 OKT3 scFv gene (blue boxes), and His tag (brown box). BCMA-TCE-encoding lentiviral vector, containing the tdTo gene following the 2A sequence from Thosea asigna virus (C). A similar lentiviral vector (D), containing the tdTo gene and the 2A sequence was designed for cloning the anti-BCMA CAR (BCMA- CAR). Genetic (E) and domain structure (F) of the second-generation CAR BCMA. (G) Schematic representation of the effectortarget (E:T) ratios used in cytotoxicity experiments using U266Lucas target, showing the proportion of transduced (CAR-T or STAb-T) and NT effector T cells. (H) Cytotoxicity induced at decreasing E:T ratios of NT- T, CAR-T, or STAb-T cells from the same donor co-cultured with U266Luctarget cells for 48 hours, measured by adding D-luciferin to detect bioluminescence. IFNy secretion (I) and sBCMA levels (J) were determined by ELISA. Data are mean ± SD of two independent experiments with triplicates (n = 6). Significance was calculated by an unpaired Student t test. (K) Real-time cell cytotoxicity assay with HEK-293BCMAtarget cells co-cultured with NT-T, CAR-T, or STAb-T cells at the indicated E:T ratios. Cell index values determined every 15 minutes for 78 hours using an impedance-based method. One representative experiment performed in duplicate of two is shown. (L) Cytotoxicity induced by NT-T, CAR-T, or STAb-T cells from the same donor co-cultured in triplicates at the indicated E:T ratios with primary human BM cells from a MM patient and PBMCs from plasma cell leukemia (PCL) patient. The number of alive (DAPI-) target MM and PCL cells was determined after 24 hours. Significance was calculated by two- way ANOVA test corrected with a Tukey’s multiple comparisons test.

[0038] Figure 2. STAb-T cells recruit bystander T cells and prevent tumor escape in vitro. (A) Cytotoxic activity induced by varying numbers of A-T cells (NT-T, CAR-T, or STAb- T) and freshly isolated NA-T cells from the same donor co-cultured with U266Luctarget cells for 48 hours, maintaining a constant 1 :1 E:T ratio, measured by adding D-luciferin to detect bioluminescence. (B) IFNy secretion was determined by ELISA. Data are mean ± SD of three independent experiments with triplicates (n = 9). Significance was calculated by two-way ANOVA test corrected with a Tukey’s multiple comparisons test. (C) the percentage of cytotoxicity was determined by luciferase assay, and (D) IFNy secretion by ELISA. Data are mean ± SD of triplicates from an experiment (n = 3). Significance was calculated by two-way ANOVA test corrected with a Tukey’s multiple comparisons test. (E) Myeloma escape from immune pressure. U266 cells were co- cultured with NT-T, CAR-T, or STAb-T cells at the indicated E:T ratios, and the expression of CD3 and BCMA was analyzed by flow cytometry. Graphs show the change over time in relative percentages of CD3+BCMA CD3'BCMA+, CD3'BCMA' and CD3+BCMA+. Data are mean ± SD of three independent experiments (n = 3). (F,G) NT- T, CAR-T, or STAb-T cells were co-cultured with U266Luctarget cells at a 1 :1 E:T ratio in the presence of increasing concentrations of purified human BCMA-Fc fusion protein; after 48 hours, (F) cytotoxic activity and (G) IFNy secretion were analyzed. Data are mean ± SD of three independent experiments with duplicates (n = 6). Significance was calculated by two-way ANOVA test corrected with a Tukey’s multiple comparisons test. Figure 3. Comparative in vivo efficacy of STAb-T and CAR-T cells. (A) Change in body weight over time (%). (B) Radiance quantification in the standard model (control, NT-T, CAR-T and STAb-T) at the indicated time points. (C) Radiance quantification in the TLD model (control, CAR-T and STAb-T) at the indicated time points. Significance was calculated by two-way ANOVA test corrected with a Tukey’s multiple comparisons test. (D) Detection by flow cytometry of MM cells (BCMA+) cells in peripheral blood (PB) at day 28, and in PB, bone marrow (BM) and spleen at endpoint (day 42). (F) Detection by flow cytometry of T cells (CD3+) cells in PB at day 28, and in PB, BM and spleen at endpoint (day 42). (G) Detection by flow cytometry of tdTo+T cells (CD3+) in PB at day 28, and in PB, BM and spleen at endpoint (day 42). (H) Relative BCMA mRNA expression in BM (spine / sternum) at endpoint. (I) Soluble BCMA (sBCMA) levels in plasma from mice at endpoint (day 42). Significance was calculated by two-way ANOVA test corrected with a Tukey’s multiple comparisons test.

[0039] Figure 4. T cell phenotypes associated with STAb-T cell therapy. (A) Radiance quantification at NSG mice receiving i.v U266Luccells followed 3 days after by i.v. infusion of 3 x 106NT-T CAR-T or STAb-T cells [therapeutic limiting dose (TLD) model] accounting for 20% tdTo+T cells. Significance was calculated by two-way ANOVA test corrected with a Tukey’s multiple comparisons test. (B) Detection by flow cytometry of CD3+T cells in PB at endpoint (day 26). (C) Relative numerical (median ± SD of 2 independent pooled sample experiments) of the different T cell subsets identified in preinfusion STAb-T and CAR-T products (grey areas) overlayed with the corresponding (color-coded) BM and spleen from STAb-T- and CAR-T -treated mice classified according to the presence vs absence of tdTo expression. EM, effector memory; TE, terminal effector; MAIT, mucosal-associated invariant T cells; iNKT, invariant natural killer T cells; Treg, regulatory T cells; TFH, follicular helper T cells; Th, (classical) T helper cells.

[0040] Fig. 5. BCMA-TCE and CAR-BCMA characterization in Jurkat T cells. (A) Expression of tdTomato (tdTo) in non-transduced (J-NT-T) and transduced (J-CAR-T and J-STAb- T) Jurkat T cells was determined by measuring the fluorescence intensity using flow cytometric analysis. (B) Representative analysis of cell surface-bound BCMA-TCE (CD3 decoration), and intracellular BCMA-TCE expression by flow cytometry in J-STAb-T cells. (C) Representative analysis of cell surface-expressed BCMA-CAR by flow cytometry in J-CAR-T cells. Western blot detection of BCMA-CAR (D) and BCMA-TCE (E) in Jurkat T cell lysates (J-NT-T, J-CAR-T and J-STAb-T). (F) Western blot detection of secreted BCMA-TCE in the conditioned media from from J-STAb-T cells by western blot. Blinatumomab (BLI) was used as positive control. (G) Detection of soluble functional BCMA-TCE in the conditioned media from J-STAb-T cells by ELISA against plastic- immobilized human BCMA-Fc chimera (BCMA-Fc) or BSA. (H) CD69 expression by J- NT-T, J-CAR-T or J-STAb-T cells cocultured with BCMA' (K562) or BCMA+(U266) target cells or plastic immobilized anti-CD3 mAb (iCD3 mAb) for 24 hours. Representative histograms are shown. (I) Detection of soluble functional BCMA-TCE by ELISA in the conditioned media from J-STAb-T cells co-cultured for 24 and 72 hours with U266 cells at an E:T=1 :1. (J) PD-1 surface expression by flow cytometry in J-CAR-T and J-STAb-T cells after co-culturing for 24, 48 and 72 hours with U266 cells at an E:T=1 :1.

[0041] Fig. 6. Target cell characterization. (A) BCMA cell surface expression in K562 and U266 cells by flow cytometric analysis. Percentages of BCMA+cells are indicated in black and mean fluorescence intensity (MFI) values in red. (B) Detection of soluble BCMA (sBCMA) secretion by ELISA in the conditioned media from K562 and U266 cells. (C) BCMA cell surface expression in untransfected HEK293 and transfected HEK293BCMAcells by flow cytometric analysis. Percentages of BCMA+cells are indicated in black and MFI values in red. (D) Detection of sBCMA secretion by ELISA he conditioned media from HEK293 and HEK293BCMAcells.

[0042] Fig. 7. Polarization of F-actin and pTyr to the immune synapse. (A-B) Each dot represents the value of individual cell interactions obtained from two independent experiments. The dashed line indicates the polarization ratio equals 1 , meaning there is no polarization. Samples were compared by a one-way ANOVA with a Tukey’s multiple comparison test. (C-D) Percentage of activating interactions in each co-culture assessed by F-actin and pTyr polarization. Fisher’s exact tests were performed for each possible comparison. Results from two experiments are shown. (E) F-actin clearance calculated from 3D reconstructions obtained at the IS established by J-CAR-T and J-STAb-T cells.

[0043] Fig. 8. Characterization of effector primary T cells. (A) Percentage of tdTomato (tdTo) expression in non-transduced (NT-T) or transduced (CAR-T and STAb-T) primary T cells determined by flow cytometric analysis (means ± SD of at eight independent experiments are shown). (B) Percentage of CD4+and CD8+T cells, and (C) percentage of naive (TN), central memory (TCM), effector memory (TEM) and effector (TE) T cells among NT-T, CAR- T and STAb-T cells 7 days after transduction (means ± SD of at least three independent experiments are shown).

[0044] Fig. 9. Cytotoxic activity and IFNy production at decreasing E:T ratios. (A) Cytotoxic activity of primary NT-T, CAR-T, and STAb-T cells co-cultured 48 hours with BCMA' K562 cells at decreasing E:T ratios, measured by adding D-luciferin to detect bioluminescence. (B) Detection of IFNy in the conditioned media by ELISA. Data are shown as mean ± SD from three replicates (n = 6). Significance was calculated by two- way ANOVA test corrected with a Tukey’s multiple comparisons test.

[0045] Fig. 10. Real-time cell cytotoxicity assay with HEK293 target cells. (A) HEK293 target cells were co-cultured with primary NT-T, CAR-T or STAb-T effector cells at a 1 :1 E:T ratio, and cell index values were determined over 79 hours with measurements taken at 15 min intervals and normalized. Results from one of two experiments performed in duplicate are shown. (B) Total lysis and spontaneous lysis controls from untransfected HEK293 and transfected HEK293BCMAtarget cells.

[0046] Fig. 11. Phenotype of human primary tumor samples. Histograms showing the BCMA expression of tumor plasma cells (CD38+CD138+) and T cells (CD45+CD3+) in primary BM cells from a MM patient (A) and in PBMCs from a patient with plasma cell leukemia (B), analyzed by flow cytometry.

[0047] Fig. 12. Cytotoxicity and IFNy production in contacting co-cultures with K562 cells. (A) Cytotoxic activity of primary A-T (NT-T, CAR-T, STAb-T) cells. Decreasing numbers of A-T cells were co-cultured with K562Luc(BCMA-) cells and increasing numbers of NA- T cells from the same donor, keeping a constant 1 :1 E:T ratio. (B) Detection of IFNy in the conditioned media by ELISA. Data are mean ± SD of three independent experiments performed in duplicate (n = 6). Significance was calculated by two-way ANOVA test corrected with a Tukey’s multiple comparisons test.

[0048] Fig. 13. Cytotoxicity and IFNy production in contacting co-cultures with ARP1 cells. (A) Bystander cytotoxic activity of decreasing numbers of primary A-T (NT-T, CAR-T, STAb-T) cells co-cultured with ARP1Luctarget cells and increasing numbers of NA-T cells from the same donor, keeping constant a 1 :1 E:T ratio. (B) Detection of IFNy in the conditioned media by ELISA. Data are mean ± SD of three independent experiments performed in triplicate (n = 9). Significance was calculated by two-way ANOVA test corrected with a Tukey’s multiple comparisons test. (C) ARP1 cells were cocultured with primary NT-T, CAR-T or STAb-T cells at the indicated E:T ratios, and the expression of CD3 and BCMA was analyzed by flow cytometry up to 7 days. Graphs showing the change overtime in relative percentages of CD3+BCMA+’ CD3'BCMA+, CD3' BCMA' and CD3+BCMA+. Data are mean ± SD of three independent experiments (n = 3).

[0049] Fig. 14. Cytotoxicity and IFNy production in non-contacting co-cultures with K562 cells. (A) Cytotoxic activity of primary A-T (NT-T, CAR-T, STAb-T BCMA) cells. K562Luctarget cells and primary NA-T cells were plated in the bottom well (1 :1 E:T ratio) and decreasing numbers primary A-T (NT-T, CAR-T, STAb-T BCMA) cells from the same donor in the insert well. (B) Detection of IFNy in the conditioned media by ELISA. Data are mean ± SD of an independent experiment performed in triplicate (n = 3). Significance was calculated by two-way ANOVA test corrected with a Tukey’s multiple comparisons test.

[0050] DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention relates to cells expressing and secreting a bispecific antibody for BCMA and CD3 which find use in the treatment of cancer. The results herein provided show that the cells are more effective in the treatment of cancer, since the cells have a higher efficiency in recruiting bystander T cells and in preventing tumor escape, when compared with BCMA CAR-T cells as well as promoting memory T cells.

[0052] Cell expressing a bispecific antibody for BCMA and CD3

[0053] The first aspect of the invention relates to a cell, characterized in that it comprises a polynucleotide that encodes a bispecific antibody comprising:

[0054] - an anti-BCMA single chain fragment variable (scFv), and

[0055] - an anti-CD3 single chain fragment variable (scFv), wherein:

[0056] - the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 1 , 2 and 3, or a functionally equivalent variant thereof, - the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 4, SAS and SEQ ID NO: 6, or a functionally equivalent variant thereof,

[0057] - the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 7, 8 and 9, or a functionally equivalent variant thereof, and

[0058] - the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 10, DTS and SEQ ID NO: 12, or a functionally equivalent variant thereof, wherein the cell is selected from a group consisting of an immune cell, a somatic cell and a progenitor cell.

