CD229 and BCMA targeting moieties for the treatment of CD229-BCMA-positive cancers

Abstract

The present invention relates to CD229 and BCMA targeting moieties, wherein the CD229 targeting moiety is an antibody, F(ab')2, Fab, scFab, or scFv. The present invention further provides CARs, nucleic acids, cells, pharmaceutical compositions, and kits comprising the CD229 and BCMA targeting moieties. Methods for treating CD229-positive cancers, preferably multiple myeloma, are also provided.

Description

[Technical Field]

[0001] The present invention provides therapeutic agents for treating CD229-positive cancers. In particular, the present invention provides CD229-targeting moieties. [Background technology]

[0002] Multiple myeloma (MM) is a clonal plasma cell (PC) malignancy that arises in a patient's bone marrow (BM) and ultimately leads to bone lesions with fractures, renal failure, BM failure, and immunodeficiency with fatal infections. Standard care for MM patients includes treatments such as proteasome inhibitors, immunomodulatory agents, autologous stem cell transplants, and monoclonal antibodies, which have substantially long-term patient survival. However, to date, MM remains an incurable disease. Chimeric antigen receptor (CAR) T cells have emerged as an effective approach for many other hematologic malignancies, and recent results from clinical trials of CAR T cells targeting BCMA in MM have demonstrated impressive overall response rates.

[0003] B-cell maturation antigen (BCMA) is the primary target used in CAR therapy for MM. The selection of BCMA targets is based on its high expression on the surface of malignant plasma cells and its restricted expression in normal tissues / cells, excluding mature B cells. However, many other targets are being investigated and emerging from preclinical studies in the context of adoptive cell therapy (ACT) for MM. Most studies have used anti-BCMA targeting, either alone or in combination with a second target. The second most studied target has been SLAMF7 (CD319), which has been investigated in clinical trials. Other antigens investigated and described here include CD44v6, CD38, CD138, MUC1, CD56, CD19, Igk light chain, Lewis Y, CD229, and GPRC5D.

[0004] CD229 / LY9 is a cell surface receptor present on B and T lymphocytes that is ubiquitously and strongly expressed on MM plasma cells. CAR-based therapies have been developed against CD229 and have shown remarkable activity. However, no defined CAR-based therapy is yet available that is effective in all patients with low toxicity and side effects. Therefore, efforts to develop new CARs against new targets are still needed. Summary of the Invention

[0005] In a first aspect, the present invention relates to a CD229 targeting moiety, wherein the CD229 targeting moiety is an antibody, F(ab')2, Fab, scFab or scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2 and HCDR3 polypeptides; -LCDR1 consists only of SEQ ID NO: 4 [SSVSY], LCDR2 consists solely of SEQ ID NO: 5 [DTS], LCDR3 consists solely of SEQ ID NO: 6 [CQQWSSYPPTF], HCDR1 consists solely of SEQ ID NO: 7 [GYTFTHYY], HCDR2 consists solely of SEQ ID NO: 8 [IFPESGST], - HCDR3 consists solely of SEQ ID NO: 9 [CARGTGFFDYW].

[0006] Preferably, the CD229 targeting moiety is - a VL domain comprising a sequence having at least 85%, 90%, 92%, 94%, preferably 95%, 98% or 100% sequence identity with SEQ ID NO: 1, wherein the VL domain comprises CDR regions consisting only of LCDR1 as set forth in SEQ ID NO: 4, LCDR2 as set forth in SEQ ID NO: 5 and LCDR3 as set forth in SEQ ID NO: 6; - a VH domain comprising a sequence having at least 85%, 90%, 92%, 94%, preferably 95%, 98% or 100% sequence identity to SEQ ID NO: 2, wherein the VH domain comprises a CDR region consisting only of HCDR1 as set forth in SEQ ID NO: 7, HCDR2 as set forth in SEQ ID NO: 8 and HCDR3 as set forth in SEQ ID NO: 9.

[0007] Preferably, the VL domain comprises a sequence having at least 85%, 90%, preferably 95% sequence identity with SEQ ID NO: 1, and the VL domain comprises a CDR region consisting only of LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6; the VH domain comprises a sequence having at least 85%, 90%, preferably 95% sequence identity with SEQ ID NO: 2, and the VH domain comprises a CDR region consisting only of HCDR1 shown in SEQ ID NO: 7, HCDR2 shown in SEQ ID NO: 8, and HCDR3 shown in SEQ ID NO: 9.

[0008] Preferably, the CD229 targeting moiety is an antibody, F(ab')2, Fab, scFab or scFv comprising a VL domain and a VH domain, wherein the VL domain consists solely of SEQ ID NO: 1 and the VH domain consists solely of SEQ ID NO: 2. Preferably, the CD229 targeting moiety is an scFV, more preferably an scFV consisting solely of SEQ ID NO: 17.

[0009] In a preferred embodiment, the first aspect comprises: an extracellular domain comprising a CD229 targeting moiety, wherein the CD229 targeting moiety is as defined in the first aspect or any of its embodiments; -transmembrane domain; and -Providing a CAR comprising an intracellular signaling domain.

[0010] Preferably, the transmembrane domain comprises the transmembrane domain of CD8. Preferably, the intracellular signaling domain comprises the intracellular domain of CD3ζ. Preferably, the costimulatory signaling domain comprises the intracellular domain of CD137.

[0011] Preferably, the CAR consists solely of SEQ ID NO:18.

[0012] A second aspect provides a nucleic acid encoding a CAR according to the first aspect.

[0013] A third aspect provides a cell, preferably a T cell, comprising a nucleic acid according to the second aspect.

[0014] A fourth aspect provides a pharmaceutical composition comprising a plurality of cells according to the third aspect.

[0015] A fifth embodiment provides a CD229 targeting moiety according to any of the preceding embodiments for use in a method of treating a CD229-positive cancer, the method comprising administering the CD229 targeting moiety, CAR, nucleic acid, or cell to a patient in need thereof, wherein the CD229-positive cancer is multiple myeloma. Preferably, the use is in combination with anti-BCMA therapy, more preferably the anti-BCMA therapy comprises administering a CAR directed against BCMA.

[0016] A sixth aspect provides a combination therapy comprising or consisting of the administration of a CD229 targeting moiety according to any of aspects 1 to 4 or any of their embodiments, in combination with a BCMA targeting moiety. Preferably, this aspect includes the following combination:

[0017] - a CD229 targeting moiety according to the first aspect or any of its embodiments, comprising a BCMA targeting moiety;

[0018] a CAR anti-CD229 according to the first aspect or any of its embodiments, with a CAR anti-BCMA,

[0019] - a nucleic acid as defined in the second aspect or any of its embodiments, comprising a nucleic acid encoding or expressing a BCMA targeting moiety and / or a CAR anti-BCMA,

[0020] a cell, preferably a T cell, according to the third aspect or any of its embodiments, comprising a nucleic acid encoding or expressing a BCMA targeting moiety and / or a CAR anti-BCMA,

[0021] - A composition according to the fourth aspect or any of its embodiments, having a composition comprising a plurality of cells, preferably T cells, comprising a nucleic acid encoding or expressing a BCMA targeting moiety and / or a CAR anti-BCMA.

[0022] A method of use for the treatment of combination therapy is also included in the sixth aspect.

[0023] In a seventh aspect, the present invention provides kits according to any of the preceding aspects and uses thereof. [Brief explanation of the drawings]

[0024] [Figure 1] Diagram of four anti-CD229 CAR constructs. hLy9 1.84 antibody was used to generate VH-VL-41BB-CD3z (GL001) and VL-VH-41BB-CD3z (GL002) CARs. hLy 10.169 was used to generate VH-VL-41BB-CD3z (GL005) and VL-VH-41BB-CD3z (GL006) CARs. [Figure 2] Detection of CAR proteins on the cell surface. B. Detection of GL001 (hLy9 1.84 VH-VL-41BB-CD3z), GL002 (hLy9 1.84 VL-VH-41BB-CD3z), and ARI002h (anti-BCMA) on the surface of AT cells (both CD4 and CD8). C. Detection of GL005 (hLy9 10.169 VH-VL-41BB-CD3z) and GL006 (hLy9 10.169 VL-VH-41BB-CD3z) on the surface of HEK293-T cells. Detection of chimeric proteins was performed by flow cytometry. [Figure 3]Cytotoxicity of CAR T cells against different target cells. Killing capacity of GL001, GL002, and ARI002h CAR-T cells co-incubated with K562 WT (BCMA- and CD229-), MM.1S WT (BCMA+ and CD229+), and U266 WT (BCMA+CD229+) tumor cells at different ratios for 4 and 24 hours. Shown are the mean and ±SD of three experiments using three different donors. [Figure 4] CAR T cell proliferation. GL001 (left) and GL002 (right) T cell proliferation in vitro as measured by CFSE assay at 72 hours. Panels show representative flow cytometry images. [Figure 5] CAR T cell cytokine secretion. Cytokine production (IFNg and IL-2) of GL001, GL002 and ARI002h cells in co-culture with K562, MM.1S and U266 cells at 24 hours as measured by ELISA. [Figure 6] Bioluminescence imaging showing differences in tumor progression in mice treated with different CAR T cells (UTD, GL001, GL002, and ARI002h). Diagram of experimental design and quantification of disease progression by weekly bioluminescence imaging and overall survival of mice in different groups. Mice in the in vivo experimental design were injected with 5x106 GFP-ffLuc-expressing U266 cells. On day 14, mice were randomized to treatment with 3.0x106 untransduced (UTD), GL001, GL002, or ARI0002h. A. Tumor progression was measured by taking weekly bioluminescence images. B. Quantification of tumor burden by bioluminescence imaging over time of tumor cell (U266 WT) injection. Lines indicate the median and 95% confidence interval from all mice in that group. C. Overall survival of mice from each group. *p<0.01; **p<0.001. [Figure 7]Cytotoxicity of CAR-T cells against target cells with different BCMA antigen expression. A: Flow cytometry of MM.1S BCMA KO cells showing BCMA antigen expression. B: Killing ability of GL002 and ARI002h CAR-T cells co-incubated at different ratios with MM.1S WT (BCMA+ and CD229+) and MM.1S BCMA KO (BCMA-CD229+) tumor cells for 4 and 24 hours. Mean and ±SD of three experiments using three different donors are shown. [Figure 8] Bioluminescence imaging showing differences in tumor progression between mixed populations of specific antigen-expressing and non-expressing tumor cells in mice treated with different CAR-Ts (UTD, GL002, and ARI002h). In vivo experimental design: Mice were injected with 2 x 10 GFP-ffLuc-expressing MM.1S cells containing a population of BCMA-KO cells (15%). On day 14, mice were randomized to treatment with 1.0 x 10 untransduced (UTD), GL002, or ARI0002h. A. Tumor progression was measured by taking weekly bioluminescence images. B. Quantification of tumor burden by bioluminescence imaging over time of tumor cell (MM.1Swt and MM.1S BCMAKO) injection. Lines indicate the median and 95% confidence interval from all mice in that group. C. Overall survival of mice from each group. **p<0.001; ***p<0.0001. [Figure 9] BCMA antigen expression in bone marrow from mice. BCMA antigen expression on bone marrow MM.1S myeloma tumor cells harvested from mice treated with UTD, GL002, or ARI002h CAR T cells. Antigen expression detected by flow cytometry. [Figure 10]Diagram of two anti-CD229 / BCMA bicistronic CAR constructs. A. Schematic of the bicistronic vector in which both CARs are cloned into the same plasmid. Subsequently, upon infection of T lymphocytes, the virus expresses both CARs on their surface. B. The new bicistronic CARs, GL002[T2A]ARI2h (GL007) and ARI2h[T2A]GL002 (GL008), are shown. The T2A peptide sequence was added to allow ribosome skipping, allowing the encoding of CARs that dissociate into individual CAR proteins during translation. Two linkers were added before and after the T2A to stabilize the protein. [Figure 11] Detection of both CAR proteins on the cell surface. Detection of both CARs (GL002 [CD229 CAR] and ARI2h [ARI2h]) on the surface of the same T cells when infected with lentivirus containing CARs GL007 and GL008. Detection of the chimeric proteins was performed by flow cytometry. [Figure 12] Cytotoxicity of CAR T cells against different target cells. Killing capacity of GL007 and GL008 CAR-T cells co-incubated with K562 WT (BCMA- and CD229-), MM.1S WT (BCMA+ and CD229+), U266 WT (BCMA+CD229+), OPM2 WT (BCMA+ and CD229+), MM.1S BCMA KO (BCMA- and CD229+), OPM2 BCMA KO (BCMA- and CD229+), and K562 BCMA (BCMA+ and CD229-) tumor cells at different ratios for 24 hours. Shown are the mean and ±SD of three experiments using three different donors. [Figure 13] CAR T cell proliferation. In vitro GL007 (left) and GL008 (right) T cell proliferation measured by CFSE assay at 72 hours after co-culture with K562, K562 BCMA, MM.1S, and MM.1S BCMA KO. Negative controls were performed with the addition of media alone. Positive controls were performed with the addition of IL-2. Panels show representative flow cytometry images. [Figure 14]CAR T cell cytokine secretion. Cytokine production (IFNg and IL-2) of GL007 and GL008 cells in co-culture with K562, K562 BCMA, MM.1S, MM.1S BCMA KO, OPM2, OPM2 BCMA KO and U266 cells at 24 hours as measured by ELISA. [Figure 15] Bioluminescence imaging showing differences in tumor progression in mice treated with different CAR T cells (UTD, ARI002h, GL007, and GL008). Diagram of experimental design and quantification of disease progression by weekly bioluminescence imaging and overall survival of mice in different groups. Mice in the in vivo experimental design were injected with 2x106 GFP-ffLuc-expressing MM.1S cells. On day 14, mice were randomized to treatment with 3.0x106 untransduced (UTD), ARI002h, GL007, or GL008. A. Tumor progression was measured by taking weekly bioluminescence images. B. Quantification of tumor burden by bioluminescence imaging over time of tumor cell (MM.1S WT) injection. Lines indicate the median and 95% confidence interval from all mice in that group. C. Overall survival of mice from each group. *p<0.01; **p<0.001; NS: not significant. [Figure 16] Bioluminescence imaging showing differences in tumor progression between mixed populations of specific antigen-expressing and non-expressing tumor cells in mice treated with different CAR-Ts (UTD, GL002, ARI002h, GL007, and GL008). In vivo experimental design. Mice were injected with 2 x 10 GFP-ffLuc-expressing MM.1S cells containing a population of BCMA-KO cells (15%). On day 14, mice were randomized to treatment with 1.0 x 10 untransduced (UTD), GL002, ARI002h, GL007, or GL008. A. Tumor progression was measured by taking weekly bioluminescence images. B. Quantification of tumor burden by bioluminescence imaging over time of tumor cell (MM.1Swt and MM.1S BCMAKO) injection. Lines indicate the median and 95% confidence interval from all mice in that group. C. Overall survival of mice from each group. **p<0.001; ***p<0.0001. NS: not significant. [Figure 17] BCMA and CD229 antigen expression in bone marrow from mice. BCMA and CD229 antigen expression on bone marrow MM.1S myeloma tumor cells harvested from mice treated with UTD, GL007, or GL008 CAR T cells. Antigen expression detected by flow cytometry. [Figure 18] Generation of cell lines with reduced BCMA expression. A. MM.1S BCMAdim tumor cell line with reduced BCMA expression obtained by fluorescence-activated cell sorting of tumor cells with minimal BCMA expression from the MM.1S BCMAKO line. B. Flow cytometry of MM.1S WT, MM.1S BCMA KO, and MM.1S BCMAdim cells showing BCMA antigen expression and mean fluorescence intensity (MFI). [Figure 19] Cytotoxicity of CAR-T cells against target cells with different BCMA antigen expression. Killing capacity of ARI002h, GL002, GL007, and GL008 CAR-T cells co-incubated with MM.1S WT (BCMA+ and CD229+), MM.1S BCMA KO (BCMA-CD229+), and MM.1S BCMAdim (BCMAdim CD229+) tumor cells at different ratios for 24 hours. Shown are the mean and ±SD of three experiments using three different donors. [Figure 20]Bioluminescence imaging showing differences in tumor progression between mixed populations of specific antigen-expressing and -reduced tumor cells in mice treated with different CAR-Ts (UTD, GL002, ARI002h, and GL007). In vivo experimental design: Mice were injected with 2 x 10 GFP-ffLuc-expressing MM.1S cells containing a population of BCMAdim cells (50%). On day 14, mice were randomized to treatment with 1.0 x 10 untransduced (UTD), GL002, ARI002h, or GL007. A. Tumor progression was measured by taking weekly bioluminescence images. B. Quantification of tumor burden by bioluminescence imaging over time of tumor cell (MM.1Swt and MM.1S BCMAdim) injection. Lines indicate the median and 95% confidence interval from all mice in that group. C. Overall survival of mice from each group. **p<0.001; ***p<0.0001. NS: Not significant. DETAILED DESCRIPTION OF THE INVENTION

[0025] General definition "Administering" a medication to a patient or "administration of" a medication to a patient (and grammatical equivalents of this phrase) refers to direct administration, which can be administration to a patient by a medical professional, and / or indirect administration, which can be the act of prescribing a medication. For example, a physician who instructs a patient to self-administer a medication or provides a patient with a prescription for a medication is administering a medication to a patient.

[0026] The term "affibody" refers to proteins derived from the Z domain of Protein A and engineered to bind to specific targets (see Frejd & Kim, 2017. Exp Mol Med. 49(3):e306).

[0027] The term "antibody" refers to a molecule containing at least one immunoglobulin domain that binds to or is immunologically reactive with a specific target. The term includes whole antibodies and any antigen-binding portion thereof, or single chains thereof, and combinations thereof. For example, the term "antibody" specifically includes bivalent antibodies and bivalent, bispecific antibodies.

[0028] A typical type of antibody comprises at least two heavy chains (“HC”) and two light chains (“LC”) interconnected by disulfide bonds.

[0029] Each "heavy chain" comprises a "heavy chain variable domain" (abbreviated herein as "VH") and a "heavy chain constant domain" (abbreviated herein as "CH"). The heavy chain constant domain typically comprises three constant domains, CH1, CH2, and CH3.

[0030] Each "light chain" contains a "light chain variable domain" (abbreviated herein as "VL") and a "light chain constant domain" ("CL"). The light chain constant domain (CL) can be of kappa or lambda type. The VH and VL domains can be further subdivided into regions of hypervariability called complementarity-determining regions ("CDRs"), interspersed with more conserved regions called "framework regions" ("FW"). In molecular biology, the framework region is part of the variable region (Fab) of an antibody. The variable region is composed of seven regions, four of which are framework regions and three of which are hypervariable regions. The framework region constitutes approximately 85% of the variable region and acts as a scaffold for the CDR regions. These CDRs are in direct contact with the antigen and are involved in antigen binding, while the framework region supports the binding of the CDRs to the antigen and may help maintain the overall structure of the four variable domains on the antibody.

[0031] Each VH and VL is composed of three CDRs and four FWs arranged from amino terminus to carboxy terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. This disclosure presents, inter alia, VH and VL sequences and subsequences corresponding to CDR1, CDR2, and CDR3.