[0059] Another aspect of the invention relates to a cell, characterized in that it comprises a polynucleotide that encodes a bispecific antibody comprising:

[0060] - an anti-BCMA single chain fragment variable (scFv), and

[0061] - an anti-CD3 single chain fragment variable (scFv), wherein:

[0062] - the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 1 , 2 and 3, or a functionally equivalent variant thereof,

[0063] - the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 4, SAS and SEQ ID NO: 6, or a functionally equivalent variant thereof,

[0064] - the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 7, 8 and 9, or a functionally equivalent variant thereof, and

[0065] - the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 10, DTS and SEQ ID NO: 12, or a functionally equivalent variant thereof.

[0066] As used herein, the term “cell” or “engineered cells” 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, somatic cells and progenitor cells. The isolated immune cells, somatic cells or progenitor cells that contain the DNA or RNA sequences encoding a bispecific antibody specific for BCMA and CD3, and that secrete said bispecific antibody are also included in the definition of “cell” or “engineered cells”. Cells that encode for, express and secrete a bispecific antibody are also known in the art as “secretion of T-cell engager antibodies cells” or “STAb cells”. In a preferred embodiment of the first aspect of the invention the cell is further characterized in that it secretes the bispecific antibody.

[0067] In some instances, the cell is an immortalized cell line. 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. The 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.

[0068] The cell 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 reintroduced into the individual. 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. The term "xenogeneic" refers to a graft derived from an animal of a different species. 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.

[0069] In a particular embodiment, the cell according to the invention is an autologous cell, obtained from a subject to whom the cells are later to be administered, after ex vivo modification and expansion. As used herein, the terms “subject” and “individual” are used interchangeably and refer to a mammal, preferably a human of any gender, race or age.

[0070] The term “polynucleotide” or “nucleic acid” 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.

[0071] In a particular embodiment, the polynucleotide according to the first aspect of the invention is operationally linked with a promoter.

[0072] 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.

[0073] 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-1 a (CHEF-1) (Ebadat, et al, PLoS One, 2017, 12(10):e018596), Human elongation factor- 1 alpha (EFla) (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. In a particular embodiment, the polynucleotide according to the first aspect of the invention is operationally linked with a promoter. In a particular embodiment, the polynucleotide according to the first aspect of the invention is operationally linked with a promoter selected from human cytomegalovirus promoter (CMV), Chinese Hamster Elongation Factor-1a (CHEF-1), Human Phosphoglycerate Kinase (hPGK), and Human elongation factor-1 alpha (EFla), preferably Human elongation factor-1 alpha (EF-1a).

[0074] 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.

[0075] The term “antibody” (Ab), in the context of the present invention, refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system. As indicated above, the term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that are antigen-binding fragments which have been previously defined. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique.

[0076] The first aspect of the invention refers to a cell characterized in that it comprises a polynucleotide that encodes a bispecific antibody. The term “bispecific antibody” refers to, in the context of the present invention, an antibody having two different antigenbinding regions defined by different antibody sequences. Bispecific antibodies are able to bind to two different antigens simultaneously. The term “bispecific antibody” encompasses multispecific antibodies, which in addition to the first and second domains contain one or more other domains binding to other antigens and derived from or homologous to variable domains of natural antibodies. The term “bispecific antibody” also encompasses an antibody containing a first binding domain derived from or homologous to a variable domain of a natural antibody, and a second binding domain derived from another type of protein, e.g., the extracellular domain of a receptor, (a "bispecific antibody-immunoadhesin"). Examples of bispecific antibody formats include but are not limited to Bispecific T cell engager (BiTE), F(ab')2, F(ab')-ScFv2, di-scFv, diabody, minibody, scFv-Fc, DART, TandAb, ScDiabody, ScDiabody-CH3, Diabody- CH3, triple body, miniantibody, minibody, TriBi minibody, ScFv-CH3 KIH (knobs in holes), Fab-ScFv, SCFv-CH-CL-scFv, scFv-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, intrabody, dock and lock antibodies, ImmTAC, HSAbody, ScDiabody-HAS, humabody and Tandem ScFv-toxic.

[0077] In a particular embodiment, the bispecific antibody is a humanized bispecific antibody.

[0078] As it is used herein, the term "functionally equivalent variant of a CDR sequence" refers to a sequence variant of a particular CDR 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 as the ScFv described herein. For example, a functionally equivalent variant of a CDR 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. In another embodiment, the CDR1 , CDR2, and / or CDR3 regions according to SEQ ID NO: 1-4, 6-10, and 12, and according to the sequences SAS and DTS, comprise at least 1 , at least 2, at least 3 or more conservative mutations. In another embodiment, the FR1 , FR2, FR3, and / or FR4 regions according to SEQ ID NO: 13-28 comprise at least 1 , at least 2, at least 3, at least 4, at least 5 or more conservative mutations. 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. The following six groups each contain amino acids that are conservative substitutions for one another:

[0079] 1) Alanine (A), Serine (S), Threonine (T);

[0080] 2) Aspartic acid (D), Glutamic acid (E);

[0081] 3) Asparagine (N), Glutamine (Q);

[0082] 4) Arginine (R), Lysine (K);

[0083] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0084] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0085] Functionally equivalent variants of a CDR sequence according to the invention include CDR sequences having at least 70%, at least 75%, at least 80%, at least 85%, 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 corresponding amino acid sequences shown in one of the above reference sequences. It is also contemplated that functionally equivalent variants of a CDR sequence comprise additions consisting of at least 1 amino acid, or at least 2 amino acids, or at least 3 amino acids, or at least 4 amino acids, or at least 5 amino acids, or at least 6 amino acids, or at least 7 amino acids, or at least 8 amino acids, or at least 9 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 shown in one of above referenced sequences. 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 3 amino acids, or at least 4 amino acids, or at least 5 amino acids, or at least 6 amino acids, or at least 7 amino acids, or at least 8 amino acids, or at least 9 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 shown in one of the above mentioned sequences.

[0086] Functionally equivalent variants of a CDR sequence according to the invention will preferably maintain at least 70%, at least 75%, at least 80%, at least 85%, 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%, at least 99%, at least 100%, at least 105%, at least 1 10%, at least 1 15%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 200% or more of the capacity of the corresponding amino acid sequence shown in one of SEQ ID NOs: 1 to 12 to bind to its cognate antigen when being part of an antibody fragment or antigen-binding domain such as the ScFv of the CAR 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.

[0087] The bispecific antibody which is encoded by the polypeptide expressed by the cells of the first aspect of the invention comprises: an anti-BCMA single chain fragment variable (scFv). The term “BCMA” refers to the B cell maturation antigen, also known as CD269 or TNFRSF17 (tumor necrosis factor receptor superfamily member 17) (SwissProt Q02223), a member of the tumor necrosis factor receptor (TNFR) superfamily, and an anti-CD3 single chain fragment variable (scFv). The term “CD3” refers to the human CD3 protein complex, which is composed of six distinct chains (a CD3y chain (SwissProt P09693), a CD35 chain (SwissProt P04234), two CD3E chains (SwissProt P07766), and one CD3 zeta chain homodimer (SwissProt P20963) (E y: E 5:< )), 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.

[0088] 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.

[0089] The variable regions of each pair of light and heavy chains form the binding site of the antibody. They are characterized by the same general structure constituted by relatively preserved regions called frameworks (FR) joined by three hyper-variable regions called complementary determining regions (CDR). 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. 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.

[0090] 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. In a particular embodiment, the cell according to the first aspect of the invention, wherein the bispecific antibody is characterized in that:

[0091] - the FR1 , FR2, FR3 and FR4 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 13, 14, 15 and 16, or a functionally equivalent variant thereof,

[0092] - the FR1 , FR2, FR3 and FR4 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 17, 18, 19 and 20, or a functionally equivalent variant thereof,

[0093] - the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 21 , 22, 23 and 24, or a functionally equivalent variant thereof, and

[0094] - the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 25, 26, 27 and 28, or a functionally equivalent variant thereof. As it is used herein, the term "functionally equivalent variant of a FR sequence" refers to a sequence variant of a particular FR 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 or antibody-binding domains described herein. For example, a functionally equivalent variant of a FR sequence may be a polypeptide sequence derivative of said sequence comprising the addition, deletion or substitution of one or more amino acids.

[0095] Functionally equivalent variants of a FR sequence according to the invention include FR sequences having at least approximately 70% , at least 75%, at least 80%, at least 85%, 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 corresponding amino acid sequences shown in one of the above reference sequences. It is also contemplated that functionally equivalent variants of a FR sequence comprise additions consisting of at least 1 amino acid, or at least 2 amino acids, or at least 3 amino acids, or at least 4 amino acids, or at least 5 amino acids, or at least 6 amino acids, or at least 7 amino acids, or at least 8 amino acids, or at least 9 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 shown in one of above referenced sequences. 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 3 amino acids, or at least 4 amino acids, or at least 5 amino acids, or at least 6 amino acids, or at least 7 amino acids, or at least 8 amino acids, or at least 9 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 shown in one of the above mentioned sequences.

[0096] Functionally equivalent variants of a FR sequence according to the invention will preferably maintain at least 70%, at least 75%, at least 80%, at least 85%, 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%, at least 99%, at least 100%, at least 105%, at least 1 10%, at least 1 15%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 200% or more of the capacity of the corresponding amino acid sequence shown in one of SEQ ID NOs: 13-28 to bind to its cognate antigen when being part of an antigen-binding domain 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.

[0097] In a particular embodiment the functionally equivalent variant of the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 49, 50, 51 , and 52. In another particular embodiment the functionally equivalent variant of the FR1 , FR2, FR3 and FR4 regions within the anti- CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 53, 54, 55, and 56.

[0098] In another particular embodiment, the polynucleotide which is expressed by the cell according to the first aspect of the invention further comprises a region encoding a signal peptide preceding and in the same reading frame as the bispecific antibody.

[0099] As used herein, the term “reading frame” refers to a sequence of nucleotide consecutive no-overlapping triplets that is potentially translatable into a polypeptide and that is determined by the placement of a codon that initiates the translation.

[0100] The term "polypeptide" or "peptide" or "protein (if single chain)" are used interchangeably herein and generally refer to amino acid polymers of any length. The polymer can be linear or branched, it can contain modified amino acids, or it can be interrupted by nonamino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with labeled components). Polypeptides can be isolated from natural sources, can also be produced from eukaryotic or prokaryotic hosts by recombinant technology, or can be artificially synthesized products, as long as they can be expressed.

[0101] The term “signal peptide”, “signal seguence” or “signal seguence peptide”, also known as “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. “Signal peptide” include, but are not limited to, human kappa light chain signal peptide, human lambda light chain signal peptide, human heavy chain signal peptide (VH1 , VH2, VH3, VH4, VH5, VH6 or VH7), oncostatin M signal peptide, human interleukin signal peptides, such as, but not limited to, IL-1 signal peptide, IL-2 signal peptide, IL-3 signal peptide, IL-7 signal peptide, IL-15 signal peptide and IL-21 signal peptide; immunoglobulin signal peptide, for example, but not limited to, an IgG signal peptide and IgE signal peptide; etc.

[0102] In a preferred embodiment, the signal peptide according is the human kappa light chain signal peptide, which comprises the sequence SEQ ID NO: 35.

[0103] In another embodiment, the bispecific antibody which is encoded by the polynucleotide expressed by the cells according to the first aspect of the invention is characterized in that:

[0104] - the anti-BCMA heavy chain variable domain VHBCMA comprises the amino acid sequence SEQ ID NO: 29, or a functionally equivalent variant thereof,

[0105] - the anti-BCMA light chain variable domain VLBCMA comprises the amino acid sequence SEQ ID NO: 30, or a functionally equivalent variant thereof,

[0106] - the anti-CD3 heavy chain variable domain VHCD3 comprises the amino acid sequence SEQ ID NO: 31 or 57, or a functionally equivalent variant thereof, and

[0107] - the anti-CD3 light chain variable domain VLCD3 comprises the amino acid sequence SEQ ID NO: 32 or 58, or a functionally equivalent variant thereof.

[0108] In another embodiment, the anti-BCMA scFv forming part of the antibody expressed by the cells according to the first aspect of the invention is located N-terminal with respect to the anti-CD3 scFv.

[0109] In a preferred embodiment, the VH region of the anti-BCMA scFv (VHBCMA) according to the first aspect of the invention is located N-terminal with respect to the VL ( LBCMA), and / or wherein the VH region of the anti-CD3 scFv (VHCD3) is located N-terminal with respect to the VL (VLCD3). In a preferred embodiment, the VH region of the anti-BCMA scFv (VHBCMA) according to the first aspect of the invention is located C-terminal with respect to the VL (VLBCMA), and / or wherein the VH region of the anti-CD3 scFv ( HCD3) is located C-terminal with respect to the L (VLCD3).

[0110] In a particular embodiment, the variable heavy chain regions (VH) and the variable light chain regions (VL) regions according to the first aspect of the invention are arranged, from N-terminus to C-terminus, in the order VHBCMA-VLBCMA-VHCD3-VLCD3.

[0111] In a particular embodiment, the variable heavy chain regions (VH) and the variable light chain regions (VL) regions according to the first aspect of the invention are arranged, from N-terminus to C-terminus, in the order VLBCMA-VHBCMA-VLCD3-VHCD3.

[0112] In a more particular embodiment, the bispecific antibody according to the first aspect of the invention is characterized in that the anti-BCMA single chain fragment variable (scFv) comprises the sequence SEQ ID NO: 33, and the anti-CD3 single chain fragment variable (scFv) comprises the sequence SEQ ID NO: 34.

[0113] In a more particular embodiment, the bispecific antibody according to the first aspect of the invention is characterized in that the anti-BCMA single chain fragment variable (scFv) comprises the sequence SEQ ID NO: 33, and the anti-CD3 single chain fragment variable (scFv) comprises the sequence SEQ ID NO: 59.

[0114] In another embodiment, the VH and VL regions of the anti-BCMA scFv, the VH and VL regions of the anti-CD3 scFv, and / or the anti-BCMA scFv and anti-CD3 scFv according to the first aspect of the invention are connected by a peptide linker.

[0115] The term “peptide linker”, “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.

[0116] The peptide linker 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.

[0117] 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.

[0118] In a particular embodiment, the linker according to the first aspect of the invention is selected from SEQ ID NO: 36 or 38. In another particular embodiment, the linker according to the first aspect of the invention has a sequence according to (GGGGS)n, wherein n is equal to or bigger than 1 , 2, 3, 4, 5.

[0119] In a more particular embodiment, the bispecific antibody according to the first aspect of the invention comprises the amino acid sequence SEQ ID NO: 39 or 60.

[0120] In another embodiment, the bispecific antibody according to the first aspect of the invention further contains a polypeptide tag. Concretely, the tag is located at the C- terminus of the bispecific antibody.

[0121] The term “tag” are also known as “markers” or “labels” which may improve or facilitate certain properties of the protein, i.e. protein stability or resistance to degradation, or provide technical advantages during production, i.e., facilitate expression and / or purification. Examples of such tags are biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, 2A peptides, fluorescent protein tag and the like. Said tags can be fused or conjugated to the amino-terminal or the carboxyl-terminal a polypeptide, such as the bispecific antibody expressed by the immune cell of the invention. His tags or “polyhistidines” sequence of histidine amino acids bound by peptide bonds which are fused or conjugated to the amino-terminal end of the bispecific antibody according to the first aspect of the invention, and / or to the carboxyl terminal end of the bispecific antibody according to the first aspect of the invention. 2A peptides are a class of 18-22 aa-long peptide tags, which can induce ribosomal skipping during translation of a protein in a biological cell and which are fused or conjugated to the amino-terminal end of the bispecific antibody according to the first aspect of the invention, and / or to the carboxyl terminal end of the bispecific antibody according to the first aspect of the invention.

[0122] Examples of fluorescence proteins tags which can be fused to the bispecific antibody according to the first aspect of the invention are, without limitation, GFP, YFP, CFP, RFP, mCherry, BFP, tdTomato, in both their wild type forms and their enhanced forms (eGFP for example). In a preferred embodiment the polypeptide tag is a fluorescence protein, preferably tdTomato.

[0123] In a preferred embodiment the polypeptide tag is a polyhistidine tag and / or a 2A peptide tag. In a preferred embodiment the polyhistidine region contains at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 histidine residues. Concretely, in a more preferred embodiment, the tag is a hexahistidine tag. In another preferred embodiment the 2A peptide tag is selected from the group consisting of; 2A sequence from foot-and-mouth disease virus 18 (F2A) according to SEQ ID NO: 40, 2A sequence from equine rhinitis A virus (E2A) according to SEQ ID NO: 41 , 2A sequence from porcine teschovirus-1 (P2A) according to SEQ ID NO: 42, and 2A sequence from Thosea asigna virus (T2A) according to SEQ ID NO: 43. In a more preferred embodiment the 2A peptide tag is SEQ ID NO: 43 (T2A).

[0124] In another preferred embodiment, the polypeptide tag comprises one, two, three or more polypeptide tags. In another preferred embodiment the bispecific antibody comprises a polypeptide tag with a hexahistidine tag, a T2A tag and a tdTomato tag, wherein the polypeptide tag is fused to the fused or conjugated to the amino-terminal end of the protein of the invention, and / or to the carboxyl terminal end of the protein of the invention.

[0125] 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. 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.

[0126] Chemical means for introducing a polynucleotide into a host cell include 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). 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 / DN A 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.

[0127] 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 ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL).

[0128] 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.

[0129] In another embodiment, the polynucleotide according to the first aspect of the invention that encodes the bispecific antibody has been introduced into the cell by transducing said cell with a viral vector that contains said polynucleotide.

[0130] The term “transduction” refers to a process by which an exogenous polynucleotide is introduced into a host cell. A “transduced” cell is one which has been transduced with an exogenous polynucleotide. The cell includes the primary subject cell and its progeny.

[0131] As used herein, the term “viral vector” is widely used to refer either to a nucleic acid molecule that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell, or to a viral particle that mediates nucleic acid transfer. Viral particles typically include viral components, and sometimes also host cell components, in addition to nucleic acid(s). Viral vectors that can be used in the disclosure include, for example, retrovirus vectors (including lentivirus vectors), adenovirus vectors, and adeno-associated virus vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.). Retroviral vectors used herein contain structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. Retroviral lentivirus vectors contain structural and functional genetic elements, or portions thereof including LTRs, that are primarily derived from a lentivirus (a sub-type of retrovirus).

[0132] In a particular embodiment, the cell according to the first aspect of the invention, wherein the polynucleotide that encodes the bispecific antibody is an RNA that has been introduced into the cell by transfecting said cell with said RNA. The term "RNA molecule" or "ribonucleic acid molecule" refers to a polynucleotide having a ribose sugar rather than deoxyribose sugar and typically uracil rather than thymine as one of the pyrimidine bases. An RNA molecule of the invention is generally single- stranded, but can also be double-stranded. In the context of an RNA molecule from an RNA sample, the RNA molecule can include the single-stranded molecules transcribed from DNA in the cell nucleus, mitochondrion or chloroplast, which have a linear sequence of nucleotide bases that is complementary to the DNA strand from which it is transcribed.

[0133] As used herein the term "RNA polynucleotide" relates to any macromolecule comprising two or more ribonucleotides. Ribonucleotides typically contain a nucleobase, a ribose sugar and at least one phosphate group. The nucleobases are typically adenine, guanine, cytosine and uracil. RNA polynucleotides are typically single-stranded molecules, can, however, also be provided in a double- stranded form by partial complementary base pairing. The RNA polynucleotide typically does not form long double helical stretches. The RNA polynucleotide may be naturally occurring or be artificial. It may comprise, in addition to the elements mentioned above, modifications such as oxidized or methylated nucleotides. The RNA polynucleotide may also, in certain embodiments, comprise artificial additions such as tags or labels.

[0134] The RNA polynucleotide may be of any possible origin, e.g. prokaryotic, eukaryotic, archaeal or viral. The RNA polynucleotide to be characterized according to the present invention may have any known possible biological or cellular function. For example it may be any naturally occurring or synthetic polynucleotide such as messenger RNA (mRNA), ribosomal RNA (rRNA), heterogenous nuclear RNA (hnRNA), transfer RNA (tRNA), transfer messenger RNA (tmRNA), micro RNA (miRNA), small nuclear RNA (snRNA), spliced leader RNA (siRNA), small nucleolar RNA (snoRNA), antisense RNA (asRNA), guide RNA (gRNA), long noncoding RNA (IncRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA) and trans-acting RNA (taRNA).

[0135] The term “transfection” of a cell as used herein means that genetic material is introduced into a cell for the purpose of genetically modifying the cell. Transfection can be accomplished by a variety of means known in the art, such as transduction or electroporation. Methods of introducing exogenous polynucleotides into a host cell include the use of DNA or RNA vectors.

[0136] In a more particular embodiment, the cell according to the invention is selected from a group consisting of an immune cell, a somatic cell and a progenitor cell. According to the first aspect of the invention the cell is selected from a group consisting of an immune cell, a somatic cell and a progenitor cell.

[0137] As used herein, the term “immune cell” refers to a cell that can elicit an immune response, including but not limited to T cells, B cells, NK cells, NKT cells, DNT cells, neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells, mast cells, etc., their respective precursor cells and progenies. Immune response cells can also refer to cells of the lymphoid or bone marrow lineage. The term "immune cell" and other grammatical forms thereof may refer to immune cells of any origin. For example, immune cells may be derived from blood, such as autologous T cells, allogeneic T cells, autologous NK cells, xenogeneic NK cells, or may be derived from cell lines, such as NK cell line prepared by infection with EBV virus, NK cells and NK92 cell line induced from embryonic stem cells and iPSC. The term "immune cell" may also be of human or non-human.

[0138] 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.

[0139] 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. In a preferred embodiment, the immune cell according to the first aspect of the invention 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.

[0140] 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.

[0141] “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.

[0142] 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 co-express 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.

[0143] 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.

[0144] 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 HLA-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+.

[0145] 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.

[0146] In a more preferred embodiment, the cell according to the invention is selected from a T cell and a precursor thereof. 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).

[0147] In a particular embodiment, T cell according to the invention is a primary T cell.

[0148] 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.

[0149] Method for obtaining a cell expressing a bispecific antibody

[0150] The second aspect of the invention relates to an ex vivo method for obtaining a cell expressing a bispecific antibody, the method comprising transducing the cell or precursor thereof with a polynucleotide encoding said bispecific antibody, or a vector comprising the same, wherein said bispecific antibody comprises:

[0151] - an anti-BCMA single chain fragment variable (scFv), and

[0152] - an anti-CD3 single chain fragment variable (scFv), wherein:

[0153] - the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 1 , 2 and 3, or a functionally equivalent variant thereof,

[0154] - the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 4, SAS and SEQ ID NO: 6, or a functionally equivalent variant thereof, - the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 7, 8 and 9, or a functionally equivalent variant thereof, and

[0155] - the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 10, DTS and SEQ ID NO: 12, or a functionally equivalent variant thereof, wherein the cell is selected from a group consisting of an immune cell, a somatic cell and a progenitor cell.

[0156] Another aspect of the invention relates to an ex vivo method for obtaining a cell expressing a bispecific antibody, the method comprising transducing the cell or precursor thereof with a polynucleotide encoding said bispecific antibody, or a vector comprising the same, wherein said bispecific antibody comprises:

[0157] - an anti-BCMA single chain fragment variable (scFv), and

[0158] - an anti-CD3 single chain fragment variable (scFv), wherein:

[0159] - the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 1 , 2 and 3, or a functionally equivalent variant thereof,

[0160] - the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 4, SAS and SEQ ID NO: 6, or a functionally equivalent variant thereof,

[0161] - the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 7, 8 and 9, or a functionally equivalent variant thereof, and

[0162] - the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 10, DTS and SEQ ID NO: 12, or a functionally equivalent variant thereof.

[0163] In a particular embodiment, the method according to the second aspect of the invention is characterized in that:

[0164] - the FR1 , FR2, FR3 and FR4 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 13, 14, 15 and 16, or a functionally equivalent variant thereof, - the FR1 , FR2, FR3 and FR4 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 17, 18, 19 and 20, or a functionally equivalent variant thereof,

[0165] - the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 21 , 22, 23 and 24, or a functionally equivalent variant thereof, and

[0166] - the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 25, 26, 27 and 28, or a functionally equivalent variant thereof.

[0167] In a particular embodiment the functionally equivalent variant of the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 49, 50, 51 , and 52. In another particular embodiment the functionally equivalent variant of the FR1 , FR2, FR3 and FR4 regions within the anti- CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 53, 54, 55, and 56.

[0168] In a more particular embodiment, the polynucleotide according to the second aspect of the invention further comprises a region encoding a signal peptide preceding and in the same reading frame as the bispecific antibody.

[0169] In another embodiment, the signal peptide according to the second aspect of the invention is the human kappa light chain signal peptide, which comprises the sequence SEQ ID NO: 35.

[0170] In another embodiment, the bispecific antibody which is encoded by the polynucleotide expressed by the cells according to the first aspect of the invention is characterized in that:

[0171] - the anti-BCMA heavy chain variable domain VHBCMA comprises the amino acid sequence SEQ ID NO: 29, or a functionally equivalent variant thereof,

[0172] - the anti-BCMA light chain variable domain VLBCMA comprises the amino acid sequence SEQ ID NO: 30, or a functionally equivalent variant thereof, - the anti-CD3 heavy chain variable domain VHCD3 comprises the amino acid sequence SEQ ID NO: 31 or 57, or a functionally equivalent variant thereof, and

[0173] - the anti-CD3 light chain variable domain VLCD3 comprises the amino acid sequence SEQ ID NO: 32 or 58, or a functionally equivalent variant thereof.

[0174] In a particular embodiment, the anti-BCMA scFv according to the second aspect of the invention is located N-terminal with respect to the anti-CD3 scFv. In a particular embodiment, the anti-BCMA scFv according to the second aspect of the invention is located C-terminal with respect to the anti-CD3 scFv.

[0175] In a more particular embodiment, the VH region of the anti-BCMA scFv (VHBCMA) according to the second aspect of the invention is located N-terminal with respect to the VL (VLBCMA), and / or wherein the VH region of the anti-CD3 scFv ( HCD3) is located N- terminal with respect to the L (VLCD3).

[0176] In a preferred embodiment, the VH region of the anti-BCMA scFv (VHBCMA) according to the first aspect of the invention is located C-terminal with respect to the VL (VLBCMA), and / or wherein the VH region of the anti-CD3 scFv (VHCD3) is located C-terminal with respect to the VL (VLCD3).

[0177] In another embodiment, the variable heavy chain regions (VH) and the variable light chain regions (VL) regions according to the second aspect of the invention are arranged, from N-terminus to C-terminus, in the order VHBCMA-VLBCMA-VHCD3-VLCD3.

[0178] In a particular embodiment, the variable heavy chain regions (VH) and the variable light chain regions (VL) regions according to the first aspect of the invention are arranged, from N-terminus to C-terminus, in the order VLBCMA-VHBCMA-VLCD3-VHCD3.