[0032] The precise amino acid sequence boundaries of a given CDR can be determined using any of several well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the "Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (the "Chothia" numbering scheme).

[0033] Therefore, those skilled in the art will understand that the sequences FW1, FW2, FW3, and FW4 are equally disclosed. For a particular VH, FW1 is a subsequence between the N-terminus of VH and the N-terminus of H-CDR1, FW2 is a subsequence between the C-terminus of H-CDR1 and the N-terminus of H-CDR2, FW3 is a subsequence between the C-terminus of H-CDR2 and the N-terminus of H-CDR3, and FW4 is a subsequence between the C-terminus of H-CDR3 and the C-terminus of VH. Similarly, for a particular VL, FW1 is a subsequence between the N-terminus of VL and the N-terminus of L-CDR1, and FW2 is a subsequence between the C-terminus of L-CDR1 and the N-terminus of L-CDR2. FW3 is a subsequence between the C-terminus of L-CDR2 and the N-terminus of L-CDR3, and FW4 is a subsequence between the C-terminus of L-CDR3 and the C-terminus of VL.

[0034] The variable domains of the heavy and light chains contain regions that interact with binding targets, and these regions that interact with binding targets are also referred to herein as "antigen-binding sites" or "antigen binding sites." The constant domains of antibodies can mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Exemplary antibodies of the present disclosure include not only typical antibodies but also bivalent fragments such as F(ab')2 and modifications thereof.

[0035] As used herein, the term "antibody" includes intact polyclonal antibodies, intact monoclonal antibodies, bivalent antibody fragments (such as F(ab')2), multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and any other modified immunoglobulin molecule containing an antigen-binding site.

[0036] The term "humanized antibody" refers to forms of antibodies containing sequences derived from non-human (e.g., murine) and human antibodies. Humanized antibodies may contain conservative amino acid substitutions or non-naturally occurring residues from the same or different species that do not significantly alter their binding and / or biological activity. Such antibodies are chimeric antibodies containing minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the recipient's complementarity-determining regions (CDRs) are replaced with residues from a CDR (donor antibody) of a non-human species such as mouse, rat, camel, cow, goat, or rabbit that possesses the desired properties. Furthermore, humanized antibodies can contain residues found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. Thus, in general, a humanized antibody can comprise all or substantially all of at least one, and in one embodiment, two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin sequence. A humanized antibody can also optionally comprise at least a portion of an immunoglobulin constant region (Fc), or at least a portion of that of a human immunoglobulin. The process of humanizing antibodies involves CDR grafting technology, which involves the use of a "donor" antibody containing antigen-specific CDRs and an "acceptor" antibody that serves as the framework for the hybrid molecule. Typically, the "donor" antibody is of non-human origin, such as a murine antibody, and the "acceptor" antibody is a human antibody. As a result of the humanization process, the resulting humanized or hybrid antibody retains the CDR sequences in both the VL and VH domains of the "donor" antibody, but the framework sequences are altered relative to those of the "acceptor" antibody.

[0037] Antibodies can be of any of the five major classes of immunoglobulins (isotypes): IgA, IgD, IgE, IgG, and IgM, or their subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, called alpha, delta, epsilon, gamma, and mu, respectively. Different classes of immunoglobulins have different, well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules (such as therapeutic or diagnostic agents) to form immunoconjugates.

[0038] The term "anticalin" refers to proteins derived from lipocalins and engineered to bind to specific targets (Skerra, 2008. FEBS J. 275(11):2677-83).

[0039] The term "antigen-binding fragment" or "Fab" refers to an antibody fragment containing one constant domain and one variable domain of each of the heavy and light chains. Fab fragments can be obtained by digesting an intact monoclonal antibody with papain.

[0040] The term "cancer" refers to a group of diseases that can be defined as abnormal benign or malignant new growth of tissue that has no physiological function, results from uncontrolled, usually rapid, cell proliferation, and has the potential to invade or spread to other parts of the body.

[0041] The term "CD229" or "cluster of differentiation 229" refers to the T lymphocyte surface antigen Ly-9, a protein encoded by the LY9 gene in humans. Exemplary sequences and data related to human CD229 have been deposited in the UniProtKB database under ID numbers Q9HBG7 and Q5VYI1. CD229 has four Ig-like domains, while the remaining SLAMF receptors contain two Ig-like domains.

[0042] "CD229-positive" cancers, including "CD229-positive" cancerous diseases, are those containing cells that have CD229 present on their cell surface. The term "CD229-positive" also refers to cancers that produce sufficient levels of CD229 on the cell surface such that the CAR-containing cells of the present invention have a therapeutic effect mediated by the binding of the CAR to CD229. In some embodiments, the CD229-positive cancer is multiple myeloma cancer.

[0043] The term "CD229 targeting moiety" or "CD229 targeting moiety" refers to a substance capable of binding to CD229, preferably human CD229. In the context of a CAR, the CD229 targeting moiety targets T cells to CD229-positive cells, preferably cancer cells. In the context of a CAR, it should be understood that the CD229 targeting moiety can be genetically encoded.

[0044] The term "B-cell maturation antigen" (BCMA or BCM), also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), is a protein that in humans is encoded by the TNFRSF17 gene.

[0045] The term "BCMA-targeting moiety" or "BCMA targeting moiety" refers to a substance capable of binding to BCMA, preferably human BCMA. In the context of a CAR, the BCMA-targeting moiety targets T cells to BCMA-positive cells, preferably cancer cells. In the context of a CAR, it should be understood that the BCMA-targeting moiety can be genetically encodable.

[0046] "BCMA-positive" cancers, including "BCMA-positive" cancerous diseases, are those that contain cells that have BCMA present on their cell surface. The term "BCMA-positive" also refers to cancers that produce sufficient levels of BCMA on the surface of their cells. In some embodiments, the BCMA-positive cancer is multiple myeloma cancer.

[0047] The term "multiple myeloma," also known as plasma cell myeloma, is a cancer of plasma cells, a type of white blood cell that usually produces antibodies. Often, there are no symptoms at first. Progression can lead to bone pain, bleeding, frequent infections, and anemia. Complications can include amyloidosis.

[0048] The term "chimeric antigen receptor" or "CAR" refers to a synthetic receptor that targets T cells to a selected antigen and reprograms T cell function, metabolism, and persistence. Similarly, the term "CART" refers to a T cell containing a CAR.

[0049] As used herein, the terms "combination therapy," "in combination with," or "in conjunction with" refer to any form of concurrent, parallel, simultaneous, sequential, or intermittent treatment with at least two distinct treatment modalities (i.e., compounds, components, targeted agents, or therapeutic agents). Thus, the terms refer to the administration of one treatment modality before, during, or after the administration of another treatment modality to a subject. The combined modalities can be administered in any order. Therapeutically active modalities are administered together (e.g., simultaneously in the same or separate compositions, formulations, or unit dosage forms) or separately (e.g., on the same or different days, in any order according to appropriate administration protocols for the separate compositions, formulations, or unit dosage forms) in a manner and dosage regimen prescribed by a healthcare professional or in accordance with regulatory authorities. Generally, each treatment modality can be administered at a dose and / or time schedule determined for that treatment modality. Optionally, three or more modalities may be used in combination therapy. Additionally, the combination therapy provided herein may be used in conjunction with other types of treatments, for example, other anti-cancer treatments may be selected from the group consisting of chemotherapy, surgery, radiotherapy (radiation), and / or hormone therapy, among other treatments associated with the current standard of care for the subject.

[0050] "Complete response" or "complete remission" or "CR" refers to the disappearance of all target lesions, as defined by the RECIST v1.1 guideline. This does not necessarily mean that the cancer has been cured.

[0051] The term "costimulatory signaling domain" refers to a signaling moiety that provides a signal to a T cell that mediates T cell responses, including, but not limited to, activation, proliferation, differentiation, cytokine secretion, and the like, in addition to the primary signal provided, for example, by the CD3ζ chain of the TCR / CD3 complex. Costimulatory domains can include, but are not limited to, all or a portion of CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, 1COS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds CD83. In some embodiments, a costimulatory signaling domain is an intracellular signaling domain that interacts with other intracellular mediators to mediate cellular responses, including, but not limited to, activation, proliferation, differentiation, cytokine secretion, and the like.

[0052] The term "designed ankyrin repeat protein" or "DARPin" refers to proteins derived from ankyrin repeats that have been engineered to bind to specific targets (Pluckthun, 2015. Annu Rev Pharmacol Toxicol. 55:489-511).

[0053] "Disease-free survival" (DFS) refers to the length of time during and after treatment that a patient remains disease-free.

[0054] As used herein, the term "effective amount" of a therapeutic agent, such as a drug, e.g., CART cells, is an amount sufficient to produce beneficial or desired results, e.g., clinical results, and therefore "effective amount" depends on the context in which it is applied. For example, in the context of administering a therapeutic agent to treat T-ALL, an effective amount can reduce the number of cancer cells; reduce tumor size or burden; inhibit (i.e., slow to some extent, in certain embodiments, stop) the infiltration of cancer cells into peripheral organs; inhibit (i.e., slow to some extent, in certain embodiments, stop) tumor metastasis; inhibit tumor growth to some extent; alleviate to some extent one or more symptoms associated with cancer; and / or produce a favorable response, such as an increase in progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS), a complete response (CR), a partial response (PR), or in some cases, stable disease (SD), a decrease in progressive disease (PD), a shortened time to progression (TTP), or any combination thereof. The term "effective amount" may be used interchangeably with "effective dose," "therapeutically effective amount," or "therapeutically effective dose."

[0055] The term "Fynomer" refers to proteins derived from the SH3 domain of human Fyn kinase that have been engineered to bind to specific targets (see Bertschinger et al., 2007. Protein Eng Des Sel. 20(2):57-68).

[0056] The terms "individual," "patient," or "subject" are used interchangeably in this application to designate a human and are not meant to be limiting in any way. An "individual," "patient," or "subject" may be of any age, sex, and physical condition. The term "patient in need thereof" generally refers to a patient suffering from a CD229-positive cancer. Preferably, the term "patient in need thereof" may also refer to a patient suffering from a CD229-positive / BCMA-negative cancer or a CD229-positive / BCMA-positive cancer.

[0057] "Infusion" or "infusing" refers to the introduction of a therapeutic agent-containing solution into the body through a vein for therapeutic purposes. Generally, this is accomplished via an intravenous bag.

[0058] As used herein, the term "intracellular signaling domain" refers to all or a portion of one or more domains of a molecule (here, a chimeric receptor molecule) that activates lymphocytes. The intracellular domain of such a molecule mediates signals by interacting with cellular mediators, resulting in proliferation, differentiation, activation, and other effector functions. Examples of intracellular signaling domains for use in the CARs of the present invention include the intracellular sequence of the CD3 zeta chain and / or co-receptors that act in concert to initiate signaling after CAR engagement, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capabilities. T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation and provide T cell receptor-like signals (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner and provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as receptor tyrosine-based activation motifs or ITAMs. Examples of primary cytoplasmic signaling sequences containing ITAMs include those derived from CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0059] The term "monobody" refers to a protein derived from a fibronectin type III domain that has been engineered to bind to a specific target (see Koide et al., 2013. J Mol Biol. 415(2):393-405).

[0060] The term "nanobody" refers to a protein comprising a soluble single antigen-binding V domain of a heavy chain antibody, preferably a camelid heavy chain antibody (see Bannas et al., 2017. Front Immunol. 8:1603).

[0061] "Overall survival" (OS) refers to the time from patient enrollment to death or censoring at the date the patient is last known to be alive. OS includes an increase in life expectancy compared to naive or untreated individuals or patients. Overall survival refers to the situation in which a patient survives for a specified period of time, such as 1 year, 5 years, etc., from the time of diagnosis or treatment.

[0062] "Partial response" or "PR" refers to at least a 30% reduction in the sum of the diameters of the target lesions in response to treatment, relative to the baseline sum diameter, as defined by RECIST v1.1 guidelines.

[0063] The term "peptide aptamer" refers to a short 5-20 amino acid residue sequence that can bind to a specific target. Peptide aptamers are typically inserted into the loop region of a stable protein scaffold (see Reverdatto et al., 2015. Curr Top Med Chem. 15(12):1082-101).

[0064] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable diluent" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to, the following: additional buffering agents; preservatives; cosolvents; antioxidants, including ascorbic acid and methionine; chelating agents, such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; salt-forming counterions, such as sodium, polyhydric sugar alcohols; amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; lactitol, stachyose, and the like. Organic sugars or sugar alcohols such as sucrose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol, etc.; sulfur-containing reducing agents such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [α]-monothioglycerol, sodium thiosulfate, etc.; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers such as polyvinylpyrrolidone. Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), may be included in the pharmaceutical compositions described herein, so long as they do not adversely affect the desired properties of the pharmaceutical composition.

[0065] "Progressive disease" or "advanced disease" refers to the appearance of another new lesion or tumor and / or definite progression of an existing non-target lesion, as defined by the RECIST v1.1 guideline. Progressive disease or advanced disease can also refer to tumor growth of more than 20% since treatment began, due to either an increase in mass or tumor spread.

[0066] "Progression-free survival" (PFS) refers to the time from enrollment to disease progression or death. PFS is commonly measured using the Kaplan-Meier method and the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 standard. In general, progression-free survival refers to the period in which a patient remains alive without their cancer worsening.

[0067] The term "RECIST" refers to Response Evaluation Criteria in Solid Tumors. RECIST guidelines, criteria, or standards describe a standard approach to measuring solid tumors and definitions for the objective assessment of changes in tumor size for use in adult and pediatric cancer clinical trials. RECIST v1.1 refers to the revised RECIST guidelines, version 1.1, published in European Journal of Cancers 45 (2009) 228-247.

[0068] The term "lipibody" refers to proteins derived from leucine-rich repeat modules and engineered to bind to specific targets (Lee et al., 2012. PNAS. 109(9):3299-3304).

[0069] The term "respond favorably" generally refers to causing a beneficial state in a subject. In the context of cancer treatment, the term refers to bringing about a therapeutic effect in a subject. A positive therapeutic effect in cancer can be measured in several ways. For example, tumor growth inhibition, expression of molecular markers, expression of serum markers, and molecular imaging techniques can all be used to evaluate the therapeutic efficacy of anti-cancer therapeutic agents. A favorable response can be evaluated, for example, by an extension of progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), a complete response (CR), a partial response (PR), or in some cases, stable disease (SD), a reduction in progressive disease (PD), a shortened time to progression (TTP), or any combination thereof.

[0070] The term "sequence identity" refers to the percentage value obtained when comparing two sequences using a pairwise sequence alignment tool. In this case, the sequence identity is obtained using the global alignment tool "EMBOSS Needle" (Rice et al., 2000. Trends Genet. 16(6):276-7; Li et al., 2015. Nucleic Acids Res. 43(W1):W580-4) using default settings. The global alignment tool is available at https: / / www.ebi.ac.uk / Tools / psa / .

[0071] The term "single-chain antigen-binding fragment" or "scFab" refers to a fusion protein containing one variable domain and one constant domain of an antibody light chain linked to one variable domain and one constant domain of an antibody heavy chain, the heavy and light chains being linked together via a short peptide.

[0072] The term "single-chain variable fragment" or "scFv" refers to a fusion protein comprising the variable domains of the heavy and light chains of an antibody linked together by a peptide linker. The term also includes disulfide-stabilized Fvs (dsFvs).

[0073] "Stable disease" refers to disease without progression or recurrence as defined by RECIST v1.1 guidelines. Stable disease does not involve sufficient tumor shrinkage to qualify as a partial response or sufficient tumor growth to qualify as progressive disease.

[0074] "Time to tumor progression" (TTP) is defined as the time from enrollment to disease progression. TTP is generally measured using RECIST v1.1 criteria.

[0075] As used in this application, the terms "treatment" and "therapy" refer to a set of hygienic, pharmacological, surgical, and / or physical measures used to cure and / or alleviate a disease and / or symptoms with the aim of improving a health problem. The terms "treatment" and "therapy" both include preventative and curative methods, as they are directed to maintaining and / or re-establishing the health of an individual or animal. The administration of appropriate medications to alleviate and / or cure a health problem, regardless of the cause of the symptoms, disease, and disorder, should be construed as a form of treatment or therapy within the context of this application.

[0076] Description of the embodiment Four different CD229 CAR sequences (designated GL001, GL002, GL005, and GL006) based on two different antibodies (hLy9 1.84 and hLy9 10.169) were generated and tested herein. GL001 and GL002 are based on hLy9 1.84 scFV (see Figure 1) and share CDRs in both the VL and VH chains, but the order of the VH and VL chains is reversed. On the other hand, GL005 and GL006 are based on hLy9 10.169, thereby also sharing their CDRs, but with different VH and VL chain orders (see Figure 1).

[0077] First, we tested the expression of the newly developed CAR sequences in HEK293T cells and T cells. As shown in Figure 2, only GL001 and GL002 (VH-VL-41BB-CD3z and VL-VH-41BB-CD3z) based on hLy9 1.84 scFV could be detected, whereas GL005 and GL006 (VH-VL-41BB-CD3z and VL-VH-41BB-CD3z) were not. Considering these results, the following tests were performed only on GL001 and GL002 CARS.

[0078] The efficacy of GL001 and GL002 cells against myeloma cell lines was tested by co-culturing T cells and myeloma cell lines. GL001 and GL002 CAR T cells conferred specific cytolytic activity against U266 and MM.1S myeloma cell lines in a dose-response relationship from high to low E:T ratios, indicating that GL001 and GL002 CAR T cells exhibit potent and specific cytotoxic effects against CD229-positive cells in vitro (see Figure 3).

[0079] To better characterize the GL001 and GL002 cell responses upon CD229 binding, cell proliferation was measured. As shown in Figure 4, GL001 and GL002 cells proliferated in contact with the CD229+ MM.1S cell line and in response to interleukin-2 (IL-2). Some proliferation was observed in the absence of stimulation or in contact with a CD229-negative cell line (K562), confirming that cell proliferation was mediated by CD229 recognition (see Figure 4).

[0080] Next, we measured GL001 and GL002 cell production of cytokines in the supernatants of effector-target cell cocultures. We observed that GL001 and GL002 cells showed a significant increase in IFNg and IL-2 pro-inflammatory cytokines when cocultured with CD229-positive cells (U266 and MM.1S) (see Figure 5).

[0081] Finally, to evaluate the efficacy of GL001 and GL002 cells in vivo, we performed xenograft experiments in NOD.Cg-Prkdcscid Il2rdtm1Wjl / SzJ (NSG) mice. As shown in Figure 6, disease progression was clearly observed after U266 cell injection in the UTD cell group. The groups administered the U266 tumor cell line and GL002 showed better disease control compared to the UTD and GL001 groups. These data suggest that GL002 is more effective than GL001 in terms of efficacy.

[0082] To recapitulate human MM, we generated a BCMA knockout (KO) MM.1S myeloma cell line, modeling established BCMA-positive heterogeneous disease. GL001 and GL002 CAR T cells were able to lyse these BCMA-KO cells (see Figure 7B). Furthermore, as shown in Figure 8, GL002 outperformed ARI002h, a CAR directed against the BCMA antigen used as a positive control. Importantly, these results demonstrate that GL002 outperforms anti-BCMA CARs in heterogeneous tumors where a population of BCMA-negative cells is present.