[0179] In preferred embodiment, the bispecific antibody according to the second aspect of the invention is characterized in that: the anti-BCMA single chain fragment variable (scFv) comprises the sequence SEQ ID NO: 33, and the anti-CD3 single chain fragment variable (scFv) comprises the sequence SEQ ID NO: 34 or SEQ ID NO: 59.

[0180] In a particular embodiment, the VH and VL regions of the anti-BCMA scFv, the VH and VL regions of the anti-CD3 scFv, and / or the anti-BCMA scFv and anti-CD3 scFv according to the second aspect of the invention are connected by a peptide linker.

[0181] In a more particular embodiment, the linker according to the second aspect of the invention is selected from SEQ ID NO: 36 or 38. In another particular embodiment, the linker according to the first aspect of the invention has a sequence according to (GGGGS)n, wherein n is equal to or bigger than 1 , 2, 3, 4, 5.

[0182] In a particular embodiment, the bispecific antibody according to the second aspect of the invention comprises the amino acid sequence SEQ ID NO: 39 or SEQ ID NQ:60.

[0183] In a preferred embodiment, the bispecific antibody according to the second aspect of the invention further contains a polypeptide tag. Preferably, the tag is located at the C- terminus of the bispecific antibody. More preferably, the tag is a hexahistidine tag, a 2A peptide according to SEQ ID NO: 43 (T2A), and / or a tdTomato protein.

[0184] In another embodiment, the polynucleotide according to the second aspect of the invention that encodes the bispecific antibody has been introduced into the cell by transducing said cell with a viral vector that contains said polynucleotide.

[0185] In a preferred embodiment, the polynucleotide according to the second aspect of the invention that encodes the bispecific antibody is an RNA polynucleotide that has been introduced into the cell by transfecting said cell with said RNA polynucleotide.

[0186] In a more preferred embodiment, the cell according to the invention is selected from a group consisting of an immune cell, a somatic cell and a progenitor cell. Preferably, 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. More preferably, the cell is selected from a T cell and a precursor thereof. Preferably, T cell is a primary T cell.

[0187] All the terms and embodiments described in the first aspect of the invention are equally applicable to the third aspect of the invention.

[0188] Cell obtained by the method of the invention and pharmaceutical composition comprising the cell of the invention.

[0189] The third aspect of the invention relates to a cell obtainable by the method of the second aspect of the invention.

[0190] The fourth aspect of the invention relates to a pharmaceutical composition comprising the cell according to the first aspect of the invention and the third aspect of the invention, and at least one pharmaceutically acceptable excipient.

[0191] 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.

[0192] Pharmaceutical compositions and formulations as described herein can be prepared by mixing the active ingredients having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22nd edition, 2012), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and 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 nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutralactive hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.).

[0193] The expression “pharmaceutically acceptable excipient”, as used herein, includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible with the cells and the bispecific antibody according to the invention (e.g., SCE, mAbs, nucleic acids, vectors).

[0194] All the terms and embodiments described in any of the previous aspects of the invention are equally applicable to the third and fourth aspects of the invention.

[0195] Medical uses of the cells according to the invention

[0196] In a particular embodiment, the cell according to the first aspect of the invention and the third aspect of the invention, for use as a medicament.

[0197] The term “medicament” as used herein refers to a composition comprising a therapeutically effective amount of an agent, preferably an anti-cancer agent, more preferably a DNA damaging agent. The medicament also comprises at least one pharmaceutically acceptable excipient or carrier. In a particular embodiment, the cell according to the first aspect of the invention and the third aspect of the invention, for use in the treatment of cancer.

[0198] As used herein, the terms "treat", "treatment" or "amelioration". The term refers 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.

[0199] The term "cancer" 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 signalling, 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. 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, leukemia, 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, hematological malignancy

[0200] In a particular embodiment, the cell according to the first aspect of the invention and the third aspect of the invention, for use in the treatment of an hematological malignancy.

[0201] As used herein, the term “malignancy” refers to tumor capacity to spread to or invade the tissue in which it is located, the adjacent or distal tissues, being life-threatening to the subject having the tumor.

[0202] As used herein, the term “hematological malignancy” refers to any type of cancer that affects blood, bone marrow, and / or lymph nodes. As the three are intimately connected, a disease affecting one of the three will often affect the others as well. Although lymphoma is technically a disease of the lymph nodes, it often spreads to the bone marrow, affecting the blood. Hematological malignancies include, but are not limited to multiple myeloma, leukemias, and lymphomas.

[0203] In a more particular embodiment, the cell according to the first aspect of the invention and the third aspect of the invention, for use in the treatment of an hematological malignancy, wherein the hematological malignancy is characterized by the presence of malignant cells which exhibit BCMA expression on their surface.

[0204] In a preferred embodiment, the cell for use according to the third aspect of the invention wherein said hematological malignancy is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute B cell lymphoblastic leukemia, minimal residual disease (MRD)-positive ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), lymphoplasmacytic lymphoma (LPL), B cell lymphoma, diffuse large B- cell lymphoma (DLBCL), Burkitt’s lymphoma (BL), primary mediastinal large B-cell lymphoma (PMBL), marginal zone B cell lymphoma, Hodgkin’s lymphoma (HL), nonHodgkin’s lymphoma (NHL), NK- and T-cell neoplasms, histiocytic neoplasms, mantle cell lymphoma (MCL), hairy cell leukemia (HCL), plasma cell myeloma (PCM), plasma cell leukemia (PCL), and multiple myeloma (MM).

[0205] In a preferred embodiment, the cell for use according to the third aspect of the invention wherein the cell is administrated in a therapeutic effective dose. In another preferred embodiment, the therapeutic effective dose of cells is between about 0.5x104to about 1 xio10, preferably between about 0.5X105to about 1 X 109, between about 0.5x106to about 1 xio8.

[0206] All the terms and embodiments described in any of the previous aspects of the invention are equally applicable to medical uses of the invention.

[0207] Bispecific antibodies

[0208] In another aspect the invention relates to a bispecific antibody comprising:

[0209] - an anti-BCMA single chain fragment variable (scFv), and

[0210] - an anti-CD3 single chain fragment variable (scFv), wherein:

[0211] - the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 1 , 2 and 3, or a functionally equivalent variant thereof,

[0212] - the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 4, SAS and SEQ ID NO: 6, or a functionally equivalent variant thereof,

[0213] - the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 7, 8 and 9, or a functionally equivalent variant thereof, and

[0214] - the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 10, DTS and SEQ ID NO: 12, or a functionally equivalent variant thereof.

[0215] In a preferred embodiment, the bispecific antibody is characterized in that:

[0216] - the FR1 , FR2, FR3 and FR4 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 13, 14, 15 and 16, or a functionally equivalent variant thereof,

[0217] - the FR1 , FR2, FR3 and FR4 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 17, 18, 19 and 20, or a functionally equivalent variant thereof,

[0218] - the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 21 , 22, 23 and 24, or a functionally equivalent variant thereof, and

[0219] - the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 25, 26, 27 and 28, or a functionally equivalent variant thereof.

[0220] In a particular embodiment the functionally equivalent variant of the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 49, 50, 51 , and 52. In another particular embodiment the functionally equivalent variant of the FR1 , FR2, FR3 and FR4 regions within the anti- CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 53, 54, 55, and 56. In another embodiment, the bispecific antibody is characterized in that:

[0221] - the anti-BCMA heavy chain variable domain VHBCMA comprises the amino acid sequence SEQ ID NO: 29, or a functionally equivalent variant thereof,

[0222] - the anti-BCMA light chain variable domain VLBCMA comprises the amino acid sequence SEQ ID NO: 30, or a functionally equivalent variant thereof,

[0223] - the anti-CD3 heavy chain variable domain VHCD3 comprises the amino acid sequence SEQ ID NO: 31 or 57, or a functionally equivalent variant thereof, and

[0224] - the anti-CD3 light chain variable domain VLCD3 comprises the amino acid sequence SEQ ID NO: 32 or 58, or a functionally equivalent variant thereof.

[0225] In another embodiment, the bispecific antibody is characterized in that the anti-BCMA scFv is located N-terminal with respect to the anti-CD3 scFv.

[0226] In another embodiment, the bispecific antibody is characterized in that the VH region of the anti-BCMA scFv (VHBCMA) is located N-terminal with respect to the VL ( LBCMA), and / or wherein the VH region of the anti-CD3 scFv (VHCD3) is located N-terminal with respect to the VL (VLCD3). In another preferred embodiment, the VH region of the anti- BCMA scFv (VHBCMA) according to the first aspect of the invention is located C-terminal with respect to the VL (VLBCMA), and / or wherein the VH region of the anti-CD3 scFv (VHCD3) is located C-terminal with respect to the VL (VLCD3).

[0227] In another embodiment, the bispecific antibody is characterized in that the variable heavy chain regions (VH) and the variable light chain regions (VL) regions are arranged, from N-terminus to C-terminus, in the order VHBCMA-VLBCMA-VHCD3-VLCD3. In a particular embodiment, the variable heavy chain regions (VH) and the variable light chain regions (VL) regions according to the first aspect of the invention are arranged, from N-terminus to C-terminus, in the order VHBCMA-VLBCMA-VHCD3-VLCD3.

[0228] In another embodiment, the bispecific antibody is characterized in that the:

[0229] - the anti-BCMA single chain fragment variable (scFv) comprises the sequence SEQ ID NO: 33, and the anti-CD3 single chain fragment variable (scFv) comprises the sequence SEQ ID NO: 34 or SEQ ID NO:59.

[0230] In another embodiment, the bispecific antibody is characterized in that the VH and VL regions of the anti-BCMA scFv, the VH and L regions of the anti-CD3 scFv, and / or the anti-BCMA scFv and anti-CD3 scFv are connected by a peptide linker.

[0231] In another embodiment, the bispecific antibody is characterized in that the linker is selected from SEQ ID NO: 36 or 38. In another particular embodiment, the linker according to the first aspect of the invention has a sequence according to (GGGGS)n, wherein n is equal to or bigger than 1 , 2, 3, 4, 5.

[0232] In yet another embodiment, the bispecific antibody is characterized in that it comprises the amino acid sequence SEQ ID NO: 39 or SEQ ID NQ:60.

[0233] In yet another embodiment, the bispecific antibody is characterized in that it further contains a polypeptide tag. Preferably, the tag is located at the C-terminus of the bispecific antibody. More preferably, the tag is a hexahistidine tag, a 2A peptide according to SEQ ID NO: 43 (T2A), and / or a tdTomato protein.

[0234] The terms ” bispecific antibody”, “ScFv” “CDR”, “FR”, “variable region, “functionally equivalent variants of a CDR”, “functionally equivalent variants of a FR”, “peptide linker”, “tag” have been defined above in the context of the cells of the invention and are equally applicable to the antibodies of the invention as defined in the present section.

[0235] Polynucleotides encoding the bispecific antibodies and expression vectors

[0236] In another aspect, the invention relates to polynucleotide encoding the bispecific antibody according to the invention and as defined in the previous section. In one embodiment the polynucleotides according to the invention further comprises a region encoding a signal peptide preceding and in the same reading frame as the bispecific antibody. In yet another embodiment, the signal peptide is the human kappa light chain signal peptide, which comprises the sequence SEQ ID NO: 35. The terms “polynucleotide” and “signal peptide”, have been defined above in the context of the cells of the invention and are equally applicable to the polynucleotides of the invention as defined in the present section.

[0237] In one embodiment, the polynucleotide according to the invention is an RNA molecule. The term “RNA molecule” has been defined in the above in the context of the cells of the invention and are equally applicable to the polynucleotides of the invention as defined in the present section.

[0238] In yet another embodiment, the polynucleotide of the invention forms part of an expression vector. In one embodiment, the expression vector is a viral expression vector. In yet another embodiment, the viral expression vector is a lentiviral vector.

[0239] The terms “viral vector” and ’’lentiviral vector” have been defined above in the context of the cells of the invention and are equally applicable to the polynucleotides of the invention as defined in the present section.

[0240] Pharmaceutical compositions comprising the antibodies and polynucleotides of the invention and medical uses of the antibodies and polynucleotides of the invention

[0241] In another aspect, the invention relates to a pharmaceutical composition comprising the bispecific antibody of the invention, the polynucleotide according to the invention or the expression vector according to the invention and at least one pharmaceutically acceptable excipient.

[0242] The terms ’’pharmaceutical composition” and ’’pharmaceutically acceptable excipient” have been defined above in the context of the cells of the invention and are equally applicable to the pharmaceutical compositions of the invention as defined in the present section.

[0243] In another aspect, the invention relates to a pharmaceutical composition comprising the bispecific antibody of the invention, the polynucleotide according to the invention or the expression vector according to the invention for use as a medicament. In another aspect, the invention relates to a pharmaceutical composition comprising the bispecific antibody of the invention, the polynucleotide according to the invention or the expression vector according to the invention for use in the treatment of cancer.

[0244] In another aspect, the invention relates to a pharmaceutical composition comprising the bispecific antibody of the invention, the polynucleotide according to the invention or the expression vector according to the invention for use in the treatment of an hematological malignancy.

[0245] In a preferred embodiment, the hematological malignancy is characterized by the presence of malignant cells which exhibit BCMA expression on their surface. In another embodiment, the hematological malignancy is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute B cell lymphoblastic leukemia, minimal residual disease (MRD)-positive ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), lymphoplasmacytic lymphoma (LPL), B cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt’s lymphoma (BL), primary mediastinal large B- cell lymphoma (PMBL), marginal zone B cell lymphoma, Hodgkin’s lymphoma (HL), nonHodgkin’s lymphoma (NHL), NK- and T-cell neoplasms, histiocytic neoplasms, mantle cell lymphoma (MCL), hairy cell leukemia (HCL), plasma cell myeloma (PCM), plasma cell leukemia (PCL), and multiple myeloma (MM).

[0246] The terms “medicament”, “treatment”, “cancer", “malignancy” have been defined above in the context of the cells of the invention and are equally applicable to the therapeutic methods of the antibodies, polynucleotides and vectors of the present invention.

[0247] The invention is described below by means of the following examples which are to be construed as merely illustrative and not limitative of the scope of the invention.

[0248] EXAMPLES

[0249] Materials and Methods

[0250] Experimental design

[0251] The study was designed to develop and characterize engineered T cells secreting BCMAxCD3 TCEs (STAb-T BCMA) and to conduct a comprehensive preclinical study comparing their therapeutic potential with T cells expressing anti-BCMA CARs (CAR-T BCMA), in a T cell-limiting experimental setting mimicking the conditions found in R / R MM patients. The study was performed with human cell lines and human primary T cells. We used in vitro and in vivo models to evaluate the ability of STAb-T BCMA cells to prevent tumor scape and perform potent cytotoxic responses at low E:T ratios. Every experiment was performed comparing STAb-T BCMA cells activity to CAR-T BCMA. Non-transduced T cells were used as negative control. The number of replicates for each in vitro experiment to ensure adequate statistical power are indicated in the figure legends. For in vivo experiments using NSG mice, animals were randomly assigned to each treatment group.

[0252] Cell lines and culture conditions

[0253] Cell lines used are detailed in Table 1. Cells were purchased from American Tissue Culture Collection (ATCC, Manassas, VA, USA). ARP1 cell line was provided by Multiple Myeloma Research Center (Little Rock, AK, USA). Suspension cell lines were maintained in RPMI-1640 (Cat. No. BE12-167F, Lonza, Walkersville, MD, USA) supplemented with 2 mM L-glutamine, heat-inactivated 10% FBS, and antibiotics. Adherent cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) (Cat. No. BE12-709F, Lonza) supplemented with 2 mM L-glutamine (Cat. No. 25030081 , Life Technologies, Paisley, UK), 10% (vol / vol) heat inactivated fetal bovine serum (FBS, Cat. No. F7524) and antibiotics (100 units / mL penicillin, 100 pg / mL streptomycin, Cat. No. P4333, both from Sigma-Aldrich, St. Louis, MO, USA).

[0254] Table 1. Cell lines Vector construction

[0255] The pCCL-BCMA-4-1 BB-CD3z-T2A-tdTomato vector (BCMA-CAR) has been previously described and clinically validated. For the lentiviral vector pCCL-BCMA-OKT3-His-T2A- tdTo construction, the humanized anti-BCMA scFv (J22.9) was subcloned in pCDNA3.1 expression vector. OKT3-m / h26, from pCR3.1-Ega1-OKT3 vector was subcloned as Not\ / Xba\ into pcDNA3.1-hBCMA-ScFv vector resulting in pCDNA3.1-BCMA-OKT3-m / h. To obtain the lentiviral vector pCCL-BCMA-OKT3-His-T2A-tdTo, a synthetic gene encoding the C-terminal polyHis (HHHHHH - SEQ ID NO: 44) tag, followed by 2A sequence from Thosea asigna virus (T2A) and the reporter red fluorescent protein tdTomato (tdTo) was synthesized by Geneart AG (ThermoFisher Scientific, Regensburg, Germany), and BCMA-OKT3 encoding fragment was subcloned as Bgl\\ / BstB\ in this vector and then, the whole fragment encoding BCMA-OKT3-His-T2A-tdTo was subcloned into lentiviral pCCL vector as BamH\ / BstB\ to obtain the final pCCL-BCMA- OKT3-His-T2A-tdTo vector.

[0256] Lentiviral particle production and titration

[0257] To produce lentiviral particles for pre-clinical in vitro and in vivo studies, HEK293T cells were transfected with transfer vectors together with packaging plasmids pMDLg / pRRE (Cat. No. PF1083, Plasmid Factory, Bielefeld, Nordrhein-Westfalen, Germany), pRSVrev (Cat. No. PF1084, Plasmid Factory), and envelope plasmid pMD2.G (Cat. No. PF096, Plasmid Factory), using linear polyethyleneimine of molecular weight 25,000 (Cat. No. 23966-1 , Polysciences, Warrington, PA, USA). After 72 hours, viral supernatants were collected, clarified by centrifugation, filtrated using a 0.45 pm-pore filter, and ultracentrifuged for 2.5 hours at 26,000 rpm. Pellets containing the lentiviral particles were resuspended in RPMI-1640 (Lonza), aliquoted and stored at -80 °C until use. Functional titers of BCMA-TCE and BCMA-CAR encoding lentiviral vectors were determined by limiting dilution in Jurkat and HEK293T cells and analyzed using tdTo expression by flow cytometry.

[0258] T cell transduction

[0259] Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of volunteer healthy donors by density gradient centrifugation using Lymphoprep™ (Cat. No. AXS-1114544, Axis-Shield, Oslo, Norway). All donors provided written informed consent in accordance with the Declaration of Helsinki and approved by the Institutional Research Ethics Committees of the hospitals and research centers involved (HCB / 2019 / 0450, HCB / 2018 / 0030). Cells were plate-coated activated with 1 pg / ml anti- CD3 (OKT3 clone, Cat. No. 555336, BD Biosciences, Franklin Lakes, NJ, USA) and 1 pg / ml anti-CD28 (CD28.2 clone, Cat. No. 555725, BD Biosciences) mAbs for 2 days and transduced at 10 or 20 in vitro and in vivo assays respectively) of multiplicity of infection (MOI) with BCMA CAR- or BCMA TCE-encoding lentiviral vectors in the presence of 10 ng / ml interleukin (IL)-7 (Cat. No. 30-095-367, Miltenyi Biotec, Bergisch Gladbach, Germany) and 10 ng / ml IL-15 (Cat. No. 30-095-764 Miltenyi Biotec). As negative controls, non-transduced primary T cells (NT-T) were used. A period of cell expansion of 6-8 days was carried out before conducting experiments. Jurkat T cell transduction is detailed in Supplementary Materials and Methods.

[0260] Flow cytometry

[0261] Antibodies used for flow cytometry analysis are detailed in Table 2. Cell surface expression of BCMA-CAR was analyzed by incubation with recombinant human BCMA / TNFRSF17 Fc Chimera Protein (Cat. No. RND-193-BC-050; R&D Systems, Minneapolis, MN, USA) followed incubation by Brilliant Violet (BV421)-conjugated antihuman IgG-Fc specific antibody (Cat. No. 410704; Biolegend, San Diego, CA, USA), alternatively, it was estimated based on tdTo fluorescent protein expression. BCMA-TCE surface decoration and intracellular expression was assessed using an A PC-conjugated anti- His mAb (Cat. No. 130-119-782; Miltenyi Biotec) alternatively, it was estimated based on tdTo expression. Intracellular BCMA-TCE was detected using the Inside Stain Kit (Cat. No. 130-090-477, Miltenyi Biotec) following manufacturer’s instructions. Cell acquisition was performed in a BD FACSCAnto II flow cytometer using BD FACSDiva software (BD Biosciences, San Jose, CA, USA). Alternatively, a DxFIex flow cytometer (Beckman Coulter, Brea, CA, USA) was used. Analysis was performed using FlowJo V10 software (Tree Star, Ashland, OR, USA).

[0262] Table 2. Antibodies for flow cytometry (FC), and immunofluorescence (IF) microscopy

[0263]

[0264] Cytotoxicity assays

[0265] For cytotoxicity assays, activated T (A-T) cells, either non-transduced (NT-T), or transduced (CAR-T or STAb-T), were co-cultured with or without freshly isolated non- activated T cells (NA-T) and luciferase-expressing tumor target cells (U266Luc, ARP1Lucor K562Luc) at the indicated E:T ratios. After 48 hours, supernatants were collected and stored at -20 °C for further IFNy secretion analysis, and D-luciferin (Cat. No. E1602, Promega, Madison, Wl, USA) was added to cells to a final concentration of 20 pg / ml before bioluminescence quantification in relative light units (RLU) using a Victor luminometer (PerkinElmer, Waltham, MA, USA). The value for spontaneous lysis was obtained by incubating the target cells with NT-T or with NA-T effector cells only. Percent tumor cell viability was calculated as the mean bioluminescence of each sample divided by the mean bioluminescence of the input number of control target cells times 100. Specific lysis was established as 100% cell viability. For cytotoxic studies in non- contacting transwell systems, polycarbonate filter inserts (4.26 mm diameter) with 0.4 pm pores (Cat. No. CLS3381-1 EA; Corning, Kennebunk, Me, USA) were used. In this experiments, luciferase-expressing targets cells (5 x 104) were plated on bottom wells with 5 x 104ThO cells (1 :1 E:T ratio), and A-T cells (NT-T, CAR-T, STAb-T cells) at the indicated ratios were added to transwell inserts. After 48 hours bioluminescence was quantified as described in Methods. For cytotoxicity assays in presence of soluble BCMA, U266Luccells (5 x 104) were plated at 1 :1 E:T ratio with 5 x 104A-T cells (NT-T, CAR-T, STAb-T) and increasing concentrations of recombinant human BCMA / TNFRSF17 Fc Chimera Protein (Cat. No. RND-193-BC-050; R&D Systems, Minneapolis, MN, USA) were added in the culture medium (from 0 to 5ug / mL). After 48 hours bioluminescence was quantified as described in Methods. For real-time cytotoxicity assays, the xCELLigence RTCA DP system (Acea BioSciences, San Diego, CA) was used. 1x104wild-type HEK293 or stably transfected BCMA-expressing HEK293 cells (HEK293BCMA+) were plated in an E-Plate 16 (Cat. No. 05469813001 , Acea Biosciences, San Diego, CA) and cultured at 37 °C and 5% CO2. After 20 hours, NT-T, CAR-T BCMA or STAb-T cells were added at different E:T ratios and cell index values were measured every 15 min for 48 hours using RTCA Software 2.0 (Acea Biosciences, San Diego, CA). In another set of experiments, transduced (CAR-T or STAb-T) or NT activated T cells were co-cultured with primary MM BM cells o PBMCs from a patient diagnosed with an aggressive PCL at the indicated E:T ratios. After 24 hours, cells were stained for 30 minutes at 4 °C with CD2-PE, CD3-PE-Cy5.5, BCMA-AlexaFluor647, CD38-APC-Cy7, CD138-PE-Cy7, CD56-FITC, CD45-V500 ( Table 2), and DAPI (catalog no. D9542-10MG, Sigma) in 50 pL PBS-0.5% FBS, in TruCount Absolute Counting Tubes (Cat. No. 340334, BD Biosciences). Then, samples were diluted by adding 450 pL of PBS and gently mixed before proceeding to FACS analysis. Cytotoxicity was determined by analyzing the residual live (DAPI-negative) target cells.

[0266] Cytokine secretion analysis

[0267] IFNy secretion was analyzed by ELISA (Cat. No. 950.000.096; Diaclone, Besancon Cedex, France).

[0268] Immunofluorescence and confocal microscopy

[0269] Jurkat effector T cells (J-NT-T, J-CAR-T or J-STAb-T) were incubated at 37 °C with U266 target cells at an E:T ratio 1 :1 for 15 minutes. As a positive control, a co-culture of 1.5 x 105J-NT-T cells and 1.5 x 105Raji SEE-loaded cells (pre-incubated with CFSE 1 pM) were used. Jurkat / U266 conjugates (1.5 x 105cells each) were fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS during 5 minutes at room temperature. Cells were then permeabilized with 0.1 % Triton X-100 (Sigma-Aldrich) during 5 minutes at room temperature followed by blocking with 10 pg / mL human y-globulin for 20 minutes at room temperature. Samples were stained with the antibodies indicated in Table 2 for 1 hour at room temperature. Then, cells were washed with TBS (Tris 20 mM, NaCI 150 mM) and incubated with Alexa Fluor™ 405 a-rabbit secondary antibody and phalloidin- Alexa Fluor™ 647 at room temperature for 30 minutes. Finally, the coverslips were washed twice with TBS and once with distilled water before being mounted with Mowiol medium. Confocal sections of fixed samples were acquired using a Leica SP-8 confocal scanning laser microscopy with a 60X / 1.35 oil immersion objective. Alexa Fluor™ 405 and phalloidin-Alexa Fluor™ 647 were excited with 405 and 633 nm laser lines, respectively. Image acquisition was automatically optimized with the Leica SP-8 confocal scanning laser microscopy software to get an image resolution of 58 nm / pixel. Analysis of images was conducted with Imaged freeware (National Institutes of Health). The polarization of pTyr and F-actin at the immunological synapse (IS) was estimated with the Synapse Measure plugin of Imaged. Actin clearance value from each individual synapse is calculated as the ratio between the central actin cleared area and the total area of the interface obtained from the 3D reconstruction.

[0270] Xenograft animal model studies

[0271] NSG mice were irradiated (2 Gy) and infused intravenously (i.v.) with 1 x 106U266Luccells. After 3 days mice were randomly allocated to six different groups and received either 5 x 106or 3 x 106NT-T, CAR-T (20% BCMA-CAR+) or STAb-T (20% BCMA-TCE+) cells. Tumor growth was evaluated weekly by bioluminescence imaging as previously described (29). Briefly, 150 mg / Kg of D-Luciferin (Cat. No. E1605, Promega, Madison, Wl, USA) was administrated intraperitoneally in 200 pL of sterile PBS. Animals were imaged 10 minutes after D-Luciferin injection using the Xenogen I VIS Lumina II imaging system (Caliper Life 374 Sciences, Waltham, MA, USA). The photon flux emitted by the luciferase-expressing cells was measured as an average radiance (photons / sec / cm2 / sr). In vivo studies were carried out at the Barcelona Biomedical Research Park (PRBB) in accordance with the guidelines of the Animal Experimentation Ethics Committee. All procedures were performed in compliance with the institutional animal care committee of the PRBB (DAAM9624). Tumor burden, T cell persistence and % of tdTo+T cells in peripheral blood were analyzed by flow cytometry at day 28 and / or at euthanization. Bone marrow (spine / sternum and femur) and spleen samples were collected after euthanization and analyzed by flow cytometry. Body weight was monitored over time. Animals showing endpoint clinical disease or GVHD signs were euthanized. Human BCMA relative gene expression in BM was analyzed by qRT-PCR. Soluble BCMA was assessed in mice plasma samples collected at endpoint.