[0083] Preferably, the first aspect relates to a CD229 targeting moiety comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2 and HCDR3 polypeptides; - LCDR1 comprises, consists of, or consists essentially of [SSVSY] (SEQ ID NO: 4) or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4; - LCDR2 comprises, consists of, or consists essentially of [DTS] (SEQ ID NO: 5) or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5; - LCDR3 comprises, consists of, or consists essentially of [CQQWSSYPPTF] (SEQ ID NO: 6), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6; - HCDR1 comprises, consists of, or consists essentially of [GYTFTHYY] (SEQ ID NO: 7), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7; - HCDR2 comprises, consists of, or consists essentially of [IFPESGST] (SEQ ID NO: 8), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8; and - HCDR3 comprises, consists of, or consists essentially of [CARGTGFFDYW] (SEQ ID NO: 9), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 9.

[0084] In some embodiments, the CD229 targeting moiety is an antibody, anticalin, lipibody, monobody, scFv, Fab, scFab, affibody, fynomer, DARPin, nanobody, or peptide aptamer that specifically binds to CD229.

[0085] In some embodiments, the CD229 targeting moiety is an antibody, F(ab')2, Fab, preferably scFv, or scFab comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2, and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2, and HCDR3 polypeptides, wherein LCDR1 consists solely of [SSVSY] (SEQ ID NO: 4), LCDR2 consists solely of [DTS] (SEQ ID NO: 5), LCDR3 consists solely of [CQQWSSYPPTF] (SEQ ID NO: 6), HCDR1 consists solely of [GYTFTHYY] (SEQ ID NO: 7), HCDR2 consists solely of [IFPESGST] (SEQ ID NO: 8), and HCDR3 consists solely of [CARGTGFFDYW] (SEQ ID NO: 9).

[0086] Preferably, the CD229 targeting moiety is an antibody, F(ab')2, Fab, preferably scFv, or scFab, comprising a VL domain and a VH domain, wherein the VL domain comprises, consists of, or consists essentially of SEQ ID NO:1, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1; and the VH domain comprises, consists of, or consists essentially of SEQ ID NO:2, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2. In some embodiments, the CD229 targeting moiety is an antibody, scFv, Fab or scFab comprising a VL domain and a VH domain, wherein the VL domain consists solely of SEQ ID NO:1 and the VH domain consists solely of SEQ ID NO:2.

[0087] The VL and VH of the CD229 targeting moieties described herein can be humanized. In the humanization process, the framework regions of the variable regions of the targeting moiety can be altered, while the CDRs remain constant. Thus, in some embodiments, the CD229 targeting moiety is an antibody, F(ab')2, Fab, preferably scFv, or scFab, comprising a VL domain and a VH domain, wherein the VL domain has a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1. the VH domain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, and the CDRs contained in the VH are kept constant and consist only of SEQ ID NOs: 7, 8 and 9. "Kept constant" as used herein refers to the fact that the framework regions of the targeting moiety may vary (e.g., they may differ depending on whether it is a murine ScFv or a humanized ScFv), but the sequences belonging to the CDRs are not changed or remain unchanged.

[0088] Thus, in one embodiment, a CD229 targeting moiety of the invention comprises a VL domain and a VH domain, - the VL domain comprises a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, and the VL domain comprises CDR regions consisting only of LCDR1 [SSVSY] (SEQ ID NO: 4), LCDR2 [DTS] (SEQ ID NO: 5) and LCDR3 [CQQWSSYPPTF] (SEQ ID NO: 6); - the VH domain comprises a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, wherein the VH domain comprises CDR regions consisting only of HCDR1 [GYTFTHYY] (SEQ ID NO: 7), HCDR2 [IFPESGST] (SEQ ID NO: 8) and HCDR3 [CARGTGFFDYW] (SEQ ID NO: 9); Preferably, the CD229 targeting moiety is capable of binding to human CD229, preferably as measured by surface plasmon resonance (SPR).

[0089] Preferably, the CD229 targeting moiety comprises, from N-terminal to C-terminal, a VL domain and a VH domain, - the VL domain comprises a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, and the VL domain comprises CDR regions consisting only of LCDR1 [SSVSY] (SEQ ID NO: 4), LCDR2 [DTS] (SEQ ID NO: 5) and LCDR3 [CQQWSSYPPTF] (SEQ ID NO: 6); - the VH domain comprises a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, wherein the VH domain comprises CDR regions consisting only of HCDR1 [GYTFTHYY] (SEQ ID NO: 7), HCDR2 [IFPESGST] (SEQ ID NO: 8) and HCDR3 [CARGTGFFDYW] (SEQ ID NO: 9); Preferably, the CD229 targeting moiety is capable of binding to human CD229, preferably as measured by surface plasmon resonance (SPR).

[0090] Preferably, the VL domain is located upstream of the VH domain. "Located upstream" as used herein refers to the fact that in the CD229 targeting moiety, the VL domain is located toward the N-terminus of the moiety, and the VH domain is located toward the C-terminus of the moiety. In other words, the VL domain is located at the N-terminus of the VH domain.

[0091] In one embodiment, a CD229 targeting moiety of the invention is capable of binding to human CD229, wherein the CD229 targeting moiety comprises a VL domain and a VH domain, - the VL domain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1; - the VH domain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2; LCDR1, 2 and 3 contained in the VL domain consist only of SEQ ID NOs: 4, 5 and 6, respectively, and HCDR1, 2 and 3 contained in the VH domain consist only of SEQ ID NOs: 7, 8 and 9, respectively.

[0092] "Capable of binding to human CD229" means that the CD229 targeting moiety has binding affinity for human CD229. Preferably, the binding affinity is specific, i.e., CD229 can specifically bind to human CD229 (its target molecule) in a manner that distinguishes it from binding to non-target molecules, i.e., other molecules that are not CD229. Thus, the CD229 targeting moiety does not bind to non-target molecules, or exhibits negligible or substantially reduced (e.g., background) binding to non-target molecules (compared to the target). The CD229 targeting moiety specifically recognizes CD229. Specific binding of the CD229 targeting moiety to human CD229 can be assessed by a number of techniques, such as surface plasmon resonance, flow cytometry, or ELISA assays.

[0093] In preferred embodiments, the VL and VH domains comprised in the CD229 targeting moiety are linked by a peptide linker. In some embodiments, the linker comprises at least 5 amino acids, preferably 5-25, preferably 10-20, and most preferably 20 amino acids. Preferably, the amino acids are G and / or S. Preferably, the peptide linker comprises, consists of, or consists essentially of SEQ ID NO: 14, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14. Preferably, the amino acids are G and / or S. Preferably, the peptide linker comprises, consists of, or consists essentially of SEQ ID NO: 16, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 16. Preferably, the VL and VH of GL001 (SEQ ID NOs: 1 and 2) are linked with the peptide linker of SEQ ID NO: 14, as shown in SEQ ID NO: 3. Preferably, the VL and VH of GL002 (SEQ ID NOs: 1 and 2) are linked with the peptide linker of SEQ ID NO: 16, as shown in SEQ ID NO: 17.

[0094] In a preferred embodiment, the CD229 targeting moiety further comprises a signal peptide, preferably located at the N-terminal region of the CD229 targeting moiety. Preferably, the signal peptide comprises, consists of, or consists essentially of SEQ ID NO: 15, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 15.

[0095] Preferably, the CD229 targeting moiety comprises, consists of, or consists essentially of SEQ ID NO: 3 (GL001 signal peptide + VH + linker + VL), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3. Most preferably, the CD229 targeting moiety consists solely of SEQ ID NO: 3 (GL001 signal peptide + VH + linker + VL).

[0096] Preferably, the CD229 targeting moiety comprises, consists of, or consists essentially of SEQ ID NO: 17 (GL002 signal peptide + VL + linker + VH), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17. Most preferably, the CD229 targeting moiety consists solely of SEQ ID NO: 17 (GL002 signal peptide + VL + linker + VH).

[0097] In some embodiments, the CD229 targeting moiety is an scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2, and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2, and HCDR3 polypeptides; - LCDR1 comprises, consists of, or consists essentially of [SSVSY] (SEQ ID NO: 4) or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4; - LCDR2 comprises, consists of, or consists essentially of [DTS] (SEQ ID NO: 5) or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5; - LCDR3 comprises, consists of, or consists essentially of [CQQWSSYPPTF] (SEQ ID NO: 6), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6; - HCDR1 comprises, consists of, or consists essentially of [GYTFTHYY] (SEQ ID NO: 7) or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 7; - HCDR2 comprises, consists of, or consists essentially of [IFPESGST] (SEQ ID NO: 8), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8; and - HCDR3 comprises, consists of, or consists essentially of [CARGTGFFDYW] (SEQ ID NO: 9), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 9.

[0098] In some embodiments, the CD229 targeting moiety is an scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2 and HCDR3 polypeptides, wherein LCDR1 consists solely of [SSVSY] (SEQ ID NO: 4), LCDR2 consists solely of [DTS] (SEQ ID NO: 5), LCDR3 consists solely of [CQQWSSYPPTF] (SEQ ID NO: 6), HCDR1 consists solely of [GYTFTHYY] (SEQ ID NO: 7), HCDR2 consists solely of [IFPESGST] (SEQ ID NO: 8), and HCDR3 consists solely of [CARGTGFFDYW] (SEQ ID NO: 9).

[0099] Preferably, the CD229 targeting moiety is an scFv comprising a VL domain and a VH domain, wherein the VL domain comprises, consists of, or consists essentially of SEQ ID NO:1, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1; and the VH domain comprises, consists of, or consists essentially of SEQ ID NO:2, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2. In some embodiments, the CD229 targeting moiety is an scFv comprising a VL domain and a VH domain, wherein the VL domain consists solely of SEQ ID NO:1 and the VH domain consists solely of SEQ ID NO:2.

[0100] Preferably, the CD229 targeting moiety is an scFv comprising, consisting of, or consisting essentially of SEQ ID NO: 3 (GL001 signal peptide + VH + linker + VL), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3. Most preferably, the CD229 targeting moiety is an scFv consisting only of SEQ ID NO: 3 (GL001 signal peptide + VH + linker + VL).

[0101] Preferably, the CD229 targeting moiety is a scFv comprising the sequence of SEQ ID NO: 17 (GL002 signal peptide + VL + linker + VH), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17, or consisting of only such a sequence, or consisting essentially of only such a sequence. Most preferably, the CD229 targeting moiety is a scFv consisting of only the sequence of SEQ ID NO: 17 (GL002 signal peptide + VL + linker + VH).

[0102] CAR anti-CD229 The CD229 targeting moiety defined above can be part of a chimeric antigen receptor (CAR). Thus, in a preferred embodiment of the first aspect, the present invention provides a) an extracellular domain comprising a CD229 targeting moiety as defined in any of the first aspect or the embodiments disclosed above, b) a transmembrane domain; and c) a chimeric antigen receptor comprising an intracellular signaling domain.

[0103] Each element of the CAR according to this embodiment of the first aspect is further developed below.

[0104] a) An extracellular domain comprising a CD229 targeting moiety as defined in any of the first aspect or its embodiments. In some embodiments, the CD229 targeting moiety is a scFv comprising a VL domain and a VH domain, the VL domain comprising LCDR1, LCDR2 and LCDR3 polypeptides, the VH domain comprising HCDR1, HCDR2 and HCDR3 polypeptides, - LCDR1 comprises [SSVSY] (SEQ ID NO: 4), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4, or consisting of only such a sequence, or consisting essentially of only such a sequence; - LCDR2 comprises, consists of, or consists essentially of [DTS] (SEQ ID NO: 5) or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5; - LCDR3 comprises, consists of, or consists essentially of [CQQWSSYPPTF] (SEQ ID NO: 6), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6; - HCDR1 comprises, consists of, or consists essentially of [GYTFTHYY] (SEQ ID NO: 7), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7; - HCDR2 comprises, consists of, or consists essentially of [IFPESGST] (SEQ ID NO: 8), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8; and - HCDR3 comprises, consists of, or consists essentially of [CARGTGFFDYW] (SEQ ID NO: 9), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 9.

[0105] In some embodiments, the CD229 targeting portion is an scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2 and HCDR3 polypeptides, wherein LCDR1 consists solely of SEQ ID NO: 4, LCDR2 consists solely of SEQ ID NO: 5, LCDR3 consists solely of SEQ ID NO: 6, HCDR1 consists solely of SEQ ID NO: 7, HCDR2 consists solely of SEQ ID NO: 8, and HCDR3 consists solely of SEQ ID NO: 9.

[0106] In one embodiment of the first aspect, the extracellular domain comprising the CD229 targeting moiety is an scFv comprising a VL domain and a VH domain, wherein the VL domain comprises, consists of, or consists essentially of SEQ ID NO:1, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO:1; and the VH domain comprises, consists of, or consists essentially of SEQ ID NO:2, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2.

[0107] Most preferably, the CD229 targeting moiety is an scFv comprising, consisting of, or consisting essentially of SEQ ID NO: 3, or a sequence (GL001 signal peptide + VH + linker + VL) having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3. Most preferably, the CD229 targeting moiety is an scFV consisting solely of SEQ ID NO:3.

[0108] Most preferably, the CD229 targeting moiety is an scFv comprising, consisting of, or consisting essentially of SEQ ID NO: 17, or a sequence (GL002 signal peptide + VH + linker + VL) having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17. Most preferably, the CD229 targeting moiety is an scFv consisting of SEQ ID NO:17.

[0109] In one embodiment, a CD229 targeting moiety of the invention is capable of binding to human CD229, wherein the CD229 targeting moiety is an scFv and comprises a VL domain and a VH domain, - the VL domain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1; - the VH domain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2; LCDR1, 2 and 3 contained in the VL domain consist only of SEQ ID NOs: 4, 5 and 6, respectively, and HCDR1, 2 and 3 contained in the VH domain consist only of SEQ ID NOs: 7, 8 and 9, respectively.

[0110] In a preferred embodiment, the VL and VH domains of the scFv are linked by a peptide linker, which preferably comprises, consists of, or consists essentially of SEQ ID NO: 14 or 16, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14 or 16. Preferably, the VL and VH of scFV GL001 are linked by a peptide linker comprising, consisting of, or consisting essentially of SEQ ID NO: 14, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14. Preferably, the VL and VH of scFV GL002 are linked by a peptide linker comprising, consisting of, or consisting essentially of SEQ ID NO: 16, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 16.

[0111] In a preferred embodiment, the CD229 targeting moiety is an scFv and further comprises a signal peptide located in the N-terminal region of the CD229 targeting moiety. Preferably, the signal peptide comprises, consists of, or consists essentially of SEQ ID NO: 15, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 15.

[0112] b) Transmembrane domain The transmembrane domain can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane region can include at least the transmembrane region(s) of the α, β, or ζ chain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.

[0113] The transmembrane domain may be a synthetic transmembrane domain or a variant of a naturally occurring transmembrane domain, hi some embodiments, the synthetic or variant transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine.

[0114] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a variant thereof, wherein the variant has 95% sequence identity.

[0115] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a variant thereof, wherein the variant has 98% sequence identity.

[0116] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.

[0117] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD8 or a variant thereof, wherein the variant has 95% sequence identity.

[0118] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD8 or a variant thereof. Preferably, the transmembrane domain consists of, or consists essentially of, SEQ ID NO: 10, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10.

[0119] In one embodiment, the transmembrane domain is linked to a hinge domain, which is preferably located N-terminal to the transmembrane domain. Preferably, the hinge domain consists of or consists essentially of SEQ ID NO: 37 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 37.

[0120] Thus, in a preferred embodiment, the transmembrane domain linked to the hinge domain consists solely or essentially of SEQ ID NO:19 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:19.

[0121] c) Intracellular signaling domain The intracellular signaling domain, upon binding to a ligand expressed on a tumor cell, results in the activation of at least one function of the cell expressing the CAR. In some embodiments, the intracellular signaling domain contains one or more intracellular signaling domains. In some embodiments, the intracellular signaling domain is a portion and / or variant of an intracellular signaling domain that results in the activation of at least one function of the CAR-containing cell.

[0122] In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3zeta (or CD3z), FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66b, or a variant thereof, wherein the variant has 95% sequence identity. In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3zeta, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66b, or a variant thereof, wherein the variant has 98% sequence identity. In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3zeta, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66b.

[0123] In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3ζ or a variant thereof, wherein the variant has 95% sequence identity. In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3ζ or a variant thereof, wherein the variant has 98% sequence identity. In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3ζ.

[0124] In a preferred embodiment, the intracellular signaling domain consists solely of, or consists essentially of, SEQ ID NO:11, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:11.

[0125] In some embodiments, the intracellular signaling domain comprises SEQ ID NO:11, or a sequence with 95% sequence identity to SEQ ID NO:11. In some embodiments, the intracellular signaling domain comprises SEQ ID NO:11, or a sequence with 98% sequence identity to SEQ ID NO:11. In some embodiments, the intracellular signaling domain comprises SEQ ID NO:11, or a sequence with 99% sequence identity to SEQ ID NO:11. In some embodiments, the intracellular signaling domain comprises SEQ ID NO:11. In some embodiments, the intracellular signaling domain consists solely of SEQ ID NO:11.

[0126] Optionally, at least a costimulatory signaling domain may also be present in the CAR according to the first aspect or any of its embodiments.

[0127] d) Costimulatory Signaling Domain In some embodiments, the CAR may further comprise a costimulatory signaling domain, hi some embodiments, the costimulatory signaling domain comprises the intracellular domain of CD27, CD28, CD137, CD134, CD30, CD40, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, CD276, or a variant thereof, wherein the variant has 95% sequence identity.

[0128] In some embodiments, the costimulatory signaling domain comprises the intracellular domain of CD27, CD28, CD137, CD134, CD30, CD40, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, CD276, or a variant thereof, wherein the variant has 98% sequence identity.

[0129] In some embodiments, the costimulatory signaling domain comprises the intracellular domain of CD27, CD28, CD137 or 4-1BB, CD134, CD30, CD40, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or CD276.

[0130] In some embodiments, the costimulatory signaling domain comprises the intracellular domain of CD137 or a variant thereof, wherein the variant has 95% sequence identity. In some embodiments, the costimulatory signaling domain comprises the intracellular domain of CD137 or 4-1BB or a variant thereof, wherein the variant has 98% sequence identity. In some embodiments, the costimulatory signaling domain comprises the intracellular domain of CD137 or 4-1BB.

[0131] In some embodiments, the costimulatory signaling domain comprises SEQ ID NO: 12, or a sequence with 95% sequence identity to SEQ ID NO: 12. In some embodiments, the costimulatory signaling domain comprises SEQ ID NO: 12, or a sequence with 98% sequence identity to SEQ ID NO: 12. In some embodiments, the costimulatory signaling domain comprises SEQ ID NO: 12, or a sequence with 99% sequence identity to SEQ ID NO: 12. In some embodiments, the costimulatory signaling domain consists solely of SEQ ID NO: 12.

[0132] In preferred embodiments, the costimulatory signaling domain consists solely of, or consists essentially of, SEQ ID NO:12, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:12.

[0133] Full sequence CAR according to the first aspect of the invention In some embodiments, the CAR comprises: (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2, and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2, and HCDR3 polypeptides, wherein LCDR1 consists solely of SEQ ID NO: 4, LCDR2 consists solely of SEQ ID NO: 5, LCDR3 consists solely of SEQ ID NO: 6, HCDR1 consists solely of SEQ ID NO: 7, HCDR2 consists solely of SEQ ID NO: 8, and HCDR3 consists solely of SEQ ID NO: 9; (ii) a transmembrane domain comprising SEQ ID NO: 19, or a sequence having 95% sequence identity to SEQ ID NO: 19; (iii) an intracellular signaling domain comprising SEQ ID NO: 11, or a sequence having 95% sequence identity to SEQ ID NO: 11; and (iv) a costimulatory signaling domain comprising SEQ ID NO: 12, or a sequence having 95% sequence identity to SEQ ID NO: 12.

[0134] In some embodiments, the CAR comprises: (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2, and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2, and HCDR3 polypeptides, wherein LCDR1 consists solely of SEQ ID NO: 4, LCDR2 consists solely of SEQ ID NO: 5, LCDR3 consists solely of SEQ ID NO: 6, HCDR1 consists solely of SEQ ID NO: 7, HCDR2 consists solely of SEQ ID NO: 8, and HCDR3 consists solely of SEQ ID NO: 9; (ii) a transmembrane domain comprising SEQ ID NO: 19 or a sequence having 98% sequence identity to SEQ ID NO: 19; (iii) an intracellular signaling domain comprising SEQ ID NO: 11, or a sequence having 98% sequence identity to SEQ ID NO: 11; and (iv) a costimulatory signaling domain comprising SEQ ID NO: 12, or a sequence having 98% sequence identity to SEQ ID NO: 12.

[0135] In some embodiments, the CAR comprises: (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2, and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2, and HCDR3 polypeptides, wherein LCDR1 consists solely of SEQ ID NO: 4, LCDR2 consists solely of SEQ ID NO: 5, LCDR3 consists solely of SEQ ID NO: 6, HCDR1 consists solely of SEQ ID NO: 7, HCDR2 consists solely of SEQ ID NO: 8, and HCDR3 consists solely of SEQ ID NO: 9; (ii) a transmembrane domain comprising SEQ ID NO: 19, or a sequence having 99% sequence identity to SEQ ID NO: 19; (iii) an intracellular signaling domain comprising SEQ ID NO: 11, or a sequence having 99% sequence identity to SEQ ID NO: 11; and (iv) a costimulatory signaling domain comprising SEQ ID NO: 12, or a sequence having 99% sequence identity to SEQ ID NO: 12.

[0136] In some embodiments, the CAR comprises: (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2, and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2, and HCDR3 polypeptides, wherein LCDR1 consists solely of SEQ ID NO: 4, LCDR2 consists solely of SEQ ID NO: 5, LCDR3 consists solely of SEQ ID NO: 6, HCDR1 consists solely of SEQ ID NO: 7, HCDR2 consists solely of SEQ ID NO: 8, and HCDR3 consists solely of SEQ ID NO: 9; (ii) a transmembrane domain comprising SEQ ID NO: 19; (iii) an intracellular signaling domain comprising SEQ ID NO: 11; and (iv) a costimulatory signaling domain comprising SEQ ID NO: 12.

[0137] In some embodiments, the CAR comprises: (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2, and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2, and HCDR3 polypeptides, wherein LCDR1 consists solely of SEQ ID NO: 4, LCDR2 consists solely of SEQ ID NO: 5, LCDR3 consists solely of SEQ ID NO: 6, HCDR1 consists solely of SEQ ID NO: 7, HCDR2 consists solely of SEQ ID NO: 8, and HCDR3 consists solely of SEQ ID NO: 9; (ii) a transmembrane domain consisting solely of SEQ ID NO: 19; (iii) an intracellular signaling domain consisting solely of SEQ ID NO: 11; and (iv) A costimulatory signaling domain consisting solely of SEQ ID NO: 12.

[0138] In some embodiments, the CAR comprises: (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain consists solely of SEQ ID NO: 1 and the VH domain consists solely of SEQ ID NO: 2; (ii) a transmembrane domain comprising SEQ ID NO: 19, or a sequence having 95% sequence identity to SEQ ID NO: 19; (iii) an intracellular signaling domain comprising SEQ ID NO: 11, or a sequence having 95% sequence identity to SEQ ID NO: 11; and (iv) a costimulatory signaling domain comprising SEQ ID NO: 12, or a sequence having 95% sequence identity to SEQ ID NO: 12.

[0139] In some embodiments, the CAR comprises: (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain consists solely of SEQ ID NO: 1 and the VH domain consists solely of SEQ ID NO: 2; (ii) a transmembrane domain comprising SEQ ID NO: 19 or a sequence having 98% sequence identity to SEQ ID NO: 19; (iii) an intracellular signaling domain comprising SEQ ID NO: 11, or a sequence having 98% sequence identity to SEQ ID NO: 11; and (iv) a costimulatory signaling domain comprising SEQ ID NO: 12, or a sequence having 98% sequence identity to SEQ ID NO: 12.

[0140] In some embodiments, the CAR comprises: (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain consists solely of SEQ ID NO: 1 and the VH domain consists solely of SEQ ID NO: 2; (ii) a transmembrane domain comprising SEQ ID NO: 19, or a sequence having 99% sequence identity to SEQ ID NO: 19; (iii) an intracellular signaling domain comprising SEQ ID NO: 11, or a sequence having 99% sequence identity to SEQ ID NO: 11; and (iv) a costimulatory signaling domain comprising SEQ ID NO: 12, or a sequence having 99% sequence identity to SEQ ID NO: 12.

[0141] In some embodiments, the CAR comprises: (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain consists solely of SEQ ID NO: 1 and the VH domain consists solely of SEQ ID NO: 2; (ii) a transmembrane domain comprising SEQ ID NO: 19; (iii) an intracellular signaling domain comprising SEQ ID NO: 11; and (iv) a costimulatory signaling domain comprising SEQ ID NO: 12.

[0142] In some embodiments, the CAR comprises: (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain consists solely of SEQ ID NO: 1 and the VH domain consists solely of SEQ ID NO: 2; (ii) a transmembrane domain consisting solely of SEQ ID NO: 19; (iii) an intracellular signaling domain consisting solely of SEQ ID NO: 11; and (iv) A costimulatory signaling domain consisting solely of SEQ ID NO: 12.

[0143] In some embodiments, the CAR comprises: (i) an scFv consisting only of SEQ ID NO: 3 or 17; (ii) a transmembrane domain comprising SEQ ID NO: 19, or a sequence having 95% sequence identity to SEQ ID NO: 19; (iii) an intracellular signaling domain comprising SEQ ID NO: 11, or a sequence having 95% sequence identity to SEQ ID NO: 11; and (iv) a costimulatory signaling domain comprising SEQ ID NO: 12, or a sequence having 95% sequence identity to SEQ ID NO: 12.

[0144] In some embodiments, the CAR comprises: (i) an scFv consisting only of SEQ ID NO: 3 or 17; (ii) a transmembrane domain comprising SEQ ID NO: 19 or a sequence having 98% sequence identity to SEQ ID NO: 19; (iii) an intracellular signaling domain comprising SEQ ID NO: 11, or a sequence having 98% sequence identity to SEQ ID NO: 11; and (iv) a costimulatory signaling domain comprising SEQ ID NO: 12, or a sequence having 98% sequence identity to SEQ ID NO: 12.

[0145] In some embodiments, the CAR comprises: (i) an scFv consisting only of SEQ ID NO: 3 or 17; (ii) a transmembrane domain comprising SEQ ID NO: 19, or a sequence having 99% sequence identity to SEQ ID NO: 19; (iii) an intracellular signaling domain comprising SEQ ID NO: 11, or a sequence having 99% sequence identity to SEQ ID NO: 11; and (iv) a costimulatory signaling domain comprising SEQ ID NO: 12, or a sequence having 99% sequence identity to SEQ ID NO: 12.

[0146] In some embodiments, the CAR comprises: (i) an scFv consisting only of SEQ ID NO: 3 or 17; (ii) a transmembrane domain comprising SEQ ID NO: 19; (iii) an intracellular signaling domain comprising SEQ ID NO: 11; and (iv) a costimulatory signaling domain comprising SEQ ID NO: 12.

[0147] In some embodiments, the CAR comprises: (i) an scFv consisting only of SEQ ID NO: 3 or 17; (ii) a transmembrane domain consisting solely of SEQ ID NO: 19; (iii) an intracellular signaling domain consisting solely of SEQ ID NO: 11; and (iv) A costimulatory signaling domain consisting solely of SEQ ID NO: 12.

[0148] In some embodiments, the CAR comprises or consists of SEQ ID NO: 13, or a sequence having 95% sequence identity to SEQ ID NO: 13. In some embodiments, the CAR comprises or consists of SEQ ID NO: 13, or a sequence having 98% sequence identity to SEQ ID NO: 13. In some embodiments, the CAR comprises or consists of SEQ ID NO: 13, or a sequence having 99% sequence identity to SEQ ID NO: 13. In some embodiments, the CAR comprises or consists of SEQ ID NO: 13.

[0149] In some embodiments, the CAR comprises or consists of SEQ ID NO: 18, or a sequence having 95% sequence identity to SEQ ID NO: 18. In some embodiments, the CAR comprises or consists of SEQ ID NO: 18, or a sequence having 98% sequence identity to SEQ ID NO: 18. In some embodiments, the CAR comprises or consists of SEQ ID NO: 18, or a sequence having 99% sequence identity to SEQ ID NO: 18. In some embodiments, the CAR comprises or consists of SEQ ID NO: 18.

[0150] nucleic acid In a second aspect, the present invention provides a nucleic acid encoding any one of the CD229-targeting moieties of the present invention, including any one of the CARs disclosed above. The nucleic acid sequence encoding the chimeric receptor links together several modular components that can be excised and replaced with other components to customize the chimeric receptor for efficient T cell activation and recognition of CD229.

[0151] In some embodiments, the nucleic acid is suitable for transducing or transforming cells, hi some embodiments, the nucleic acid is suitable for transducing or transforming T cells for use in adoptive immunotherapy.

[0152] In some embodiments, the nucleic acid is codon-optimized for expression in mammalian cells. Codon optimization methods are known in the art.

[0153] The nucleic acid of the present invention can be included in a gamma-retroviral or lentiviral vector that can be used to transduce or transform T cells. The nucleic acid can also be inserted into cells by using DNA transposon, RNA transfection, or genome editing techniques such as TALEN, ZFN and CRISPR / Cas9.

[0154] cell In a third aspect, the invention provides a cell comprising a nucleic acid of the invention and / or a CAR of the invention. In some embodiments, the cell is a T cell (referred to as a CART).

[0155] In some embodiments, the cells are naive T cells, memory stem T cells, or central memory T cells, which are currently believed to be more suitable for adaptive immunotherapy.

[0156] In some embodiments, the cells are autologous T cells. The term "autologous cells" refers to cells obtained from the same patient being treated using any one of the methods of the present invention.

[0157] In some embodiments, the cell is an allo-tolerant T cell. The term "allo-tolerant cell" refers to a cell that has been engineered to reduce the risk of graft-versus-host disease. In some embodiments, this is achieved by genome editing-mediated deletion of TCR and / or β2-microglobulin. Allo-tolerant cells are known in the art.

[0158] In some embodiments, the T cells are CD3+ T cells. In some embodiments, the T cells are CD8+ T cells. In some examples, they may be human cells.

[0159] In some embodiments, the cells are lymphoid progenitor cells, embryonic stem cells or induced pluripotent stem cells that have the capacity to differentiate into mature T cells.

[0160] Pharmaceutical Composition In a fourth aspect, the present invention provides a pharmaceutical composition comprising a plurality of cells of the present invention and a pharmaceutically acceptable carrier or diluent.

[0161] The pharmaceutical compositions described herein may also contain other substances. These substances include, but are not limited to, cryoprotectants, surfactants, antioxidants, and stabilizers. As used herein, the term "cryoprotectant" includes agents that stabilize CART against freezing-induced stress. Non-limiting examples of cryoprotectants include sugars such as sucrose, glucose, trehalose, mannitol, mannose, and lactose; polymers such as dextran, hydroxyethyl starch, and polyethylene glycol; surfactants such as polysorbates (e.g., PS-20 or PS-80); and amino acids such as glycine, arginine, leucine, and serine. Cryoprotectants that exhibit low toxicity in biological systems are commonly used.

[0162] In some embodiments, cells are formulated by first harvesting them from their culture medium, then washing and concentrating the cells in a therapeutically effective amount in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier). Suitable infusion media can be any isotonic medium formulation, typically saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), although 5% dextrose in water or lactated Ringer's can also be utilized. Infusion media can be supplemented with human serum albumin, fetal bovine serum, or other human serum components.

[0163] Treatment method In a fifth aspect, the present invention provides a CD229-targeting moiety according to the first aspect, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect for use as a medicament or in therapy. In a preferred embodiment, the use is in a method of treating a CD229-positive cancer, comprising administering to a patient in need thereof a CD229-targeting moiety of the first aspect, a nucleic acid of the second aspect, a cell of the third aspect, and / or a pharmaceutical composition of the fourth aspect. In some embodiments, the CD229-positive cancer is multiple myeloma (MM) cancer. In one embodiment, the multiple myeloma is selected from the group consisting of light chain myeloma, non-secretory myeloma, solitary plasmacytoma, extramedullary plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), immunoglobulin D (IgD) myeloma, and immunoglobulin E (IgE) myeloma. In a preferred embodiment, the MM cancer comprises a heterogeneous tumor comprising cells that are negative for the BCMA receptor. Thus, in a preferred embodiment, the cells or compositions of the invention are used to treat BCMA-negative tumors.

[0164] In one embodiment, the CD229-positive cancer is a lymphoma, preferably selected from the group consisting of chronic lymphocytic leukemia (CLL), classical mantle cell lymphoma (MCL), follicular lymphoma (FL), and / or diffuse large B-cell lymphoma (DLBCL), or any combination thereof. Preferably, the CD229-positive cancer is a B-cell lymphoma, preferably marginal zone lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), and splenic marginal zone lymphoma (SMZL).

[0165] In some embodiments, the CD229 targeting moiety, nucleic acid, cell or pharmaceutical composition is administered intravenously, intraperitoneally, intrathecally, intralymph node, and / or into the cerebrospinal fluid.

[0166] Thus, disclosed is a method for treating multiple myeloma, comprising administering to a subject in need thereof an effective amount of a CD229 targeting moiety, a nucleic acid, a cell (preferably a T cell) genetically modified to express one or more of the disclosed CAR polypeptides, or a pharmaceutical composition. For example, disclosed is a method for treating multiple myeloma, comprising administering an effective amount of a T cell genetically modified to express a CAR polypeptide comprising a CD229 antigen binding domain, a hinge and transmembrane domain, and an intracellular signaling domain, as described above.

[0167] In some embodiments, the method includes combination therapy. Thus, the CD229-targeting moieties and / or CAR-expressing cells described herein can be used in combination with other known drugs and therapies. As used herein, "administered in combination" means that two (or more) different treatments are delivered to a subject during the course of the subject's suffering from a disorder, for example, two or more treatments are delivered after the subject is diagnosed with a disorder and before the disorder is cured or eliminated, or before the treatments are discontinued for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second treatment begins, so there is an overlap in administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments, in either case, the treatments are more effective due to the combined administration. For example, the second treatment may be more effective, for example, a comparable effect may be observed with less of the second treatment, or the second treatment may reduce symptoms to a greater extent than that observed when the second treatment is administered in the absence of the first treatment, or a similar situation may be observed with the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed when one treatment is administered in the absence of the other. The effects of the two treatments may be partially additive, completely additive, or greater than additive. The delivery may be such that the effect of the first treatment being delivered is still detectable when the second treatment is delivered.

[0168] In one embodiment, the CD229 targeting moiety and / or CAR-expressing cells described herein are administered simultaneously, in the same or separate compositions, or sequentially with at least one additional therapeutic agent. For sequential administration, the CD229 targeting moiety and / or CAR-expressing cells described herein can be administered first and the additional agent can be administered second, or the order of administration can be reversed.

[0169] In some embodiments, the CD229 positive cancer is relapsed / refractory multiple myeloma.Generally, relapse of MM can occur several months or years after the first remission.However, most relapses occur within 2 years after the first treatment.Refractory is a term that implies that the patient no longer responds to at least one therapeutic strategy after relapse.

[0170] In some embodiments, patients treated with the methods of the invention are in complete or near-complete remission after treatment with another therapy. In patients with very active relapsed / refractory MM, it may be preferable or desirable to reduce the tumor burden before using the methods of the invention, as some alternative effector T cells are present. In some embodiments, patients treated with the methods of the invention have previously been treated with another therapy that resulted in a partial response, a complete response, stable disease, a decrease in progressive disease, a shortened time to tumor progression, or any combination thereof.

[0171] In some embodiments, the method further comprises administering an immune checkpoint inhibitor. In further embodiments, the method further comprises administering an immune checkpoint inhibitor and / or an IAP inhibitor. In some embodiments, the cells or pharmaceutical compositions described herein are administered in combination with a chemotherapeutic agent and / or an immunosuppressant. In one embodiment, the patient is first treated with a chemotherapeutic agent that inhibits or destroys other immune cells, followed by treatment with the cells or pharmaceutical compositions described herein. In some cases, chemotherapy may be avoided entirely. In some embodiments, the therapeutic agent may be, but is not limited to, conventional chemotherapy, including but not limited to, alkylating agents, antimetabolites, anti-microtubule agents, topoisomerase inhibitors, and cytotoxic antibiotics; high-dose chemotherapy, including but not limited to, high-dose melphalan chemotherapy with or without stem cell transplantation; proteasome inhibitors, such as but not limited to, bortezomib, ixazomib, and carfilzomib; immunomodulatory agents (IMiDS), such as but not limited to, thalidomide, lenalidomide, and pomalidomide; histone deacetylase (HDAC) inhibitors, such as but not limited to, panobinostat; monoclonal antibodies, such as but not limited to, daratumumab, isatuximab, or elotuzumab; bispecific antibodies; and immune checkpoint inhibitors, such as but not limited to, ipilimumab, nivolumab, and pembrolizumab.

[0172] Combination therapy In a sixth aspect, the present invention provides a combination therapy and uses thereof. In one embodiment, the combination therapy comprises: i) a CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect, and ii) administering a therapeutic agent capable of binding to human BCMA; The administration of i) and ii) may be simultaneous or sequential.

[0173] By "capable of binding to human BCMA" is meant that the BCMA targeting moiety has binding affinity for human BCMA. Preferably, the binding affinity is specific, i.e., BCMA is able to specifically bind to human BCMA (its target molecule) in a manner that distinguishes it from binding to non-target molecules, i.e., other molecules that are not BCMA. Thus, the BCMA targeting moiety does not bind to non-target molecules, or exhibits negligible or substantially reduced (e.g., background) binding to non-target molecules (compared to the target). The BCMA targeting moiety specifically recognizes BCMA. Specific binding of the BCMA targeting moiety to human BCMA can be assessed by a number of techniques, such as surface plasmon resonance, flow cytometry, or ELISA assays.