[0272] Quantitative real-time PCR

[0273] Total RNA from BM mice samples was isolated with the RNeasy Micro Kit (Cat. No. 74004, Qiagen, Hilden, Germany) and cDNA was synthesized using NZY First Strand cDNA Synthesis Kit (Cat. No. MB125, Nzytech, Lisbon, Portugal). qRT-PCR was performed with LightCycler480 SYBR Green I Master Kit (Cat. No. 04707516001 , Roche Diagnostics, Mannheim, Germany) on a LightCycler system (Roche Diagnostics, Basel, Switzerland). Each sample was analyzed in triplicate and fold-expression changes were calculated with the equation 2-ACt. Human succinate dehydrogenase gene expression was used to normalize. The primers used (synthetized by Roche Diagnostics, Basel, Switzerland) are detailed in Table 3.

[0274] Table 3. Quantitative real-time PCR primers

[0275] Soluble BCMA detection in culture supernatants

[0276] For soluble BCMA assessment in culture samples the human BCMA / TNFRSF17 DuoSet ELISA kit (Cat. No. DY193, R&D Systems, Minneapolis, MN, USA) was used.

[0277] Flow cytometric characterization of T lymphocyte subsets

[0278] Preinfused STAb-T and CAR-T cell products, whole BM and spleen specimens from STAb-T- and CAR-T-treated mice were stained with the 30-color T cell module of the Euroflow-lmmune monitoring (IMM) antibody panel adapted for the simultaneous measurement of tdTo-associated fluorescence of both STAb-T and CAR-T cells, and the FITC-labeled BCMA-Fc (Cat. No. CD8-HF255, ACROBiosystems). Samples were labelled using the EuroFlow standard operating procedures for staining of cell surface membrane markers-only (41), available at www. EuroFlow.org, plus the BCMA-FITC protein, aimed at simultaneous immunophenotypic identification of both transduced STAb-T (tdTo+BCMA-TCE+) or CAR-T (tdTo+BCMA-CAR+) cells and NT (tdTo’) human T cells, and their most relevant maturation-associated and functional subsets ( Table 4). Stained samples were measured in a 5-laser Aurora cell sorter (Aurora CS, Cytek Biosciences, Fremont, CA) using the SpectroFlo® software (Cytek Biosciences). For data analysis and T cell subsetting (Table 4), the Infinicyt™ software (Cytognos SL, Salamanca, Spain) was used. LIMAP (Uniform Manifold Approximation and Projection) plots of the various T cell subset profiles were built with the R (version 4.3.1) and the UWOT software packages (42).

[0279] Table 4. List of T cell populations identified and their corresponding surrogate immunophenotypic features

[0280] MAIT, mucosa-associated invariant ? cells; EM, effector memory; TE, terminal effector; iNKT, invariant natural killer T cells; Treg, regulatory T cells; TFH, T follicular helper cells; Th, (classical) T helper; TCR, T cell receptor. Statistical Analysis

[0281] Results of experiments are expressed as mean or mean ± standard deviation (SD). Statistical tests indicated in figure legends and data processing were performed using GraphPad Prism version 7 (GraphPad Prism Software, La Jolla, CA, USA). Statistical significance was calculated by one-way or two-way ANOVA test corrected with a Tukey’s multiple comparisons test. Significance was considered only when P values were less than .05 (*P < .05; **P < .01 ; ***P < .001 , ****P < .0001).

[0282] Enzyme-linked immunosorbent assay

[0283] To detect the BCMA-TCE secreted to cell culture supernatants, recombinant human BCMA:Fc chimera (hBCMA-Fc) (Cat. No. RND-193-BC-050, R&D Systems, Minneapolis, MN, USA) was immobilized (3 pg / ml) on Maxisorp plates (Cat. No. M9410- 1CS, NUNC, Roskilde, Denmark) overnight at 4 °C. After washing and blocking, conditioned media was added and incubated for 1 hour at room temperature. Then wells were washed and anti-His mAb (PentaHis, Cat. No. 34650, Quiagen Hilden, Germany) was added (1 pg / ml) and incubated for 1 hour at room temperature. After washing, horseradish peroxidase (HRP)-conjugated Goat anti-Mouse IgG, Fc specific (Cat. No.1 15-085-166; Jackson Immunoresearch, West Grove, PA, USA) was added (0,4 pg / ml) and incubated for 1 hour at room temperature, and then the plate was developed using tetramethylbenzidine (TMB) (Cat. No. T0440, Sigma-Aldrich, St. Louis, MO, USA).

[0284] HEK293BCMA+cell line generation

[0285] HEK293 cell line was transfected with a pCMV3-BCMA-ORF vector including hygromycin resistance marker for further cell selection (Cat. No. SIB-HG10620-UT, Sino Biological, Beijing, China) and cultured in DMEM 10% FBS + hygromycin (150 ug / uL). All the cell lines were grown at 37 °C and 5% CO2 and were routinely screened for mycoplasma contamination by PCR using the Mycoplasma Gel Detection Kit (Cat. No. 90.021-4542, Biotools, Madrid, Spain).

[0286] Jurkat T cell transduction and culture conditions

[0287] Jurkat T cells (1 x 105) were transduced with BCMA CAR- or BCMA TCE-encoding lentivirus at MOI of 10. As negative controls, non-transduced Jurkat T cells (J-NT-T) were used. Cells were cultured in RPMI-1640 (Cat. No. BE12-167F, Lonza, Walkersville, MD, USA) supplemented with 2 mM L-glutamine, heat-inactivated 10% FBS, and antibiotics. Western blotting

[0288] Samples were separated under reducing conditions on 10-20% Tris-glycine gels (Cat. No. 250 XP 10202 BOX, Life Technologies, Carlsbad, CA, USA), transferred onto PVDF membranes (Cat. No. IPVH00010, Merck Millipore, Tullagreen, Carrigtwohill, Ireland) and probed with anti-His mAb (PentaHis, Cat. No. 34650, Quiagen, Hilden, Germany) (200 ng / ml), followed by incubation with horseradish peroxidase (HRP)-conjugated Goat anti-mouse IgG, Fc specific (0.8 pg / ml) (Cat. No. A2554, Sigma-Aldrich). For cell lysates analysis, purified mouse anti-human CD247 (clone 8D3, Cat. No. 551034, BD Biosciences) followed by incubation with horseradish peroxidase (HRP)-conjugated Goat anti-mouse IgG was used to CD3^D detection. Visualization of protein bands was performed with Pierce ECL Western Blotting substrate (Cat. No. 32132, Invitrogen, Rockford, IL, USA) using ChemiDoc™ MP Imaging System machine (Bio-Rad Laboratories, Hercules, CA, USA). BioRad Gel Doc Imaging System.

[0289] Enzyme-linked immunosorbent assay

[0290] To detect the BCMA-TCE secreted to cell culture supernatants, recombinant human BCMA:Fc chimera (hBCMA-Fc) (Cat. No. RND-193-BC-050, R&D Systems, Minneapolis, MN, USA) was immobilized (3 pg / ml) on Maxisorp plates (Cat. No. M9410- 1CS, NUNC, Roskilde, Denmark) overnight at 4 °C. After washing and blocking, conditioned media was added and incubated for 1 hour at room temperature. Then wells were washed and anti-His mAb (PentaHis, Cat. No. 34650, Quiagen Hilden, Germany) was added (1 pg / ml) and incubated for 1 hour at room temperature. After washing, horseradish peroxidase (HRP)-conjugated Goat anti-Mouse IgG, Fc specific (Cat. No.1 15-085-166; Jackson Immunoresearch, West Grove, PA, USA) was added (0,4 ng / ml) and incubated for 1 hour at room temperature, and then the plate was developed using tetramethylbenzidine (TMB) (Cat. No. T0440, Sigma-Aldrich, St. Louis, MO, USA).

[0291] Results

[0292] STAb-T cells efficiently secrete anti-BCMA TCEs and promote the formation of canonical immunological synapses

[0293] We generated a tandem anti-BCMA x anti-CD3 bispecific antibody (BCMA-TCE) by fusing the clinically validated anti-BCMA J22.9 scFv and the anti-CD3 OKT3 scFv through a G4S sequence (SEQ ID NO: 38) (Fig. 1 , A and B), which was cloned under the control of the EF1 a promoter into a T2A-based bicistronic lentiviral vector expressing the tdTomato (tdTo) reporter protein (Fig. 1C). The vector encoding an anti-BCMA second- generation CAR (Fig. 1 D) using the same anti-BCMA scFv (BCMA-CAR) (Fig. 1 E and F). Comparable transduction efficiencies were observed according to the percentage of tdTo+cells in Jurkat T cells transduced with BCMA-TCE- or BCMA-CAR-encoding lentiviral vectors (J-STAb-T and J-CAR-T, respectively) (Fig. 5A). The intracellular expression of BCMA-TCE and its interaction with CD3 on the surface of Jurkat T cells (a process called “CD3 decoration”) were verified with an anti-His-tag mAb (Fig. 5B), and the cell surface expression of BCMA-CAR was confirmed after staining with BCMA-Fc (Fig. 5C). The intracellular expression of both BCMA-CAR and BCMA-TCE (Fig. 5D and E) and the extracellular secretion of BCMA-TCE (Fig. 5F) were further confirmed by Western blotting. The secreted BCMA-TCE specifically recognized plastic-immobilized BCMA-Fc (Fig. 5G), and induced CD69 expression in co-cultures of J-STAb-T cells with BCMA+LI266 cells (Fig. 6A) at levels similar to those obtained in co-cultures with J-CAR- T cells (Fig. 5H). Importantly, the secretion of BCMA-TCE by J-STAb-T cells is not reduced in situations of persistent activation and upregulation of exhaustion markers (Fig. 5I and J).

[0294] Immunofluorescence assays were performed to study the assembly of the immunological synapse (IS) and early signaling by staining for filamentous actin (F- actin), as a marker of the distal supramolecular activation clusters (dSMAC), and phospho-Tyrosine (pTyr), respectively. CAR-T and STAb-T Jurkat cells were co-cultured with LI266 cells for 15 min to allow the assembly of the IS and superantigen-E (SEE)- loaded Raji cells co-cultured with non-transduced (NT) Jurkat T cells were used as a positive control. Confocal microscopy images showed how both J-CAR-T and J-STAb-T cells induced the assembly of the IS with LI266 cells. F-actin and pTyr were observed to polarize towards the IS. While the pTyr ratio did not vary significantly, the F-actin polarization ratio appeared to be slightly lower in BCMA-CAR-mediated IS than in BCMA- TCE-mediated-IS (Fig. 7 A-D). Typical hypodense actin network at the central area of the IS was not properly organized in J-CAR-T in comparison with J-STAb-T cells (Fig. 7E).

[0295] STAb-T cells induce specific cytotoxic responses at low effector: target ratios

[0296] Primary T cells were transduced with BCMA-CAR- or BCMA-TCE-encoding lentiviral vectors with comparable transduction efficiencies, ranging from 17 to 30% (average 22.7 and 21%, respectively) (Fig. 8A). No differences were observed in the CD4 / CD8 ratio between BCMA-CAR+and BCMA-TCE+cells (Fig. 8B), with comparable proportions of naive, central memory, effector memory and effector subsets among NT- T, CAR-T and STAb-T cells, and an overall prevalence of effector T cells (Fig. 8C). To study their specificity and killing efficiency, CAR-T and STAb-T effector cells were cocultured with BCMA+(U266Luc) or BCMA' (K562Luc) target cells (Fig. 6A) at different effectortarget (E:T) ratios. To better understand the contribution of transduced (tdTo+) effector T cells (CAR-T+and STAb-T+), and non-transduced (tdTo-) effector T cells (CAR- T' and STAb-T') in the functional responses, both total E:T and the transduced E:T ratios are depicted in Figure 11. CAR-T cells exhibited significant cytotoxicity (>80%) at higher ratios, which was drastically reduced at E:T ratios below 1 :1(transduced E:T 0.2:1) (Fig. 1G and 1 H). In contrast, STAb-T cells induced >80% specific cytotoxicity at all ratios tested, even at the lowest ratio corresponding to one T cell per ten target cells (Fig. 1G and H). In both CAR-T and STAb-T cell co-cultures IFNy secretion was detected at E:T ratios above 1 :1 , and it is remarkable that while in CAR-T cell co-cultures there is a strong correlation between cytotoxic activity and IFNy secretion, in STAb-T cell co-cultures at very low E:T ratios maximal cytotoxicity is reached without detectable levels of IFNy (Fig. 11). No cytotoxicity or IFNy secretion was observed when K562 cells were used as targets (Fig. 9A and B). 11266 cells produce significant amounts of soluble BCMA (sBCMA) under basal conditions (Fig. 6B), and as shown in Figure 1 J in co-cultures with CAR-T cells, sBCMA levels were reduced only at the higher E:T ratios whereas in cocultures with STAb-T cells sBCMA levels were practically undetectable in all conditions studied (Fig. 1J). The sBCMA levels were not modified in co-cultures with NT-T cells (Fig. 1J).