[0174] In some embodiments, the therapeutic agent capable of binding to human BCMA is a BCMA targeting moiety, preferably a CAR anti-BCMA. In some embodiments, the therapeutic agent capable of binding to human BCMA is a nucleic acid encoding the BCMA targeting moiety and / or a nucleic acid encoding a CAR anti-BCMA. In some embodiments, the therapeutic agent capable of binding to human BCMA is a cell or pharmaceutical composition comprising said BCMA targeting moiety or said CAR anti-BCMA or said nucleic acid encoding same.

[0175] It should be noted that the combination therapy item i) is defined in detail in the above aspects and includes any of the embodiments and combinations of embodiments thereof. Therapeutic agents capable of binding to human BCMA according to item ii) are described in detail below.

[0176] Therapeutic agents capable of binding to human BCMA: In one embodiment, the combination therapy consists of, sequentially or simultaneously: i) a CD229 targeting moiety as defined in the first aspect or any of its embodiments, and ii) administering a therapeutic agent capable of binding to human BCMA, wherein the therapeutic agent is a BCMA targeting moiety.

[0177] In one embodiment, the BCMA targeting moiety comprises a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2 and HCDR3 polypeptides; - LCDR1 comprises, consists of, or consists essentially of SEQ ID NO: 36, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 36; - LCDR2 comprises, consists of, or consists essentially of SEQ ID NO:20, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:20; - LCDR3 comprises, consists of, or consists essentially of SEQ ID NO:21, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:21; - HCDR1 comprises, consists of, or consists essentially of SEQ ID NO: 22, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 22; - HCDR2 comprises, consists of, or consists essentially of SEQ ID NO:23, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:23; and - HCDR3 comprises, consists of or consists essentially of SEQ ID NO:24 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:24.

[0178] In some embodiments, the BCMA targeting moiety is an antibody, anticalin, lipibody, monobody, scFv, Fab, scFab, affibody, finomer, DARPin, nanobody, or peptide aptamer that specifically binds to BCMA. Preferably, the BCMA targeting moiety is an ScFv and comprises a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2, and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2, and HCDR3 polypeptides, wherein LCDR1 consists solely of SEQ ID NO:36, LCDR2 consists solely of SEQ ID NO:20, LCDR3 consists solely of SEQ ID NO:21, HCDR1 consists solely of SEQ ID NO:22, HCDR2 consists solely of SEQ ID NO:23, and HCDR3 consists solely of SEQ ID NO:24.

[0179] Preferably, the BCMA targeting moiety combined with item i) (i.e. the CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect) is an ScFV capable of binding to human BCMA and comprises a VL and a VH domain, - the VL domain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25; - the VH domain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 26; LCDR1, 2 and 3 contained in the VL domain consist only of SEQ ID NOs: 36, 20 and 21, respectively, and HCDR1, 2 and 3 contained in the VH domain consist only of SEQ ID NOs: 22, 23 and 24, respectively.

[0180] In one embodiment, the BCMA targeting moiety combined with item i) (i.e. the CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect) is an scFv comprising a VL domain and a VH domain, wherein the VL domain has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151% the VH domain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 26, and the CDRs contained in the VH are kept constant and consist only of SEQ ID NOs: 22, 23 and 24.

[0181] In one embodiment, the BCMA targeting moiety combined with item i) (i.e. the CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect) is an scFv comprising a VL domain and a VH domain, wherein the VL domain is SEQ ID NO: 25, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 1 the VH domain comprises, consists of or consists essentially of SEQ ID NO: 26 or a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 26. Preferably, in the BCMA targeting moiety, the VL domain is positioned upstream of the VH.

[0182] Preferably, the VL and VH domains of the BCMA targeting moiety are linked by a peptide linker comprising, consisting of, or consisting essentially of SEQ ID NO: 14 or 16, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or 16.

[0183] In a preferred embodiment, the BCMA targeting moiety is an scFv and further comprises a signal peptide located in the N-terminal region of the BCMA targeting moiety. Preferably, the signal peptide comprises, consists of, or consists essentially of SEQ ID NO: 15, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 15.

[0184] Preferably, the BCMA targeting moiety in combination with item i) (i.e. the CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect) is an scFv comprising, consisting of or consisting essentially of SEQ ID NO: 27 or 28, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 27 (VH + linker + VL) or 28 (signal peptide + VH + linker + VL), respectively. Most preferably, the BCMA targeting moiety in combination with item i) (i.e. the CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect) is an scFV consisting solely of SEQ ID NO: 27 or 28.

[0185] More preferably, the BCMA targeting moiety combined with item i) (i.e. the CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, the nucleic acid according to the second aspect, the cell according to the third aspect, and / or the pharmaceutical composition according to the fourth aspect) is that named ARI2h in PCT / EP2020 / 071831, the content of which comprises SEQ ID NO: 13 (the complete amino acid sequence of the ARI2h CAR) of the ARI2h sequence comprising SEQ ID NOs: 1 (VH domain) and 2 (VL domain) as disclosed in PCT / EP2020 / 071831 and included herein.

[0186] In one embodiment, the combination therapy consists of, sequentially or simultaneously: i) a CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect, and ii) administering a therapeutic agent capable of binding to human BCMA, wherein the therapeutic agent is a CAR anti-BCMA, the CAR anti-BCMA comprising: a) an extracellular domain comprising a BCMA targeting moiety as defined in item ii) above; b) transmembrane domain; c) an intracellular signaling domain, and d) optionally comprising at least a costimulatory signaling domain.

[0187] Preferably, the combination therapy is administered sequentially or simultaneously: i) a CAR anti-CD229 as defined in the first aspect or any of its embodiments, and ii) administering a therapeutic agent capable of binding to human BCMA, wherein the therapeutic agent is a CAR anti-BCMA, the CAR anti-BCMA comprising: a) an extracellular domain comprising a BCMA targeting moiety as defined above; b) transmembrane domain; c) an intracellular signaling domain, and d) optionally comprising at least a costimulatory signaling domain.

[0188] In some embodiments the extracellular domain of the CAR anti-BCMA comprising the BCMA targeting moiety and in combination with item i) (i.e. the CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect) is the extracellular domain of the CAR referred to as ARI2h in PCT / EP2020 / 071831.

[0189] Preferably, the extracellular domain of the CAR anti-BCMA comprising a BCMA targeting moiety and in combination with item i) (i.e. the CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, the nucleic acid according to the second aspect, the cell according to the third aspect, and / or the pharmaceutical composition according to the fourth aspect) comprises an scFv comprising a VL domain and a VH domain, wherein said VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides, and said VH domain comprises HCDR1, HCDR2 and HCDR3 polypeptides, - LCDR1 comprises, consists of, or consists essentially of SEQ ID NO: 36, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 36; - LCDR2 comprises, consists of, or consists essentially of SEQ ID NO:20, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:20; - LCDR3 comprises, consists of, or consists essentially of SEQ ID NO:21, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:21; - HCDR1 comprises, consists of, or consists essentially of SEQ ID NO: 22, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 22; - HCDR2 comprises, consists of, or consists essentially of SEQ ID NO:23, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:23; and - HCDR3 comprises, consists of or consists essentially of SEQ ID NO:24 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:24.

[0190] In some embodiments the extracellular domain of a CAR anti-BCMA comprising a BCMA targeting moiety and in combination with item i) (i.e. the CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect) comprises an scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2 and HCDR3 polypeptides, wherein LCDR1 consists solely of SEQ ID NO: 36, LCDR2 consists solely of SEQ ID NO: 20, LCDR3 consists solely of SEQ ID NO: 21, HCDR1 consists solely of SEQ ID NO: 22, HCDR2 consists solely of SEQ ID NO: 23 and HCDR3 consists solely of SEQ ID NO: 24.

[0191] In one embodiment, the extracellular domain of the CAR anti-BCMA comprising a BCMA targeting moiety and in combination with item i) (i.e. the CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect) comprises an scFv comprising a VL domain and a VH domain, wherein the VL domain is SEQ ID NO: 25, or has a similar sequence identity to SEQ ID NO: 25 at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:26; the VH domain comprises, consists of or consists essentially of SEQ ID NO:26 or a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:26.

[0192] In one embodiment, the extracellular domain of the CAR anti-BCMA comprising a BCMA targeting moiety and in combination with item i) (i.e. the CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect) comprises an ScFV (i.e. anti-BCMA) capable of binding to human BCMA, comprising a VL domain and a VH domain, - the VL domain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25; - the VH domain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 26; LCDR1, 2 and 3 contained in the VL domain consist only of SEQ ID NOs: 36, 20 and 21, respectively, and HCDR1, 2 and 3 contained in the VH domain consist only of SEQ ID NOs: 22, 23 and 24, respectively.

[0193] Most preferably, the extracellular domain of a CAR anti-BCMA comprising a BCMA targeting moiety and in combination with item i) (i.e. a CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect) comprises an scFv comprising, consisting of or consisting essentially of SEQ ID NO: 27 or 28, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 27 (VH+linker+VL) or 28 (signal peptide+VH+linker+VL), respectively. Most preferably, the extracellular domain of a CAR anti-BCMA comprising a BCMA targeting moiety and in combination with item i) (i.e. a CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect) comprises an scFV consisting only of SEQ ID NO: 27 or 28 (signal peptide+VH+linker+VL).

[0194] In a preferred embodiment, the extracellular domain of a CAR anti-BCMA comprising a BCMA targeting moiety and in combination with item i) (i.e. a CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect) comprises a signal peptide located in the N-terminal region of the extracellular domain. Preferably, the signal peptide comprises, consists of, or consists essentially of SEQ ID NO: 15, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 15.

[0195] It should be noted that the transmembrane domain b), intracellular domain c) and optional costimulatory signaling domain d) of a CAR against BCMA comprising a BCMA targeting moiety and combined with item i) (i.e. a CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect) may be the same domains as defined above for a CAR anti-CD229. Thus, all embodiments relating to the transmembrane domain, intracellular domain and costimulatory domain defined under the first aspect of the invention also apply to a CAR anti-BCMA that may be used in combination with a CAR anti-CD229.

[0196] Preferably, the combination therapy is administered sequentially or simultaneously: i) a CAR against CD229 as defined in the first aspect or any of its embodiments, and ii) administering a therapeutic agent capable of binding to human BCMA, wherein the therapeutic agent is a CAR anti-BCMA, and the CAR anti-BCMA comprises, consists of, or consists essentially of SEQ ID NO: 29, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 29 (CAR ARI2h).

[0197] Preferably, the combination therapy is administered sequentially or simultaneously: i) a CAR against CD229 (CAR GL001) comprising, consisting of, or consisting essentially of SEQ ID NO: 13, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 13, and ii) administering a therapeutic agent capable of binding to human BCMA, wherein the therapeutic agent is a CAR anti-BCMA, and the CAR anti-BCMA comprises, consists of, or consists essentially of SEQ ID NO: 29, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 29 (CAR ARI2h).

[0198] Preferably, the combination therapy is administered sequentially or simultaneously: i) a CAR against CD229 (CAR GL002) comprising, consisting of, or consisting essentially of SEQ ID NO: 18, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 18, and ii) administering a therapeutic agent capable of binding to human BCMA, wherein the therapeutic agent is a CAR anti-BCMA, and the CAR anti-BCMA comprises, consists of, or consists essentially of SEQ ID NO: 29, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 29 (CAR ARI2h).

[0199] Preferably, the combination therapy is administered sequentially or simultaneously: i) a CAR against CD229 comprising, consisting of, or consisting essentially of SEQ ID NO: 18 (CAR GL002) or 13 (CAR GL001), preferably SEQ ID NO: 18, and ii) administering a therapeutic agent capable of binding to human BCMA, wherein the therapeutic agent is a CAR anti-BCMA, and the CAR anti-BCMA comprises, consists of, or consists essentially of SEQ ID NO: 29 (CAR ARI2h).

[0200] Preferably, the combination therapy is administered sequentially or simultaneously: i) a CAR against CD229 comprising SEQ ID NO: 18 (CAR GL002) or 13 (CAR GL001), preferably SEQ ID NO: 18, and ii) administering a therapeutic agent capable of binding to human BCMA, wherein the therapeutic agent is a CAR anti-BCMA, and the CAR anti-BCMA comprises SEQ ID NO: 29 (CAR ARI2h).

[0201] Preferably, the combination therapy is administered sequentially or simultaneously: i) a CAR against CD229 consisting of SEQ ID NO: 18 (CAR GL002) or 13 (CAR GL001), preferably SEQ ID NO: 18 alone, and ii) administering a therapeutic agent capable of binding to human BCMA, said therapeutic agent being a CAR anti-BCMA, said CAR anti-BCMA consisting solely of SEQ ID NO: 29 (CAR ARI2h).

[0202] In another embodiment, the combination therapy consists of, sequentially or simultaneously: i) a CD229 targeting moiety of the first aspect comprising a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect, and ii) administering a therapeutic agent capable of binding to human BCMA, wherein the therapeutic agent is a nucleic acid encoding a BCMA targeting moiety and / or encoding a CAR against BCMA as defined in any of the above embodiments.

[0203] Thus, the combination therapy may include administering a nucleic acid encoding a BCMA targeting moiety and / or a CAR to BCMA, as defined in the paragraph above. Preferably, the combination therapy is administered subsequently or simultaneously with: i) a nucleic acid according to the second aspect encoding any one of the CD229-targeting moieties of the invention, preferably encoding a CAR anti-CD229, and ii) administering a therapeutic agent capable of binding to human BCMA, wherein the therapeutic agent is a nucleic acid encoding a BCMA targeting moiety, preferably encoding a CAR anti-BCMA as defined above.

[0204] In one embodiment, the nucleic acid administered in the combination therapy is a bicistronic nucleic acid, and thus includes both items i) and ii). Thus, a "bicistronic nucleic acid," as used herein, is defined as: i) a nucleic acid according to the second aspect encoding any one of the CD229-targeting moieties of the invention, preferably encoding a CAR anti-CD229, and ii) refers to a polynucleotide sequence comprising a therapeutic agent capable of binding to human BCMA, which polynucleotide sequence encodes a BCMA targeting moiety, preferably a nucleic acid encoding a CAR anti-BCMA as defined above; Both nucleic acids are preferably separated by a sequence encoding a "2A peptide cleavage site" or by an "internal ribosome entry site region", and both nucleic acids are preferably expressed under the control of the same promoter.

[0205] As used herein, the term "2A peptide cleavage site" refers to an autocatalytic peptide, preferably selected from the group consisting of a T2A cleavage site (EGRGSLLTCGDVEENPGP), a P2A cleavage peptide (ATNFSLLKQAGDVEENPGP), an E2A cleavage peptide (QCTNYALLKLAGDVESNPGP), or an F2A cleavage peptide (VKQTLNFDLLKLAGDVESNPGP). As known to those skilled in the art, the 2A peptide cleavage site functions by causing the ribosome to skip synthesis of the glycine and proline peptide bond at the C-terminus of the 2A peptide cleavage site, resulting in separation of the end of the 2A sequence from the downstream peptide.

[0206] "Internal ribosome entry site region" or "IRES," as used herein, refers to an RNA element that allows for translation initiation in a cap-independent manner. Preferably, the IRES region is derived from a picornavirus IRES region sequence; the IRES sequence is selected from the group consisting of enterovirus, rhinovirus, cardiovirus, and aphthovirus IRES sequences; and the IRES region is selected from the group consisting of hepatitis A virus IRES sequence, hepatitis B virus sequence, and hepatitis C virus IRES sequence.

[0207] Thus, in a preferred embodiment, the combination therapy comprises administering a bicistronic nucleic acid as defined in the present invention. Preferably, the combination therapy comprises administering a bicistronic nucleic acid comprising, in any order: i) a nucleic acid sequence encoding the amino acid sequence of the CD229-targeting moiety of the first aspect or any of its embodiments comprising a VL domain and a VH domain, in any order, wherein said VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides; and said VH domain comprises HCDR1, HCDR2 and HCDR3 polypeptides, wherein LCDR1 comprises, consists essentially of, or consists of [SSVSY] (SEQ ID NO: 4); and LCDR2 comprises, consists essentially of, or consists of [DTS] (SEQ ID NO: 5); wherein LCDR3 comprises, consists of, or consists essentially of [CQQWSSYPPTF] (SEQ ID NO: 6), HCDR1 comprises, consists of, or consists essentially of [GYTFTHYY] (SEQ ID NO: 7), HCDR2 comprises, consists of, or consists essentially of [IFPESGST] (SEQ ID NO: 8), and HCDR3 comprises, consists of, or consists essentially of [CARGTGFFDYW] (SEQ ID NO: 9), and ii) a nucleic acid sequence encoding the amino acid sequence of a BCMA targeting moiety comprising a VL domain and a VH domain, in any order, wherein the VL domain comprises LCDR1, LCDR2, and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2, and HCDR3 polypeptides; - LCDR1 comprises, consists or consists essentially of SEQ ID NO: 36; - LCDR2 comprises, consists or consists essentially of SEQ ID NO: 20; - LCDR3 comprises, consists or consists essentially of SEQ ID NO: 21; - HCDR1 comprises, consists or consists essentially of SEQ ID NO: 22; - HCDR2 comprises, consists or consists essentially of SEQ ID NO: 23; - HCDR3 comprises, consists of or consists essentially of SEQ ID NO: 41.

[0208] Preferably, the combination therapy comprises administering a bicistronic nucleic acid comprising, in any order: i) a nucleic acid sequence encoding the amino acid sequence of the CD229-targeting moiety of the first aspect or any of its embodiments thereof comprising a VL domain and a VH domain, in any order, wherein said VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides, and said VH domain comprises HCDR1, HCDR2 and HCDR3 polypeptides, wherein LCDR1 consists exclusively of [SSVSY] (SEQ ID NO: 4), LCDR2 consists exclusively of [DTS] (SEQ ID NO: 5), LCDR3 consists exclusively of [CQQWSSYPPTF] (SEQ ID NO: 6), HCDR1 consists exclusively of [GYTFTHYY] (SEQ ID NO: 7), HCDR2 consists exclusively of [IFPESGST] (SEQ ID NO: 8), and HCDR3 consists exclusively of [CARGTGFFDYW] (SEQ ID NO: 9); and ii) A nucleic acid sequence encoding the amino acid sequence of a BCMA targeting moiety comprising a VL domain and a VH domain in any order, wherein the VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2 and HCDR3 polypeptides, wherein LCDR1 consists solely of SEQ ID NO: 36; LCDR2 consists solely of SEQ ID NO: 20; LCDR3 consists solely of SEQ ID NO: 21; HCDR1 consists solely of SEQ ID NO: 22; HCDR2 consists solely of SEQ ID NO: 23; and HCDR3 consists solely of SEQ ID NO: 24.

[0209] Preferably, the combination therapy comprises administering a bicistronic nucleic acid comprising, in any order: i) a nucleic acid sequence encoding the CD229-targeting moiety of the first aspect or any of its embodiments comprising a VL domain and a VH domain in any order, - the VL domain comprises a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, and the VL domain comprises CDR regions consisting only of LCDR1 [SSVSY] (SEQ ID NO: 4), LCDR2 [DTS] (SEQ ID NO: 5) and LCDR3 [CQQWSSYPPTF] (SEQ ID NO: 6); - the VH domain comprises a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, wherein the VH domain comprises CDR regions consisting only of HCDR1 [GYTFTHYY] (SEQ ID NO: 7), HCDR2 [IFPESGST] (SEQ ID NO: 8) and HCDR3 [CARGTGFFDYW] (SEQ ID NO: 9); a nucleic acid sequence, wherein the CD229 targeting moiety is capable of binding to human CD229, preferably as measured by surface plasmon resonance (SPR); and ii) a nucleic acid sequence encoding a BCMA targeting moiety comprising a VL domain and a VH domain in any order, - the VL domain comprises a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25, and the VL domain comprises CDR regions consisting only of LCDR1 of SEQ ID NO: 36, LCDR2 of SEQ ID NO: 20 and LCDR3 of SEQ ID NO: 21; - the VH domain comprises a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 26, wherein the VH domain comprises a CDR region consisting only of HCDR1 of SEQ ID NO: 22, HCDR2 of SEQ ID NO: 23 and HCDR3 of SEQ ID NO: 24; A nucleic acid sequence wherein the BCMA targeting moiety is capable of binding to human BCMA, preferably as measured by surface plasmon resonance (SPR).

[0210] Preferably, the combination therapy comprises administering a bicistronic nucleic acid comprising, in any order: i) a nucleic acid sequence encoding the CD229-targeting moiety of the first aspect or any of its embodiments comprising a VL domain and a VH domain, in any order, wherein the VL domain comprises, consists of, or consists essentially of SEQ ID NO: 1 and the VH domain comprises, consists of, or consists essentially of SEQ ID NO: 2; ii) a nucleic acid sequence encoding a BCMA targeting moiety comprising a VL domain and a VH domain, in any order, wherein the VL domain comprises, consists of, or consists essentially of SEQ ID NO: 25 and the VH domain comprises, consists of, or consists essentially of SEQ ID NO: 26; A nucleic acid sequence, wherein the CD229 targeting moiety is capable of binding to human CD229 and the BCMA targeting moiety is capable of binding to human BCMA, preferably as measured by surface plasmon resonance (SPR). Preferably, the CD229 and BCMA targeting moieties are ScFvs.

[0211] Preferably, the combination therapy comprises administering a bicistronic nucleic acid comprising, in any order: i) a nucleic acid sequence encoding a CD229-targeting moiety, which is an ScFV and comprises, consists of, or consists essentially of SEQ ID NO: 3 (GL001); and ii) a nucleic acid sequence encoding a BCMA targeting moiety that is an ScFV and comprises, consists of, or consists essentially of SEQ ID NO: 27 or 28 (ARI2h); and A nucleic acid sequence wherein the CD229 targeting moiety is capable of binding to human CD229 and the BCMA targeting moiety is capable of binding to human BCMA, preferably as measured by surface plasmon resonance (SPR).

[0212] Preferably, the combination therapy comprises administering a bicistronic nucleic acid comprising, in any order: i) a nucleic acid sequence encoding a CD229-targeting moiety, which is an ScFV and comprises, consists of, or consists essentially of SEQ ID NO: 17 (GL002); ii) a nucleic acid sequence encoding a BCMA targeting moiety that is an ScFV and comprises, consists of, or consists essentially of SEQ ID NO: 27 or 28 (ARI2h); and A nucleic acid sequence wherein the CD229 targeting moiety is capable of binding to human CD229 and the BCMA targeting moiety is capable of binding to human BCMA, preferably as measured by surface plasmon resonance (SPR).

[0213] Preferably, the combination therapy comprises administering a bicistronic nucleic acid, the bicistronic nucleic acid being a construct designated GL007, which comprises, from 5' to 3': i) a nucleic acid sequence encoding a CD229-targeting moiety, which is an ScFV and comprises, consists of, or consists essentially of SEQ ID NO: 17 (GL002); ii) a nucleic acid sequence encoding a BCMA targeting moiety that is an ScFV and comprises, consists of, or consists essentially of SEQ ID NO: 27 or 28 (ARI2h); and A nucleic acid sequence wherein the CD229 targeting moiety is capable of binding to human CD229 and the BCMA targeting moiety is capable of binding to human BCMA, preferably as measured by surface plasmon resonance (SPR).

[0214] Preferably, the combination therapy comprises administering a bicistronic nucleic acid, the bicistronic nucleic acid comprising, in a 5' to 3' direction: i) a nucleic acid sequence encoding a BCMA targeting moiety that is an ScFV and comprises, consists of, or consists essentially of SEQ ID NO: 27 or 28 (ARI2h); and ii) A nucleic acid sequence encoding a CD229-targeting moiety which is an ScFV and comprises, consists of, or consists essentially of SEQ ID NO: 17 (GL002) or 3 (GL001), preferably SEQ ID NO: 17 (GL002).

[0215] Preferably, the nucleic acid sequences encoding the CD229-targeting moiety and the BCMA-targeting moiety are separated in the bicistronic nucleic acid by a sequence encoding an autocatalytic 2A peptide cleavage site. Preferably, the autocatalytic peptide is selected from the group consisting of porcine teschovirus-12A (P2A), foot-and-mouth disease virus 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), and tosea asiatic virus 2A (T2A) peptides, and the encoded autocatalytic peptide cleavage site can optionally include a GSG (glycine-serine-glycine) motif at its N-terminus and / or a GTS motif at its C-terminus. Preferably, P2A comprises, consists or consists essentially of SEQ ID NO: 33, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 33. Preferably, T2A comprises, consists or consists essentially of SEQ ID NO: 32, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 32. Preferably, E2A comprises, consists or consists essentially of SEQ ID NO: 34, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 34. Preferably, F2A comprises, consists or consists essentially of SEQ ID NO: 35, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 35.

[0216] Preferably, the combination therapy comprises administering a bicistronic nucleic acid, the bicistronic nucleic acid comprising, in a 5' to 3' direction: - a nucleic acid sequence encoding a BCMA targeting moiety, which is an ScFV and comprises, consists of, or consists essentially of SEQ ID NO: 28 (ARI2h); a nucleic acid sequence encoding a T2A peptide, preferably comprising or consisting of SEQ ID NO: 32, and - a nucleic acid sequence encoding a CD229-targeting moiety which is an ScFV and which comprises, consists of, or consists essentially of SEQ ID NO: 17 (GL002) or 3 (GL001), preferably SEQ ID NO: 17 (GL002).

[0217] Preferably, the combination therapy comprises administering a bicistronic nucleic acid, the bicistronic nucleic acid comprising, in a 5' to 3' direction: - a nucleic acid sequence encoding a BCMA targeting moiety which is an ScFV and comprises, consists of, or consists essentially of SEQ ID NO: 17 (GL002) or 3 (GL001), preferably SEQ ID NO: 17 (GL002), a nucleic acid sequence encoding a T2A peptide, preferably comprising or consisting of SEQ ID NO: 32, and - a nucleic acid sequence encoding a BCMA targeting moiety which is an ScFV and comprises, consists of, or consists essentially of SEQ ID NO: 28 (ARI2h).

[0218] Preferably, the combination therapy comprises administering a bicistronic nucleic acid, the bicistronic nucleic acid comprising, in a 5' to 3' direction: - a nucleic acid sequence encoding a CD229-targeting moiety, which is an ScFV and comprises, consists of, or consists essentially of SEQ ID NO: 17 (GL002), a nucleic acid sequence encoding a T2A peptide, preferably comprising or consisting of SEQ ID NO: 32, and - a nucleic acid sequence encoding a BCMA targeting moiety which is an ScFV and comprises, consists of, or consists essentially of SEQ ID NO: 27 or 28 (ARI2h), preferably SEQ ID NO: 28.

[0219] Preferably, the combination therapy comprises administering a bicistronic nucleic acid, the bicistronic nucleic acid comprising, in a 5' to 3' direction: - a nucleic acid sequence encoding a CD229-targeting moiety, which is an ScFV and comprises SEQ ID NO: 17 (GL002), a nucleic acid sequence encoding a T2A peptide, preferably comprising SEQ ID NO: 32, and - a nucleic acid sequence encoding a BCMA targeting moiety which is an ScFV and comprises SEQ ID NO: 27 or 28 (ARI2h), preferably SEQ ID NO: 28.

[0220] Preferably, the combination therapy comprises administering a bicistronic nucleic acid, the bicistronic nucleic acid comprising, in a 5' to 3' direction: - a nucleic acid sequence encoding a CD229-targeting moiety which is an ScFV and consists solely of SEQ ID NO: 17 (GL002), a nucleic acid sequence encoding a T2A peptide preferably consisting solely of SEQ ID NO: 32, and - a nucleic acid sequence which is an ScFV and encodes a BCMA targeting moiety consisting solely of SEQ ID NO: 27 or 28 (ARI2h), preferably SEQ ID NO: 28.

[0221] Preferably, the combination therapy comprises administering a bicistronic nucleic acid, the bicistronic nucleic acid comprising, in a 5' to 3' direction: - a nucleic acid sequence encoding a CAR anti-CD229 comprising, consisting of or consisting essentially of SEQ ID NO: 18 (GL002) or SEQ ID NO: 13 (GL001), preferably SEQ ID NO: 18 (GL002), a nucleic acid sequence encoding a T2A peptide, preferably comprising or consisting of SEQ ID NO: 32, and - a nucleic acid sequence encoding a CAR anti-CD229 comprising, consisting of or consisting essentially of SEQ ID NO: 29 (CAR ARI2h).

[0222] Preferably, the combination therapy comprises administering a bicistronic nucleic acid, the bicistronic nucleic acid comprising, in a 5' to 3' direction: - a nucleic acid sequence encoding a CAR anti-CD229 comprising SEQ ID NO: 18 (GL002) or SEQ ID NO: 13 (GL001), preferably SEQ ID NO: 18 (GL002), a nucleic acid sequence encoding a T2A peptide, preferably comprising SEQ ID NO: 32, and - a nucleic acid sequence encoding a CAR anti-CD229 comprising SEQ ID NO: 29 (CAR ARI2h).

[0223] Preferably, the combination therapy comprises administering a bicistronic nucleic acid, the bicistronic nucleic acid comprising, in a 5' to 3' direction: - a nucleic acid sequence encoding a CAR anti-CD229 consisting solely of SEQ ID NO: 18 (GL002) or SEQ ID NO: 13 (GL001), preferably SEQ ID NO: 18 (GL002), a nucleic acid sequence encoding a T2A peptide preferably consisting solely of SEQ ID NO: 32, and - a nucleic acid sequence encoding a CAR anti-CD229 consisting solely of SEQ ID NO: 29 (CAR ARI2h).

[0224] Preferably, the combination therapy comprises administering a bicistronic nucleic acid, the bicistronic nucleic acid comprising, in a 5' to 3' direction: - a nucleic acid sequence encoding a CAR anti-CD229 comprising, consisting of or consisting essentially of SEQ ID NO: 29 (CAR ARI2h), a nucleic acid sequence encoding a T2A peptide, preferably comprising or consisting of SEQ ID NO: 32, and - a nucleic acid sequence encoding a CAR anti-CD229 comprising, consisting of or consisting essentially of SEQ ID NO: 18 (GL002) or SEQ ID NO: 13 (GL001), preferably SEQ ID NO: 18 (GL002).

[0225] Preferably, the combination therapy comprises administering a bicistronic nucleic acid that comprises, consists of, or consists essentially of SEQ ID NO: 30, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 30 (GL007 bicistronic CAR nucleotide sequence). Preferably, the combination therapy comprises administering a bicistronic nucleic acid that comprises, consists of, or consists essentially of SEQ ID NO: 31, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 31 (GL008 bicistronic CAR nucleotide sequence). Preferably, the combination therapy comprises administering a bicistronic nucleic acid that encodes a protein comprising, consisting of, or consisting essentially of SEQ ID NO: 38, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 38 (GL007 CAR aa sequence). Preferably, the combination therapy comprises administering a bicistronic nucleic acid that encodes a protein comprising, consisting of, or consisting essentially of SEQ ID NO: 39, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 39 (GL008 CAR aa sequence).

[0226] In one embodiment, the combination therapy comprises the administration of at least two different CAR-expressing cells. In one embodiment, CAR-expressing cells, such as anti-BCMA CAR-expressing cells, can be used in combination with the CAR-expressing cells described herein. In one embodiment, a subject in need of treatment is treated with a CAR anti-BCMA, followed by or simultaneously with a CAR of the present invention that targets human CD229. This combination therapy is particularly advantageous in tumors that contain BCMA-negative cells and are therefore prone to recurrence after anti-BCMA therapy.

[0227] Thus, the combination therapy of the present invention may also be administered sequentially or simultaneously: i) a cell or a plurality of cells according to the third aspect or any of its embodiments, and ii) administering a therapeutic agent capable of binding to human BCMA, said therapeutic agent being a cell or cells comprising or expressing a BCMA targeting moiety, preferably a CAR anti-BCMA as defined above, and / or a nucleic acid encoding same.

[0228] Additionally, the combination therapy of the present invention may also include administering a plurality of cells, each of which comprises: i) a CD229 targeting moiety and / or a CAR anti-CD229 as defined in the first aspect or any of its embodiments, and ii) comprises or expresses a therapeutic agent capable of binding to human BCMA, which therapeutic agent is a BCMA targeting moiety and / or a CAR anti-BCMA as defined above.

[0229] In one embodiment, the combination therapy comprises administering a plurality of cells, each of the cells comprising: i) a CAR anti-CD229 as defined in the first aspect or any of its embodiments, preferably a CAR comprising or consisting of SEQ ID NO: 13 or SEQ ID NO: 18, and ii) comprises or expresses a therapeutic agent capable of binding to human BCMA, which is a BCMA targeting moiety and / or a CAR anti-BCMA as defined above, preferably a CAR comprising or consisting solely of SEQ ID NO: 29.

[0230] In one embodiment, the combination therapy of the invention may also include administering a plurality of cells, each of which comprises: i) a nucleic acid encoding a CD229 targeting moiety and / or a CAR anti-CD229 as defined in the first aspect or any of its embodiments, preferably a nucleic acid encoding a CAR comprising or consisting solely of SEQ ID NO: 13 or 18, and ii) including therapeutic agents capable of binding to human BCMA, which include therapeutic agents that are nucleic acids encoding a BCMA targeting moiety and / or a CAR anti-BCMA as defined above, preferably a CAR comprising or consisting solely of SEQ ID NO: 29.

[0231] In one embodiment, the combination therapy of the present invention may also comprise administering a plurality of cells, each of which comprises or expresses a bicistronic nucleic acid as defined above, preferably a bicistronic nucleic acid comprising or consisting solely of SEQ ID NO: 30 or 31, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 30 or 31, respectively; or a bicistronic nucleic acid encoding a protein comprising or consisting solely of SEQ ID NO: 38 or 39, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 38 or 39, respectively.

[0232] The components of the combination therapy, i.e., items i) and ii) as defined above, can be administered in the form of one or more pharmaceutical compositions. Thus, in one embodiment, the combination therapy is administered sequentially or simultaneously: i) a pharmaceutical composition according to the fourth aspect of the invention or any of its embodiments, ii) administering a therapeutic agent capable of binding to human BCMA, wherein the therapeutic agent is a pharmaceutical composition comprising a therapeutically effective amount of cells comprising a nucleic acid encoding a BCMA targeting moiety, preferably a CAR anti-BCMA as defined above; The pharmaceutical compositions of items i) and ii) may be the same.

[0233] Preferably, the pharmaceutical composition comprises a plurality of cells, or a therapeutically effective amount of cells, comprising or expressing a bicistronic nucleic acid as defined above, more preferably a bicistronic nucleic acid comprising or consisting solely of SEQ ID NO: 30 or 31, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 30 or 31, respectively; or a bicistronic nucleic acid encoding a protein comprising or consisting solely of SEQ ID NO: 38 or 39, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 38 or 39, respectively.

[0234] In some other embodiments, the combination therapy is administered sequentially or simultaneously: i) a pharmaceutical composition comprising a therapeutically effective amount of T cells expressing or encoding a CAR anti-CD229 as defined above, and ii) administering a therapeutic agent capable of binding to human BCMA, wherein the therapeutic agent is a pharmaceutical composition comprising a therapeutically effective amount of T cells expressing or encoding a CAR anti-CD229 as defined above; The pharmaceutical compositions of items i) and ii) may be the same (i.e., a pharmaceutical composition comprising T cells that express or contain both of the bicistronic nucleic acids defined above).

[0235] The combination therapy according to the sixth aspect or any of its embodiments can be used in medicine or for the manufacture of a medicament. Preferably, the combination therapy is for use in a method of treating CD229-positive cancer (including CD229-positive / BCMA-positive cancer), the method comprising administering the combination therapy to a patient in need thereof. Preferably, the CD229-positive cancer, including (CD229-positive / BCMA-positive cancer), is multiple myeloma. Since the combination therapy comprises, in item i), the administration of a CD229-targeting moiety of the first aspect, including a CAR anti-CD229, a nucleic acid according to the second aspect, a cell according to the third aspect, and / or a pharmaceutical composition according to the fourth aspect, the use defined in the fifth aspect or any of its embodiments is also a use of combination therapy.

[0236] kit In a seventh aspect, the present invention provides kits according to any of the preceding aspects and their use.The above-mentioned materials and other materials can be packaged together in any suitable combination as a kit useful for carrying out or assisting in carrying out the disclosed method.It is useful when the kit components in a given kit are designed and adapted for use together in the disclosed method.Kits that include one or more of the vectors disclosed herein are also disclosed.

[0237] The following provisions are also included in the present invention:

[0238] 1. A CD229 targeting moiety, wherein the CD229 targeting moiety is an antibody, F(ab')2, Fab, scFab, or scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2, and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2, and HCDR3 polypeptides; -LCDR1 consists only of SEQ ID NO: 4 [SSVSY], LCDR2 consists solely of SEQ ID NO: 5 [DTS], LCDR3 consists solely of SEQ ID NO: 6 [CQQWSSYPPTF], HCDR1 consists solely of SEQ ID NO: 7 [GYTFTHYY], HCDR2 consists solely of SEQ ID NO: 8 [IFPESGST], - A CD229 targeting moiety wherein HCDR3 consists solely of SEQ ID NO: 9 [CARGTGFFDYW].

[0239] 2. The CD229 targeting moiety according to clause 1, - the VL domain comprises a sequence having at least 85% sequence identity with SEQ ID NO: 1, and the VL domain comprises a CDR region consisting only of LCDR1 as set forth in SEQ ID NO: 4, LCDR2 as set forth in SEQ ID NO: 5, and LCDR3 as set forth in SEQ ID NO: 6; - a CD229 targeting moiety, wherein the VH domain comprises a sequence having at least 85% sequence identity to SEQ ID NO:2, and said VH domain comprises CDR regions consisting only of HCDR1 set forth in SEQ ID NO:7, HCDR2 set forth in SEQ ID NO:8, and HCDR3 set forth in SEQ ID NO:9.