[0297] We next assessed the cytotoxic activity under stressed conditions (< 1 :1 E:T) in an impedance-based real-time assay against non-transfected and BCMA-transfected HEK293 cells (HEK293 and HEK293BCMA, respectively). In co-cultures with HEK293BCMAcells, which express significant levels of cell surface BCMA and sBCMA secretion (Fig. 6, C and D), STAb-T cells were found to mediate reduction of BCMA+target cell viability even at 0.25:1 E:T ratio, whereas CAR-T cells showed no cytotoxic effect at any of the analyzed ratios (Fig. 1 K). HEK293BCMAcells co-cultured with NT-T cells (Fig. 1 K), or HEK293 cells co-cultured with NT-T, CAR-T or STAb-T cells, displayed similar viability kinetics to those of target cells cultured alone (Fig. 10, A and B). To study the activity of CAR-T and STAb-T cells under stressed conditions (< 1 :1 E:T) in a clinically relevant context, we designed ex vivo cytotoxicity assays using BM cells of a MM patient (Fig. 11 A) and PBMCs of a patient with plasma cell leukemia (PCL) (Fig. 11 B) as target cells. After 24 hours co-culture, STAb-T cells were able to eliminate 70% of the MM-derived target cells (CD38+CD138+) in all the analyzed E:T ratios (from 1 :1 to 0.1 :1) (Fig. 1 L). CAR-T cells were significantly less effective with 30% of MM cell lysis at the higher E:T ratios (Fig. 1 L). Against PCL-derived target cells, STAb-T cells were significantly more effective than CAR-T cells and were able to eliminate nearly 100% of the PCL cells at the 0.5:1 and 1 :1 E:T ratios (Fig. 1 L).

[0298] STAb-T cells recruit bystander T cells and prevent tumor escape in vitro more efficiently than CAR-T cells

[0299] To investigate the ability of STAb-T cells to recruit bystander T cells, we co-cultured activated T (A-T) cells (NT-T, CAR-T or STAb-T), or mixtures of A-T and freshly isolated non-activated T (NA-T) cells from the same healthy donor with either BCMA+(U266Lucor ARP1Luc) or BCMA' (K562Luc) targets. The A-T and target cells were mixed at decreasing A-T:target ratios (from 1 :1 to 0.0001 :1) but adding NA-T cells to keep a constant 1 :1 E:T ratio. In co-cultures with U266Luccells, STAb-T cells exhibited 100% cytotoxicity even at the 0.01 :1 A-T:target ratio, whereas CAR-T cells only induced 60% cytotoxicity at the 1 :1 ratio, which was severely reduced at the 0.1 :1 ratio (Fig. 2A). No cytotoxicity or IFNy secretion was observed when mixtures of A-T and NA-T cells were co-cultured with K562LUCcells (Fig. 12, A-B). In co-cultures with ARP1Luccells, CAR-T cells displayed only 30% cytotoxic activity at the 1 :1 A-T:Target ratio, whereas STAb-T cells showed >90% specific cytotoxicity even at the 0.1 :1 ratio (Fig. 13A). In these experimental conditions, there is a strong correlation between cytotoxic activity and IFNy secretion (Fig. 2B and Fig. 13B). To further demonstrate that STAb-T cells were able to recruit bystander NA-T cells, LI266Lucor K562Luctarget cells were plated with NA-T cells in the bottom chamber of a transwell system, and NT-T, STAb-T or CAR-T cells were plated in the insert well. Cytotoxicity and IFNy secretion were fully dependent on the presence of STAb-T cells in the insert well and LI266Lucin the bottom chamber, indicating that secreted BCMA-TCEs crossed the transwell filter and effectively redirected the effector activity of NA-T cells to BCMA+cells (Fig. 2C and D and Fig. 14). Next, we evaluated the ability of CAR-T and STAb-T cells to prevent tumor escape under stressed conditions in co-cultures with LI266 cells at decreasing E:T ratios (from 1 :1 to 0.06:1). CAR-T cells did not control the growth of MM cells at any of the ratios studied (Fig. 2E). These results are similar to those obtained with ARP1 cells (Fig. 13C). The difference with respect to that reported in previous works with the same CAR construct (20) may be attributable to the lower transduction levels used in this work (around 20%). However, STAb-T cells were able to control the growth of LI266 cells even at the 0.25:1 ratio (Fig. 2E) and that of ARP1 cells at a 0.5:1 ratio (Fig. 13C).

[0300] Soluble BCMA has no impact on STAb-T-mediated cytotoxicity

[0301] To study the effect of sBCMA on effector functions, we co-cultured CAR-T and STAb-T cells with U266Luccells at a 1 :1 E:T ratio in the presence of increasing concentrations (from 0 to 5 pg / mL) of BCMA-Fc chimera (sBCMA-Fc). The cytotoxic activity of CAR-T cells decreased as the concentration of sBCMA exceeded 0.1 pg / mL with total inhibition at concentrations of 5 pg / mL (Fig. 2F). By contrast, the cytotoxicity of STAb-T cells was not compromised and remained at 100% at all concentrations of sBCMA-Fc studied (Fig. 2F). Interestingly, IFNy secretion was reduced with increasing sBCMA-Fc concentration in both STAb-T and CAR-T cells (Fig. 2G) consistent with the lower threshold for cytolysis compared with cytokine production.

[0302] STAb-T cells are more effective than CAR-T cells in in vivo models of MM

[0303] To study the in vivo effect of STAb-T cells in a xenograft model of MM, NSG mice were i.v. infused with 1 x 106U266Luccells, followed 3 days later by i.v. injection of PBS (control), or T cells. Three groups of mice received 5 x 106NT-T, CAR-T or STAb-T cells, referred to as the standard condition, while two other groups were inoculated with a limited number (3 x 106) of CAR-T or STAb-T cells, referred to as the therapeutic limiting dose (TLD) condition. In both conditions the percentage of transduced CAR-BCMA+or TCE-BCMA+cells was 20%. Bioluminescence imaging (BLI) and body weight measurement were performed at the indicated timepoints to assess MM progression and xenogeneic graft-versus-host disease (xGvHD) (Fig. 3A). Control and NT-T-treated mice, and those receiving the lower number of CAR-T cells, rapidly developed MM (Fig. 3, B-C). Mice treated with the higher number of CAR-T cells showed a significant delay in disease progression, with two mice being MM-free at endpoint (Fig. 3, B-C). In contrast, both STAb-T-treated groups were able to control disease progression, as evidenced by BLI, with only one mouse showing some tumor burden at weeks 5 and 6, in the TLD condition group (Fig. 3, B-C). Flow cytometry analysis of PB, BM and spleen revealed complete disease control in both STAb-treated mice groups, except for one mouse in the TLD group (Fig. 3D), whereas a clear tumor infiltration in BM was observed in mice receiving CAR-T cells, especially in the TLD conditions (Fig. 3D). Regarding T cell expansion and persistence in STAb-treated mice, percentages of CD3+cells in PB and spleen were higher than those observed in the groups that received CAR-T cells (Fig. 3F). In both groups, the percentage of BCMA-CAR+and BCMA-TCE+, calculated according to the expression of tdTo ranged between 20-40% (Fig. 3G). qRT-PCR confirmed the absence of BCMA transcripts in the BM of STAb-treated mice (molecular complete remission), except in one case, while the presence of tumor cells was confirmed in most CAR-T-treated mice (Fig. 3H). These data are consistent with serum sBCMA levels, which were almost undetectable in mice receiving STAb-T cells, while significant levels of sBCMA were observed in both CAR groups, especially in the TLD condition (Fig. 3I).

[0304] STAb-T cell treatment promotes memory T cells in a murine MM model

[0305] To further characterize immunologically the in vivo expanded STAb-T and CAR-T cells, we performed additional in vivo studies under TLD conditions, where NSG mice received i.v. injections of 3 x 106NT-T, STAb-T or CAR-T cells, with a 20% of transduced (tdTo+) TCE-BCMA+or CAR-BCMA+cells . NT-T- and CAR-T-treated mice rapidly developed MM, whereas STAb-T-treated mice were able to control disease progression (Fig. 4A), which was consistent with the previous in vivo experiments (Fig. 3, B-C). At week 4 the mean percentage of CD3+cells in PB ranged from 4% in CAR-T-treated mice to 7% in mice receiving STAb-T cells (Fig. 4B). At this point mice were euthanized, and BM and spleen samples analyzed with a 30-antibody panel adapted for the simultaneous measurement of tdTo fluorescence and a FITC-labeled BCMA-Fc. The composition of the STAb-T and CAR-T preinfusion products was very similar, with only minor differences, such a higher percentage of effector CD4+T cells and a lower percentage naive CD4+T cells in both transduced (tdTo+) and non-transduced (tdTo-) STAb-T cells (Fig. 4C). Among the tdTo+T cells recovered from the STAb-T-treated mice, an expansion of CD4+T cells with predominance of memory phenotype CD4+T cells was observed in both BM and spleen, without a significant increase of CD4+Treg cells (Fig. 4C). In the non-transduced tdTo- cells from the STAb-T preparation, a predominance of effector memory CD4+T cells was also observed, which may be attributable to the bystander effect of the secreted BCMA-TCE (Fig. 40). Among the in vivo expanded CAR-T cells, the CD4 / CD8 ratio of the preinfusion product was preserved due to a lower expansion of CD4+TEM cells and a higher percentage of memory CD8+T cells (Fig. 4C). Collectively, this multimodal identification of T cell subsets in in vivo expanded CAR-T and STAb-T cells provides insights into the diversity of T cell responses following ACT with BCMA-specific CAR-T and STAb-T cells in MM.

Claims

CLAIMS1. A cell, characterized in that it comprises a polynucleotide that encodes a bispecific antibody comprising:- an anti-BCMA single chain fragment variable (scFv), and- an anti-CD3 single chain fragment variable (scFv), wherein:- the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 1 , 2 and 3, or a functionally equivalent variant thereof,- the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 4, SAS and SEQ ID NO: 6, or a functionally equivalent variant thereof,- the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 7, 8 and 9, or a functionally equivalent variant thereof, and- the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 10, DTS and SEQ ID NO: 12, or a functionally equivalent variant thereof, wherein the cell is selected from a group consisting of an immune cell, a somatic cell and a progenitor cell.

2. The cell according to claim 1 , wherein:- the FR1 , FR2, FR3 and FR4 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 13, 14, 15 and 16, or a functionally equivalent variant thereof,- the FR1 , FR2, FR3 and FR4 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 17, 18, 19 and 20, or a functionally equivalent variant thereof,- the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 21 , 22, 23 and 24, or a functionally equivalent variant thereof, and- the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 25, 26, 27 and 28, or a functionally equivalent variant thereof.

3. The cell according to claim 2 wherein the functionally equivalent variant of the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 49, 50, 51 , and 52 and / or wherein the functionally equivalent variant of the FR1 , FR2, FR3 and FR4 regions within the anti- CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 53, 54, 55, and 56.

4. The cell according to any of claims 1 to 3, wherein the polynucleotide further comprises a region encoding a signal peptide preceding and in the same reading frame as the bispecific antibody.

5. The cell according to claim 4, wherein the signal peptide is the human kappa light chain signal peptide, which comprises the sequence SEQ ID NO: 35.

6. The cell according to any of claims 1 to 5, wherein the bispecific antibody is characterized in that:- the anti-BCMA heavy chain variable domain VHBCMA comprises the amino acid sequence SEQ ID NO: 29, or a functionally equivalent variant thereof,- the anti-BCMA light chain variable domain VLBCMA comprises the amino acid sequence SEQ ID NO: 30, or a functionally equivalent variant thereof,- the anti-CD3 heavy chain variable domain VHCD3 comprises the amino acid sequence SEQ ID NO: 31 or 57, or a functionally equivalent variant thereof, and- the anti-CD3 light chain variable domain VLCD3 comprises the amino acid sequence SEQ ID NO: 32 or 58, or a functionally equivalent variant thereof.

7. The cell according to any of claims 1 to 6, wherein the anti-BCMA scFv is located N- terminal with respect to the anti-CD3 scFv.

8. The cell according to any of claims 1 to 7, wherein the VH region of the anti-BCMA scFv (VHBCMA) is located N-terminal with respect to the VL ( LBCMA), and / or wherein the VH region of the anti-CD3 scFv (VHCD3) is located N-terminal with respect to the VL (VLCD3).

9. The cell according to any of claims 1 to 8, wherein the variable heavy chain regions (VH) and the variable light chain regions (VL) regions are arranged, from N-terminus to C-terminus, in the order VHBCMA-VLBCMA-VHCD3-VLCD3.

10. The cell according to any of claims 1 to 9, wherein the bispecific antibody is characterized in that:- the anti-BCMA single chain fragment variable (scFv) comprises the sequence SEQ ID NO: 33, and- the anti-CD3 single chain fragment variable (scFv) comprises the sequence SEQ ID NO: 34 or 59.11 . The cell according to any of claims 1 to 10, wherein the VH and L regions of the anti- BCMA scFv, the VH and VL regions of the anti-CD3 scFv, and / or the anti-BCMA scFv and anti-CD3 scFv are connected by a peptide linker.

12. The cell according to claim 11 , wherein the linker is selected from SEQ ID NO: 36, 37 or 38.

13. The cell according to any of claims 1 to 12, wherein the bispecific antibody comprises the amino acid sequence SEQ ID NO: 39 or SEQ ID NQ:60.

14. The cell according to any of claims 1 to 12, wherein the bispecific antibody further contains a polypeptide tag.

15. The cell according to claim 14, wherein the tag is located at the C-terminus of the bispecific antibody.

16. The cell according to claim 15, wherein the tag is a hexahistidine tag.

17. The cell according to any of claims 1 to 16, wherein the polynucleotide that encodes the bispecific antibody has been introduced into the cell by transducing said cell with a viral vector that contains said polynucleotide.