[0240] 3. The CD229 targeting moiety of clause 1 or 2, wherein the CD229 targeting moiety is an antibody, F(ab')2, Fab, scFab or scFv comprising a VL domain and a VH domain, wherein the VL domain consists solely of SEQ ID NO: 1 and the VH domain consists solely of SEQ ID NO: 2.

[0241] 4. The CD229 targeting moiety of any one of clauses 1-3, wherein the CD229 targeting moiety is an scFv.

[0242] 5. The CD229 targeting moiety of any one of clauses 1-4, wherein the CD229 targeting moiety is an scFv consisting solely of SEQ ID NO:17.

[0243] 6. A chimeric antigen receptor (CAR), an extracellular domain comprising a CD229 targeting moiety, wherein the CD229 targeting moiety is as defined in any one of clauses 1-5; b. transmembrane domain; and c. A CAR, comprising an intracellular signaling domain.

[0244] 7. The CAR of clause 6, wherein the transmembrane domain comprises the transmembrane domain of CD8.

[0245] 8. The CAR of any one of clauses 6 or 7, wherein the intracellular signaling domain comprises the intracellular domain of CD3ζ.

[0246] 9. The CAR of any one of clauses 6-8, wherein the costimulatory signaling domain comprises the intracellular domain of CD137.

[0247] 10. The CAR of any one of clauses 6 to 9, consisting solely of SEQ ID NO: 18.

[0248] 11. A nucleic acid encoding a CAR according to any one of clauses 6 to 10.

[0249] 12. A cell comprising the nucleic acid of clause 11.

[0250] 13. The CD229 targeting moiety of any one of clauses 1-5, the CAR of any one of clauses 6-10, the nucleic acid of clause 11, or the cell of clause 12 for use in a method of treating a CD229-positive cancer, wherein the method comprises administering the CD229 targeting moiety, CAR, nucleic acid, or cell to a patient in need thereof, preferably wherein the cancer is multiple myeloma.

[0251] 14. The CD229 targeting moiety, CAR, nucleic acid, or cell for use according to clause 13, wherein the use is in combination with anti-BCMA therapy.

[0252] 15. The CD229 targeting moiety, CAR, nucleic acid, or cell for use according to clause 14, wherein the anti-BCMA therapy comprises administering a CAR against BCMA.

[0253] Sequence Listing General sequence of GL001 and GL002 antibodies: VL CDR: LCDR1: SEQ ID NO: 4: SSVSY LCDR2: SEQ ID NO: 5: DTS LCDR3: SEQ ID NO: 6: CQQWSSYPPTF VH CDR: HCDR1: SEQ ID NO: 7GYTFTHYY HCDR2: SEQ ID NO: 8IFPESGST HCDR3: SEQ ID NO: 9CARGTGFFDYW SEQ ID NO: 1: VL QIVLTQSPAIMSASPGEKVTVTCSASSSVSYMFWYQQKPGFSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPPTFGGGTKLEIK SEQ ID NO: 2: VH QMQLQESGPELVKPGASVKISCKASGYTFTHYYINWVQQRPGQGLEWIGWIFPESGSTYYSEKFKAKATLTVDKSSSTAYMLLSSLTSEDSAVYFCARGTGFFDYWGQGTTLTVSS SEQ ID NO: 32: T2A peptide EGRGSLLTCGDVEENPGP SEQ ID NO: 33: P2A peptide ATNFSLLKQAGDVEENPGP SEQ ID NO: 34: E2A peptide QCTNYALLKLAGDVESNPGP SEQ ID NO: 35: F2A peptide VKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 15: signal peptide MEAPAQLLFLLLLWLPDTTG SEQ ID NO: 37: CD8 hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI SEQ ID NO: 10: CD8 TM YIWAPLAGTCGVLLLSLVITLYC SEQ ID NO: 19: CD8 hinge + TM TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC SEQ ID NO: 12:4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 11: CD3z RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0254] Scfv GL001_mVH_VL_CD229.84_BB CAR sequence SEQ ID NO: 14: Linker (G4S) x3 GGGGSGGGGSGGGGS SEQ ID NO: 3: signal peptide + VH + linker + VL MEAPAQLLFLLLLWLPDTTGQMQLQESGPELVKPGASVKISCKASGYTFTHYYINWVQQRPGQGLEWIGWIFPESGSTYYSEKFKAKATLTVDKSSSTAYMLLSSLTSEDSAVYFCARGTGFFDYWGQ GTTLTVSSGGGGSGGGGSGGGSQIVLTQSPAIMSASPGEKVTVTCSASSSVSYMFWYQQKKPGFSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPPTFGGGTKLEIK SEQ ID NO: 13: Signal peptide + VH + linker + VL + CD8 hinge + CD8 TM + 4-1BB + CD3z MEAPAQLLFLLLLWLPDTTGQMQLQESGPELVKPGASVKISCKASGYTFTHYYINWVQQRPGQGLEWIGWIFPESGSTYYSEKFKAKATLTVDKSSSTAYMLLSSLTSEDSAVYFCARGT GFFDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTVTCSASSSVSYMFWYQQKPGFSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQ WSSYPPTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0255] Sequence of pGL002_mVL_VH_CD299.84_BB CAR SEQ ID NO: 16: Linker (G4S) x4 GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 17: signal peptide + VL + linker + VH MEAPAQLLFLLLLWLPDTTGQIVLTQSPAIMSASPGEKVTVTCSASSSVSYMFWYQQKPGFSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPPTFGGGTKLEIKGGGGS GGGGSGGGGSGGGGSQMQLQESGPELVKPGASVKISCKASGYTFTHYYINWVQQRPGQGLEWIGWIFPESGSTYYSEKFKAKATLTVDKSSSTAYMLLSSLTSEDSAVYFCARGTGFFDYWGQGTTLTVSS SEQ ID NO: 18: Signal peptide + VL + linker + VH + CD8 hinge + CD8 TM + 4-1BB + CD3z MEAPAQLLFLLLLWLPDTTGQIVLTQSPAIMSASPGEKVTVTCSASSSVSYMFWYQQKPGFSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPPTFGGGT KLEIKGGGGSGGGGSGGGGSGGGGSQMQLQESGPELVKPGASVKISCKASGYTFTHYYINWVQQRPGQGLEWIGWIFPESGSTYYSEKFKAKATLTVDKSSSTAYMLLSSLTSEDSAVYFCA RGTGFFDYWGQGTTLTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0256] Sequence of the BCMA targeting moiety ARI2h VL CDR ARI2h: LCDR1: SEQ ID NO: 36: KASQSVDSNVA LCDR2: SEQ ID NO: 20: SASLRFS LCDR3: SEQ ID NO: 21: QYNNYPLT VH CDR ARI2h: HCDR1: SEQ ID NO: 22: GIDFSRYWMS HCDR2: SEQ ID NO: 23: EINPDSSTINYAPSLKD HCDR3: SEQ ID NO: 24: LYYDYGDAMDY SEQ ID NO: 25: VL DIQMTQSPSSLSASVGDRVTITCKASQSVDSNVAWYQQKPGKAPKALIFSASLRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPLTFGGGTKVEIK SEQ ID NO: 26: VH EVQLVESGGGLVQPGGSLRLSCAASGIDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASLYYDYGDAMDYWGQGTLVTVSS SEQ ID NO: 14 linker GGGGSGGGGSGGGGS SEQ ID NO: 15 signal peptide MEAPAQLLFLLLLWLPDTTG SEQ ID NO: 27: VH + linker + VL ARI2h EVQLVESGGGLVQPGGSLRLSCAASGIDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASLYYDYGDAMDYWGQGTLVTVSSG GGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQSVDSNVAWYQQKPGKAPKALIFSASLRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPLTFGGGTKVEIK SEQ ID NO: 28: Signal peptide + VH + linker + VL ARI2h MEAPAQLLFLLLLWLPDTTGEVQLVESGGGLVQPGGSLRLSCAASGIDFSRYWMSWVRQAPGKGLEWGEINPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASLYYDYGDAMDYWG QGTLVTVSSGGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQSVDSNVAWYQQKPGKAPKALIFSASLRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPLTFGGGTKVEIK CAR scaffolding: -CD8 hinge is SEQ ID NO: 37 -CD8 TM is SEQ ID NO: 10 -CD8 hinge + TM is SEQ ID NO: 19 -CD3z is SEQ ID NO: 11 -4-1BB is SEQ ID NO: 12 SEQ ID NO: 29: CAR ARI2h (signal peptide + VL + linker + VH + CD8 hinge + CD8 TM + 4-1BB + CD3z) MEAPAQLLFLLLLWLPDTTGEVQLVESGGGLVQPGGSLRLSCAASGIDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASLYY DYGDAMDYWGQGTLVTVSSGGGGSGGGGSGGGSDIQMTQSPSSLSASVGDRVTITCKASQSVDSNVAWYQQKPGKAPKALIFSASLRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYNNYPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 30: GL007 bicistronic CAR sequence (GL002[T2A]ARI2h) nucleotide sequence (bicistronic nucleic acid): TIFF2026500149000001.tif206170 SEQ ID NO: 38: GL007 bicistronic CAR sequence (GL002[T2A]ARI2h) amino acid sequence: MEAPAQLLFLLLLWLPDTTGQIVLTQSPAIMSASPGEKVTVTCSASSSVSYMFWYQQKPGFSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPPTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSQMQLQESGPELVKPGASVKISCKASGYTFTHYYINWVQQRPGQGLEWIGWIFPESGSTYYSEKFKAKATLTVDKSSSTAYMLLSSLTSEDSAVYFCARGTGFFDYWGQGTTLTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGEGRGSLLTCGDVEENPGPGTSMEAPAQLLFLLLLWLPDTTGEVQLVESGGGLVQPGGSLRLSCAASGIDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASLYYDYGDAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQSVDSNVAWYQQKPGKAPKALIFSASLRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 31: GL008 bicistronic CAR sequence (ARI2h[T2A]GL002) nucleotide sequence (bicistronic nucleic acid): TIFF2026500149000002.tif206170 SEQ ID NO: 39: GL008 bicistronic CAR sequence (ARI2h[T2A]GL002) amino acid sequence: MEAPAQLLFLLLLWLPDTTGEVQLVESGGGLVQPGGSLRLSCAASGIDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASLYYDYGDAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQSVDSNVAWYQQKPGKAPKALIFSASLRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGEGRGSLLTCGDVEENPGPGTSMEAPAQLLFLLLLWLPDTTGQIVLTQSPAIMSASPGEKVTVTCSASSSVSYMFWYQQKPGFSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPPTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSQMQLQESGPELVKPGASVKISCKASGYTFTHYYINWVQQRPGQGLEWIGWIFPESGSTYYSEKFKAKATLTVDKSSSTAYMLLSSLTSEDSAVYFCARGTGFFDYWGQGTTLTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0257] The present invention will now be described with reference to the following examples, which are merely illustrative and are not intended to limit the scope of the present invention. [Example]

[0258] Example 1: Materials and Methods—CAR Design Production of anti-human Ly9 antibodies Anti-Ly9 (CD229) mAb was administered to 30 x 10 cells of the murine pre-B cell line 300.19 stably transfected with full-length cDNA. 6 The hybridoma was generated by fusing NS-1 myeloma cells with spleen cells from a BALB / c mouse immunized three times with NS-1 cells (clone 13). The hybridoma was subcloned twice. The antibody was further validated using cell lines and peripheral blood leukocytes.

[0259] Clone Hly9.1.84 was first described in the paper: de la Fuente MA, Tovar V, Villamor N, Zapater N, Pizcueta P, Campo E, Bosch J, Engel P. Molecular characterization and expression of a novel human leukocyte cell-surface marker homologous to mouse Ly-9.Blood.2001 Jun 1;97(11):3513-20.

[0260] Mapping of the antibody Hly91.184 to the second domain was published in the following paper: Romero X, Zapater N, Calvo M, Kalko SG, de la Fuente MA, Tovar V, Ockeloen C, Pizcueta P, Engel P. CD229 (Ly9) lymphocyte cell surface receptor interacts homophilically through its N-terminal domain and relocalizes to the immunological synapse. J Immunol. 2005 Jun 1;174(11):7033-42.

[0261] Mapping to the first or second domain was performed as described by Romero et al. (2005). Three different human Ly9 constructs were used: full-length human Ly9, a Ly9 isoform containing only domain 1, and a chimeric mouse-human molecule in which the second Ig domain of Ly9 was replaced with the corresponding mouse domain. Transfected cells were stained with different anti-human antibodies using flow cytometry. TIFF2026500149000003.tif86170

[0262] Cloning, plasmids and lentivirus production: We designed four CD229 CAR sequences containing the CD8α hinge and transmembrane domains, the 4-1BB costimulatory domain, and the CD3z signaling domain. The heavy (HV) and light variable (LV) regions of each antibody (hLy9 1.84 and hLy9 10.169) were used to generate a total of four single-chain variable fragment (scFv) domains. Linkers of 15 or 20 amino acids were added between the VH-VL or VL-VH sequences, respectively. ●HLy9 1.84 HLy9 1.84 VH-VL-41BB-CD3z, also known as GL001 HLy9 1.84 VL-VH-41BB-CD3z, also known as GL002 ●HLy9 10.169 HLy9 10.169 VH-VL-41BB-CD3z, also known as GL005 HLy9 10.169 VL-VH-41BB-CD3z, also known as GL006

[0263] The complete CAR sequence (including the single peptide, scFv, CD8 hinge, and transmembrane regions 4-1BB and CD3z) was synthesized by GenScript and cloned into the third-generation lentiviral vector pCCL under the control of the EF1a promoter.

[0264] To produce lentiviral particles for preclinical studies, HEK293T cells were transfected with our transfer vector (pCCL-EF1α-CARCD229) along with packaging plasmids pMDLg-pRRE (Addgene, 12251), pRSV-Rev (Addgene, 12253), and envelope plasmid pMD2.G (Addgene, 12259) using linear PEI molecular weight (MW) 25,000 (Polysciences, 23966-1). Briefly, 5 × 10 6 HEK293T cells were plated in 10 cm dishes 24 h before transfection. At the time of transfection, 14 μg of total DNA (6.9 μg of transfer vector, 1.7 μg of pMDLg / pRRE, 3.41 μg of pRSV-Rev, and 2 μg of pMD2.G) was diluted in serum-free DMEM. 35 μg of PEI was added to the mixture and incubated at room temperature for 20 min. After incubation, the DNA-PEI complex was added to cells cultured in 7 mL of complete DMEM. After 48 h, the viral supernatant was collected and concentrated using a LentiX-Concentrator (Clontech, Takara) according to the manufacturer's protocol. The concentrated lentivirus was stored at -80°C until use.

[0265] Lentiviral titration The number of transducing units (TU / mL) was determined by limiting dilution. Briefly, HEK293T cells were seeded 24 h before transduction. A 1:10 dilution of the viral supernatant was then prepared and added to the cells in complete DMEM + 5 μg / mL polybrene. Cells were trypsinized 48 h later and labeled with allophycocyanin (APC)-conjugated AffiniPure F(ab')2 fragment goat anti-mouse immunoglobulin G (IgG) (Jackson ImmunoResearch Laboratories, 115-136-072) and then analyzed by flow cytometry. Viral titers were calculated using the dilution corresponding to 2%–20% positive cells.

[0266] Generation of CAR-expressing primary human T cells PBMCs were isolated from healthy donor whole blood obtained from the Blood and Tissue Bank of Barcelona (BST) using Ficoll density gradient separation. Subsequently, T cells were isolated from the PBMCs using magnetically activated cell sorting (negative isolation). Isolated T cells were stimulated with CD3 / CD28 T cell-activating Dynabeads. For initial screening, T cells were lentivirally transduced 48 hours after stimulation. Dynabeads were then removed 7 days after stimulation. All T cells were grown in complete T cell medium, 5% AB human serum (Sigma, H4522), and penicillin-streptomycin (100 μg / mL) and supplemented with IL-2 every 2–3 days. CAR expression on the T cell surface was analyzed by flow cytometry. Experiments were performed after 8–10 days of T cell expansion.

[0267] Cell line generation and maintenance Multiple myeloma (MM) cell lines (U266, MM.1S, OPM2, and RPMI8226) and K562 were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA). K562, RPMI8226, and MM.1S cell lines were cultured in RPMI containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin, while U266 was cultured in 15% FBS. HEK293-T cells were cultured with DMEM containing 10% FBS and 1% penicillin-streptomycin. Tumor cell lines were transduced with lentivirus to express an EGFP-firefly luciferase (Ffluc) fusion gene. EGFP+ cells were enriched to >98% purity by fluorescence-activated cell sorting (FACS). BCMAneg tumor cells (BCMAKO) were generated by CRISPR / Cas9-mediated gene knockout. Tumor cells were nucleofected with a ribonucleoprotein consisting of a synthetic gRNA targeting BCMA complexed to purified Cas9 protein using electroporation solution and a Nucleofector 2B Device (Lonza). Cells were then bulk-sorted for the BCMAneg population by FACS.

[0268] Multiparameter flow cytometry: To analyze CAR expression, cells were labeled with recombinant Fc-tagged, biotinylated CD229 protein (G&P Biosciences) and recombinant human CD229 His (R&D Systems), followed by anti-Fc, streptavidin (BD Biosciences), or anti-His tag antibodies (Miltenyi Biotec). To assess T cell phenotype and CRISPR / Cas9-mediated gene disruption of surface proteins, CAR-T cells were stained with the following antibodies: anti-CD3, anti-CD4, anti-CD8, and anti-PD-1 (BD Biosciences); anti-TIGIT, anti-LAG-3, anti-TIM-3, anti-CCR7, anti-CD45RA, anti-CXCR3, anti-CD25, anti-CD71, anti-CD69, anti-CD28, and anti-CD27 (BioLegend). Tumor cells were stained with the following antibodies: anti-BCMA, anti-CD229, anti-CD38, and anti-CD138 (BioLegend). Live cells were gated by either forward / side scatter or negative uptake of a dye that stains dead cells, such as propidium iodide or LIVE / Dead Aqua (ThermoFisher Scientific). For each sample, at least 50,000 events were acquired on a BD FACSCanto II cytometer (BD Biosciences). FCS files were analyzed using FlowJo Software v.10 (BD Biosciences).

[0269] Functional assays: In vitro assays are performed by challenging CAR T cells with MM cell lines (U266, MM.1S, OPM2, RPMI-8226 lines and knockout cell lines).

[0270] Cytotoxicity assay: Cytolytic activity was assessed by a luminescence-based assay. 3ffluc-transduced target cells were co-cultured with CAR T cells at a predetermined effector-to-target (E:T) ratio for 4 to 24 hours. Immediately prior to analysis, 0.15 mg / mL D-luciferin firefly (Biosynth) was added to the co-culture, and luminescence was measured with an Infinite 200 Pro instrument (Tecan). Specific lysis was calculated using the standard formula:

[0271] Proliferation assay: T cells were labeled with 0.2 mmol / L carboxyfluorescein succinimidyl ester (CFSE; Invitrogen), washed, and plated in triplicate with stimulator cells in medium without exogenous cytokines. After 72 hours of incubation, CART cells were labeled and analyzed by flow cytometry to assess cell division.