18. The cell according to any of claims 1 to 16, wherein the polynucleotide that encodes the bispecific antibody is an RNA that has been introduced into the cell by transfecting said cell with said RNA.

19. The cell according to any of claims 1 to 18, wherein the immune cell is selected from a group consisting of a T cell, a B cell, a NK cell, a neutrophil, a eosinophil, a basophil,a monocyte, a macrophage, a dendritic cell and a mast cell, or a precursor of any of these cells.

20. The cell according to claim 19, wherein said cell is selected from a T cell and a precursor thereof.

21. The cell according to claim 20, wherein said T cell is a primary T cell.

22. The cell according to any of claims 1 to 21 wherein the cell secretes the bispecific antibody.

23. An ex vivo method for obtaining a cell expressing a bispecific antibody, the method comprising transducing the cell or precursor thereof with a polynucleotide encoding said bispecific antibody, or a vector comprising the same, wherein said bispecific antibody comprises:- an anti-BCMA single chain fragment variable (scFv), and- an anti-CD3 single chain fragment variable (scFv), wherein:- the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 1 , 2 and 3, or a functionally equivalent variant thereof,- the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 4, SAS and SEQ ID NO: 6, or a functionally equivalent variant thereof,- the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 7, 8 and 9, or a functionally equivalent variant thereof, and- the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 10, DTS and SEQ ID NO: 12, or a functionally equivalent variant thereof, wherein the cell is selected from a group consisting of an immune cell, a somatic cell and a progenitor cell.

24. The method according to claim 22, wherein:- the FR1 , FR2, FR3 and FR4 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 13, 14, 15 and 16, or a functionally equivalent variant thereof,- the FR1 , FR2, FR3 and FR4 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 17, 18, 19 and 20, or a functionally equivalent variant thereof,- the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 21 , 22, 23 and 24, or a functionally equivalent variant thereof, and- the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 25, 26, 27 and 28, or a functionally equivalent variant thereof.

25. The method according to claim 24 wherein the functionally equivalent variant of the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 49, 50, 51 , and 52 and / or wherein the functionally equivalent variant of the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 53, 54, 55, and 56.

26. The method according to any of claims 23 or 25, wherein the polynucleotide further comprises a region encoding a signal peptide preceding and in the same reading frame as the bispecific antibody.

27. The method according to claims 26, wherein the signal peptide is the human kappa light chain signal peptide, which comprises the sequence SEQ ID NO: 35.

28. The method according to any of claims 23 to 27, wherein the bispecific antibody is characterized in that: the anti-BCMA heavy chain variable domain VHBCMA comprises the amino acid sequence SEQ ID NO: 29, or a functionally equivalent variant thereof, the anti-BCMA light chain variable domain VLBCMA comprises the amino acid sequence SEQ ID NO: 30., or a functionally equivalent variant thereof, the anti-CD3 heavy chain variable domain VHCD3 comprises the amino acid sequence SEQ ID NO: 31 , SEQ ID NO:55 or a functionally equivalent variant thereof, andthe anti-CD3 light chain variable domain VLCD3 comprises the amino acid sequence SEQ ID NO: 32, SEQ ID NO:58 or a functionally equivalent variant thereof.

29. The method according to claims 23 to 29, wherein the anti-BCMA scFv is located N- terminal with respect to the anti-CD3 scFv.

30. The method according to claims 23 to 29, wherein the VH region of the anti-BCMA scFv (VHBCMA) is located N-terminal with respect to the VL (VLBCMA), and / or wherein the VH region of the anti-CD3 scFv ( HCD3) is located N-terminal with respect to the L (VLCD3).

31. The method according to claims 23 to 30, wherein the variable heavy chain regions (VH) and the variable light chain regions (VL) regions are arranged, from N-terminus to C-terminus, in the order VHBCMA-VLBCMA-VHCD3-VLCD3.

32. The method according to any of claims 23 to 31 , wherein the bispecific antibody is characterized in that:- the anti-BCMA single chain fragment variable (scFv) comprises the sequence SEQ ID NO: 33, and- the anti-CD3 single chain fragment variable (scFv) comprises the sequence SEQ ID NO: 34 or SEQ ID NO:59.

33. The method according to any of claims 23 to 32, wherein the VH and VL regions of the anti-BCMA scFv, the VH and VL regions of the anti-CD3 scFv, and / or the anti- BCMA scFv and anti-CD3 scFv are connected by a peptide linker.

34. The method according to any of claims 23 to 33, wherein the linker is selected from SEQ ID NO: 36, 37 or 38.

35. The method according to any of claims 23 to 34, wherein the bispecific antibody comprises the amino acid sequence SEQ ID NO: 39 or SEQ ID NQ:60.

36. The method according to any of claims 23 to 35 wherein the bispecific antibody further contains a polypeptide tag.

37. The method according to claim 36, wherein the tag is located at the C-terminus of the bispecific antibody.

38. The method according to claims 36 or 37, wherein the tag is a hexahistidine tag.

39. The method according to any of claims 23 to 38, wherein the polynucleotide that encodes the bispecific antibody is provided as a viral vector that contains said polynucleotide.

40. The method according to any of claims 23 to 38, wherein the polynucleotide that encodes the bispecific antibody is an RNA polynucleotide.41 . The method according to any of claims 23 to 40, wherein the immune cell is selected from a group consisting of a T cell, a B cell, a NK cell, a neutrophil, a eosinophil, a basophil, a monocyte, a macrophage, a dendritic cell and a mast cell, or a precursor of any of these cells.

42. The method according to any of claims 23 to 41 wherein the cell is selected from a T cell and a precursor thereof.

43. The method according to claim 42, wherein said T cell is a primary T cell.

44. A cell obtainable by the method of any of claims 23 to 43.

45. A pharmaceutical composition comprising the cell according to any of claims 1 to 22 and claim 44, and at least one pharmaceutically acceptable excipient.

46. The cell according to any of claims 1 to 22 and claim 44, for use as a medicament.

47. The cell according to any of claims 1 to 22 and claim 44, for use in the treatment of cancer.

48. The cell according to any of claims 1 to 22 and claim 44, for use in the treatment of an hematological malignancy.

49. The cell according to any of claims 1 to 22 and claim 44, for use in the treatment of an hematological malignancy, wherein the hematological malignancy is characterized by the presence of malignant cells which exhibit BCMA expression on their surface.

50. The cell for use according to any of claims 48 or 49 wherein said hematological malignancy is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute B cell lymphoblastic leukemia, minimal residual disease (MRD)-positiveALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), lymphoplasmacytic lymphoma (LPL), B cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt’s lymphoma (BL), primary mediastinal large B-cell lymphoma (PMBL), marginal zone B cell lymphoma, Hodgkin’s lymphoma (HL), nonHodgkin’s lymphoma (NHL), NK- and T-cell neoplasms, histiocytic neoplasms, mantle cell lymphoma (MCL), hairy cell leukemia (HCL), plasma cell myeloma (PCM), plasma cell leukemia (PCL), and multiple myeloma (MM).51 . A bispecific antibody comprising:- an anti-BCMA single chain fragment variable (scFv), and- an anti-CD3 single chain fragment variable (scFv), wherein:- the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 1 , 2 and 3, or a functionally equivalent variant thereof,- the CDR1 , CDR2 and CDR3 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 4, SAS and SEQ ID NO: 6, or a functionally equivalent variant thereof,- the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 7, 8 and 9, or a functionally equivalent variant thereof, and- the CDR1 , CDR2 and CDR3 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 10, DTS and SEQ ID NO: 12, or a functionally equivalent variant thereof.

52. The bispecific antibody to claim 51 , wherein:- the FR1 , FR2, FR3 and FR4 regions within the anti-BCMA scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 13, 14, 15 and 16, or a functionally equivalent variant thereof,- the FR1 , FR2, FR3 and FR4 regions within the anti-BCMA scFv light chain region comprise respectively the sequences of SEQ ID NO: 17, 18, 19 and 20, or a functionally equivalent variant thereof,- the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 21 , 22, 23 and 24, or a functionally equivalent variant thereof, and- the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 25, 26, 27 and 28, or a functionally equivalent variant thereof.

53. The bispecific antibody according to any of claims 51 or 52 wherein the functionally equivalent variant of the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv heavy chain region comprise respectively the sequences of SEQ ID NO: 49, 50, 51 , and 52 and / or wherein the functionally equivalent variant of the FR1 , FR2, FR3 and FR4 regions within the anti-CD3 scFv light chain region comprise respectively the sequences of SEQ ID NO: 53, 54, 55, and 56.

54. The bispecific antibody according to any of claims 51 to 53, wherein the bispecific antibody is characterized in that:- the anti-BCMA heavy chain variable domain VHBCMA comprises the amino acid sequence SEQ ID NO: 29, or a functionally equivalent variant thereof,- the anti-BCMA light chain variable domain VLBCMA comprises the amino acid sequence SEQ ID NO: 30, or a functionally equivalent variant thereof,- the anti-CD3 heavy chain variable domain VHCD3 comprises the amino acid sequence SEQ ID NO: 31 , SEQ ID N:57 or a functionally equivalent variant thereof, and- the anti-CD3 light chain variable domain VLCD3 comprises the amino acid sequence SEQ ID NO: 32, SEQ ID NO:58 or a functionally equivalent variant thereof.

55. The bispecific antibody according to any of claims 51 to 54, wherein the anti-BCMA scFv is located N-terminal with respect to the anti-CD3 scFv.

56. The bispecific antibody according to any of claims 51 to 55, wherein the VH region of the anti-BCMA scFv (VHBCMA) is located N-terminal with respect to the VL ( LBCMA), and / or wherein the VH region of the anti-CD3 scFv (VHCD3) is located N- terminal with respect to the VL (VLCD3).

57. The bispecific antibody according to any of claims 51 to 56, wherein the variable heavy chain regions (VH) and the variable light chain regions (VL) regions are arranged, from N-terminus to C-terminus, in the order VHBCMA-VLBCMA-VHCD3- VLCD3.

58. The bispecific antibody according to any of claims 51 to 57, wherein the bispecific antibody is characterized in that:- the anti-BCMA single chain fragment variable (scFv) comprises the sequence SEQ ID NO: 33, and- the anti-CD3 single chain fragment variable (scFv) comprises the sequence SEQ ID NO: 34 or SEQ ID NO:59.

59. The bispecific antibody according to any of claims 52 to 58, wherein the VH and L regions of the anti-BCMA scFv, the VH and VL regions of the anti-CD3 scFv, and / or the anti-BCMA scFv and anti-CD3 scFv are connected by a peptide linker.

60. The bispecific antibody according to claim 59, wherein the linker is selected from SEQ ID NO: 36 or 38.

61. The bispecific antibody according to any of claims 52 to 60, wherein the bispecific antibody comprises the amino acid sequence SEQ ID NO: 39 or SEQ ID NQ:60.

62. The bispecific antibody according to any of claims 52 to 61 , wherein the bispecific antibody further contains a polypeptide tag.

63. The bispecific antibody according to claim 62, wherein the tag is located at the C- terminus of the bispecific antibody.

64. The bispecific antibody according to claims 62 or 63, wherein the tag is a hexahistidine tag.

65. A polynucleotide encoding the bispecific antibody according to any of claims 52 to 64.

66. The polynucleotide according to claim 65 wherein the polynucleotide further comprises a region encoding a signal peptide preceding and in the same reading frame as the bispecific antibody.

67. The polynucleotide according to claims 66 wherein the signal peptide is the human kappa light chain signal peptide, which comprises the sequence SEQ ID NO: 35.

68. The polynucleotide according to claims 66 or 67 wherein the polynucleotide is a RNA molecule.

69. An expression vector comprising the polynucleotide according to claims 65 to 68.

70. The expression vector according to claim 69 which is a viral expression vector.71 . The expression vector according to claim 70 wherein the viral expression vector is a lentiviral vector.

72. A pharmaceutical composition comprising the bispecific antibody according to any of claims 51 to 64, the polynucleotide according to any of claims 65 to 68 or the expression vector according to any of claims 69 to 71 and at least one pharmaceutically acceptable excipient.

73. The bispecific antibody according to any of claims 51 to 64, the polynucleotide according to any of claims 65 to 68 or the expression vector according to any of claims 79 to 71 for use as a medicament.

74. The bispecific antibody according to any of claims 51 to 64, the polynucleotide according to any of claims 65 to 68 or the expression vector according to any of claims 79 to 71 for use in the treatment of cancer.

75. The bispecific antibody according to any of claims 51 to 64, the polynucleotide according to any of claims 65 to 68 or the expression vector according to any of claims 79 to 71 for use in the treatment of an hematological malignancy.

76. The bispecific antibody, polynucleotide or the expression vector for use according to claim 75, wherein the hematological malignancy is characterized by the presence of malignant cells which exhibit BCMA expression on their surface.

77. The bispecific antibody, polynucleotide or the expression vector for use according to claim 76 wherein said hematological malignancy is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute B cell lymphoblastic leukemia, minimal residual disease (MRD)-positive ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndromes (MDS),myeloproliferative neoplasms (MPN), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), lymphoplasmacytic lymphoma (LPL), B cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt’s lymphoma (BL), primary mediastinal large B-cell lymphoma (PMBL), marginal zone B cell lymphoma, Hodgkin’s lymphoma (HL), non- Hodgkin’s lymphoma (NHL), NK- and T-cell neoplasms, histiocytic neoplasms, mantle cell lymphoma (MCL), hairy cell leukemia (HCL), plasma cell myeloma (PCM), plasma cell leukemia (PCL), and multiple myeloma (MM).

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