[0272] Cytokine production: Cytokine secretion was measured using commercially available ELISA kits (BioLegend) at an E:T ratio of 4:1 (5 × 10 4 T cells) were analyzed in supernatants obtained from 24-hour co-cultures of T cells with target cells.

[0273] Safety Evaluation, Fratricide: CD229 will also be evaluated for fratricide by T cells along with peripheral blood cells from the same donor to assess activity against normal blood cells.

[0274] In vivo xenograft studies in NSG mice: All in vivo experiments were approved by the University of Barcelona Animal Research Committee. Male and female NOD / SCID IL-2Rc null (NSG) mice, 8-12 weeks old, were used. Mice were inoculated intravenously with MM cells (U266 and MM.1S cell lines) genetically modified to express GFP-Ffluc. This modification allows for disease tracking via D-luciferin bioluminescence.

[0275] Mice were irradiated with 2 Gy on day -1. On day 0, mice were irradiated with 1–5 x 10 6GFP-ffLuc-tumor cells were administered i.v. on day 14, with 1–5 × 10 6 Mice were randomly assigned to receive either untransduced T cells or CART cells intravenously. Bioluminescence images were taken weekly to track disease progression. In vivo imaging was recorded and analyzed using a Perkin Elmer IVIS Spectrum In Vivo Imaging System.

[0276] statistical analysis Statistical analysis was performed using Prism Software (GraphPad). Student's t-test was performed as a two-sided paired test with a 95% confidence interval, and results with a P value of less than 0.05 were considered significant. Statistical analysis of survival was performed using the log-rank test, and results with a P value of less than 0.05 were considered significant.

[0277] result In vitro evaluation of various anti-CD229 CAR T cells The scFvs of anti-CD229 CARs (HLy9 1.84 VH-VL-41BB-CD3z, HLy9 1.84 VL-VH-41BB-CD3z, HLy9 10.169 VH-VL-41BB-CD3z, and HLy9 10.169 VL-VH-41BB-CD3z) were cloned in-frame with the remaining CAR signaling domain in a lentiviral vector (pCCL) (Figure 1A). To evaluate the efficacy of the CARs, peripheral blood mononuclear cells (PBMCs) isolated from buffy coats were activated using CD3 and CD28 dynabeads and then transduced with anti-CD229-containing lentivirus.

[0278] Lentiviral transduction / CAR expression on T cells After lentiviral infection and an expansion period, expression of the anti-CD229 CAR on HEK293T cells and T cells was confirmed by flow cytometry only in the hLy9 1.84 CAR (VH-VL-41BB-CD3z[GL001] and VL-VH-41BB-CD3z[GL002]) (Figure 2A), whereas no anti-CD229 CAR was detected in the CARs produced using the hLy9 10.169 sequence (VH-VL-41BB-CD3z and VL-VH-41BB-CD3z) (Figure 2B).

[0279] Killing ability of anti-CD229 CAR T cells The efficacy of GL001 and GL002 cells against myeloma cell lines was tested by co-culturing T cells and myeloma cell lines. The cytotoxicity of GL001 and GL002 cells was measured by in vitro eradication of CD229-positive tumor cell lines (U266 and MM.1S). The K562 cell line was used as a negative control because it does not express CD229 on its surface. As a positive control, we used effective CAR T cells (ARI002h) developed in our facility (Perez-Amill, Haematologica, 2021) that are redirected against a well-known antigen expressed in myeloma, namely B-cell maturation antigen (BCMA). Both the U266 and MM.1S cell lines express BCMA on their surface. The K562 tumor cell line does not express BCMA on its surface.

[0280] We confirmed that GL001 and GL002 CAR T cells confer specific cytolytic activity against U266 and MM.1S myeloma cell lines in a dose-response relationship from high to low E:T ratios. We used untransduced (UTD) T cells as a reference and ARI002h (anti-BCMA) CAR T cells as comparator cells for specific target cell lysis (Figure 3).

[0281] All these data indicate that GL001 and GL002 CAR T cells exhibit potent and specific cytotoxic effects against CD229-positive cells in vitro.

[0282] To better characterize the GL001 and GL002 cell response to CD229 binding, cell proliferation was measured at 72 hours using a standard carboxyfluorescein diacetate succinimidyl ester (CFSE) assay. Antigen binding must be able to promote GL001 and GL002 cell proliferation in order to eliminate tumors in vivo. As shown in Figure 4, GL001 and GL002 cells proliferated in contact with the CD229+ MM.1S cell line and in response to interleukin-2 (IL-2) (positive control). Some proliferation was observed in the absence of stimulation or in contact with a CD229-negative cell line (K562), confirming that cell proliferation was mediated by CD229 recognition.

[0283] GL001 and GL002 cell production of cytokines in the supernatants of effector-target cell cocultures was measured after 24 hours and analyzed using ELISA. Cytokine levels from cocultures using GL001, GL002, and UTD were compared (Figure 5). While UTD cells did not show an increase in IFNg and IL-2, GL001 and GL002 cells showed a significant increase in these two pro-inflammatory cytokines when cocultured with CD229-positive cells (U266 and MM.1S). There was no significant increase in cytokine production in the absence of stimulation or in contact with CD229-negative cells (K562).

[0284] In vivo evaluation of the efficacy of GL001 and GL002 To evaluate the efficacy of GL001 and GL002 cells in vivo, we used NOD.Cg-Prkdc scid Il2rd tm1Wjl Xenograft experiments were performed in / SzJ(NSG) mice.

[0285] Mice were randomly assigned to receive U266+UTD cells (A), U266+GL001 (B), U266+GL002 (C), and U266+ARI002h (D). Mice were inoculated with U266-FfLuc+GFP+ (CD229+ / BCMA+) cells via the tail vein on day 1. On day 14, mice were injected with either UTD, GL001, GL002, or ARI002h cells.

[0286] As shown in Figure 6, disease progression was clearly observed after U266 cell injection with UTD cells. The groups receiving U266 tumor cell line and GL002 showed better disease control compared with the UTD and GL001 groups. However, the group receiving ARI002h had better disease control and a survival advantage compared with all remaining groups. In terms of efficacy, these data suggest that GL002 is superior to GL001.

[0287] To recapitulate human MM, we modeled an established BCMA xenogeneic disease. We generated a CRISPR / Cas9-mediated BCMA knockout (KO) MM.1S myeloma cell line, and BCMA KO was confirmed by flow cytometry and resistance to BCMA-targeted CAR T cell cytotoxicity (ARI002h) (Figure 7A). GL001 and GL002 CAR T cells were able to lyse these MM1S BCMA-KO cells (Figure 7B).

[0288] We wanted to analyze the efficacy of GL002 against ARI002h at lower doses in a model of established tumors, including BCMA-negative disease. To develop the model, we intravenously injected mice with a mixture of 15% MM.1S BCMA-KO tumor cells and 85% wild-type (WT) tumor cells with endogenous BCMA and CD229 expression. Mice were treated with a subtherapeutic medium dose (1 × 10 6) dose of CAR T cells. As expected, tumors progressed in control UTD T cell-treated mice. The group receiving ARI002h had progressive disease after 2 weeks of myeloma control. Nevertheless, there was a survival advantage in the group receiving GL002 (Figure 8). These data suggest that GL002 is superior to ARI002h when a population of BCMA-negative tumor cells is present.

[0289] Finally, MM1.S myeloma cells recovered from the bone marrow of mice at the time of euthanasia confirmed that tumor cells express BCMA + (UTD group), BCMA - (ARI002h group) and BCMA + (GL2 or CD229-VLVH-BBz group) (see Figure 9).

[0290] Example 2: Materials and Methods—Dual CAR Design Cloning, plasmids and lentivirus production:

[0291] To improve the efficacy of CAR therapy for multiple myeloma, we created a double CAR against two molecules (CD229 and BCMA). The CAR was designed using a bicistronic vector (Figure 10A). We cloned the GL002 (anti-CD229) and ARI2h (anti-BCMA) genes into a single vector and associated the 2A cleavage peptide (T2A) and linker before and after the T2A. Depending on the monocistronic CAR located in the most proximal region, two bicistronic CARs were obtained. 1. GL002[T2A]ARI2h, also known as GL007 2. ARI2h[T2A]GL002, also known as GL008

[0292] The complete CAR sequence (including the single peptide, scFv, CD8 hinge, and transmembrane regions 4-1BB and CD3z) was synthesized by GenScript and cloned into the third-generation lentiviral vector pCCL under the control of the EF1a promoter.

[0293] To produce lentiviral particles for preclinical studies, HEK293T cells were transfected with our transfer vector (pCCL-EF1α-dualCAR) along with packaging plasmids pMDLg-pRRE (Addgene, 12251), pRSV-Rev (Addgene, 12253), and envelope plasmid pMD2.G (Addgene, 12259) using linear PEI molecular weight (MW) 25,000 (Polysciences, 23966-1). Briefly, 5 × 10 6 HEK293T cells were plated in 10 cm dishes 24 h before transfection. At the time of transfection, 14 μg of total DNA (6.9 μg of transfer vector, 1.7 μg of pMDLg / pRRE, 3.41 μg of pRSV-Rev, and 2 μg of pMD2.G) was diluted in serum-free DMEM. 35 μg of PEI was added to the mixture and incubated at room temperature for 20 min. After incubation, the DNA-PEI complex was added to cells cultured in 7 mL of complete DMEM. After 48 h, the viral supernatant was collected and concentrated using a LentiX-Concentrator (Clontech, Takara) according to the manufacturer's protocol. The concentrated lentivirus was stored at -80°C until use.

[0294] Lentiviral titration The number of transducing units (TU / mL) was determined by limiting dilution. Briefly, HEK293T cells were seeded 24 h before transduction. A 1:10 dilution of the viral supernatant was then prepared and added to the cells in complete DMEM + 5 μg / mL polybrene. Cells were trypsinized 48 h later and labeled with allophycocyanin (APC)-conjugated AffiniPure F(ab')2 fragment goat anti-mouse immunoglobulin G (IgG) (Jackson ImmunoResearch Laboratories, 115-136-072) and then analyzed by flow cytometry. Viral titers were calculated using the dilution corresponding to 2%–20% positive cells.

[0295] Generation of CAR-expressing primary human T cells PBMCs were isolated from healthy donor whole blood obtained from the Blood and Tissue Bank of Barcelona (BST) using Ficoll density gradient separation. Subsequently, T cells were isolated from the PBMCs using magnetically activated cell sorting (negative isolation). Isolated T cells were stimulated with CD3 / CD28 T cell-activating Dynabeads. For initial screening, T cells were lentivirally transduced 48 hours after stimulation. Dynabeads were then removed 7 days after stimulation. All T cells were grown in complete T cell medium, 5% AB human serum (Sigma, H4522), and penicillin-streptomycin (100 μg / mL) and supplemented with IL-2 every 2–3 days. CAR expression on the T cell surface was analyzed by flow cytometry. Experiments were performed after 8–10 days of T cell expansion.

[0296] Cell line generation and maintenance Multiple myeloma (MM) cell lines (U266, MM.1S, OPM2, and RPMI8226) and K562 were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA). K562, RPMI8226, and MM.1S cell lines were cultured in RPMI containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin, while U266 was cultured in 15% FBS. HEK293-T cells were cultured with DMEM containing 10% FBS and 1% penicillin-streptomycin. Tumor cell lines were transduced with lentivirus to express an EGFP-firefly luciferase (Ffluc) fusion gene. EGFP+ cells were enriched to >98% purity by fluorescence-activated cell sorting (FACS). BCMAneg tumor cells (BCMAKO) were generated by CRISPR / Cas9-mediated gene knockout. Tumor cells were nucleofected with a ribonucleoprotein consisting of only a synthetic gRNA targeting BCMA complexed to purified Cas9 protein using electroporation solution and a Nucleofector 2B Device (Lonza). Cells were then bulk sorted for the BCMAneg population by FACS. Cell lines with low BCMA (BCMAdim) expression were generated by FACS of the bulk BCMA KO cell line with a gate to select for cells expressing low levels of BCMA. K562 BCMA knockin (KI) cells were lentivirally transduced to express the BCMA gene.

[0297] Multiparameter flow cytometry: To analyze CAR expression, cells were labeled with recombinant Fc-tagged biotinylated CD229 protein (G&P Biosciences), recombinant human CD229 His (R&D Systems), or Fc-tagged recombinant human BCMA protein (Enzo), followed by anti-Fc, streptavidin (BD Biosciences), or anti-His tag antibodies (Miltenyi Biotec). To assess T cell phenotype and CRISPR / Cas9-mediated gene disruption of surface proteins, CAR-T cells were stained with the following antibodies: anti-CD3, anti-CD4, anti-CD8, and anti-PD-1 (BD Biosciences); anti-TIGIT, anti-LAG-3, anti-TIM-3, anti-CCR7, anti-CD45RA, anti-CXCR3, anti-CD25, anti-CD71, anti-CD69, anti-CD28, and anti-CD27 (BioLegend). Tumor cells were stained with the following antibodies: anti-BCMA, anti-CD229, anti-CD38, and anti-CD138 (BioLegend). Live cells were gated by either forward / side scatter or negative uptake of a dye that stains dead cells, such as propidium iodide or LIVE / Dead Aqua (ThermoFisher Scientific). For each sample, at least 50,000 events were acquired using a BD FACSCanto II cytometer (BD Biosciences). FCS files were analyzed using FlowJo Software v.10 (BD Biosciences).

[0298] Functional assays: In vitro assays are performed by challenging CAR T cells with MM cell lines (U266, MM.1S, OPM2, knockout cell lines and BCMAdim cell lines), K562 and K562 KI BCMA.

[0299] Cytotoxicity assay: Cytolytic activity was assessed by a luminescence-based assay. 3ffluc-transduced target cells were co-cultured with CAR T cells at a predetermined effector-to-target (E:T) ratio for 4–24 h. Immediately prior to analysis, 0.15 mg / mL D-luciferin firefly (Perkin-Elmer) was added to the co-culture, and luminescence was measured with an Infinite 200 Pro instrument (Tecan). Specific lysis was calculated using the standard formula:

[0300] Proliferation assay: T cells were labeled with 0.2 mmol / L carboxyfluorescein succinimidyl ester (CFSE; Invitrogen), washed, and plated in triplicate with stimulator cells in medium without exogenous cytokines. After 72 hours of incubation, CART cells were labeled and analyzed by flow cytometry to assess cell division.

[0301] Cytokine production: Cytokine secretion was measured using commercially available ELISA kits (BioLegend) at an E:T ratio of 4:1 (5 × 10 4 T cells) were analyzed in supernatants obtained from 24-hour co-cultures of T cells with target cells.

[0302] Safety Evaluation, Fratricide: CD229 will also be evaluated for fratricide by T cells along with peripheral blood cells from the same donor to assess activity against normal blood cells.

[0303] In vivo xenograft studies in NSG mice: All in vivo experiments were approved by the University of Barcelona Animal Research Committee. Male and female NOD / SCID IL-2Rc null (NSG) mice, 8–12 weeks old, were used. Mice were inoculated intravenously with MM cells (U266, MM.1S, MM.1S BCMAKO, and MM.1S BCMAdim cell lines) genetically modified to express GFP-Ffluc. This modification allows for disease tracking by D-luciferin bioluminescence.

[0304] Mice were irradiated with 2 Gy on day -1. On day 0, mice were irradiated with 1–5 x 10 6 GFP-ffLuc-tumor cells were administered i.v. on day 14, with 1–3 × 10 6 Mice were randomly assigned to receive either untransduced T cells or CART cells intravenously. Bioluminescence images were taken weekly to track disease progression. In vivo imaging was recorded and analyzed using a Perkin Elmer IVIS Spectrum In Vivo Imaging System.

[0305] statistical analysis Statistical analysis was performed using Prism 8 Software (GraphPad). Student's t-test was performed as a two-sided paired test with a 95% confidence interval, and results with a P value of less than 0.05 were considered significant. Statistical analysis of survival was performed by the log-rank test, and results with a P value of less than 0.05 were considered significant.

[0306] result In vitro evaluation of various dual (CD229 / BCMA) CAR T cells The sequences of the GL002 and ARI2h CARs were cloned in-frame into a single lentiviral vector (pCCL) and associated with a 2A cleavage peptide (T2A). Depending on the monocistronic CAR located in the most proximal region, two bicistronic CARs were obtained: GL007:GL002[T2A]ARI2h; and GL008:ARI2h[T2A]GL002 (Figure 10B). To evaluate the efficacy of the CARs, peripheral blood mononuclear cells (PBMCs) isolated from buffy coats were activated using CD3 and CD28 dynabeads and then transduced with GL007- and GL008-containing lentiviruses.

[0307] Lentiviral transduction / CAR expression on T cells After lentiviral infection and a period of expansion, expression of anti-CD229 and anti-BCMA CARs on HEK293T cells and T cells was confirmed by flow cytometry (Figure 11).

[0308] Killing ability of anti-CD229 CAR T cells The efficacy of GL007 and GL008 cells against myeloma cell lines was tested by co-culturing T cells and myeloma cell lines. The cytotoxicity of GL007 and GL008 cells was measured by in vitro eradication of CD229- and BCMA-positive tumor cell lines (U266, OPM2, and MM.1S), the MM.1S BCMAKO and OPM2 BCMAKO cell lines, which express only CD229, and the K562 BCMAKI cell line, which expresses only BCMA. The K562 cell line was used as a negative control because it does not express CD229 or BCMA on its surface.

[0309] We confirmed that GL007 and GL008 CAR T cells conferred specific cytolytic activity against tumor cell lines expressing both antigens (U266, MM.1S, and OPM2), tumor cell lines expressing only BCMA (K562 KI), and tumor cell lines expressing only CD229 (MM.1S BCMAKO and OPM2 BCMAKO), with a dose-response relationship from high to low E:T ratios. UTD cells were used for specific lysis calculations (Figure 12).

[0310] All these data indicate that GL007 and GL008 CAR T cells exhibit potent and specific cytotoxic effects in vitro against tumor cells expressing one or both antigens.

[0311] To better characterize the GL007 and GL008 cell responses to BCMA and / or CD229 binding, cell proliferation was measured at 72 hours using a standard carboxyfluorescein diacetate succinimidyl ester (CFSE) assay. Antigen binding must be able to promote GL007 and GL008 cell proliferation in order to eliminate tumors in vivo. As shown in Figure 13, GL007 and GL008 cells proliferated in contact with tumor cells expressing both target antigens (MM.1S), BCMA only (K562 BCMAKI), or CD229 only (MM.1S BCMAKO), and in response to interleukin-2 (IL-2) (positive control). Some proliferation was observed in the absence of stimulation or in contact with a BCMA- and CD229-negative cell line (K562), confirming that cell proliferation was mediated by CD229 and BCMA recognition.

[0312] GL007 and GL008 cell production of cytokines in the supernatants of effector-target cell cocultures was measured after 24 hours and analyzed using ELISA. Cytokine levels from cocultures using GL007, GL008, and UTD were compared (Figure 14). While UTD cells did not show an increase in IFNg and IL-2, GL007 and GL008 cells showed a significant increase in these two pro-inflammatory cytokines when cocultured with BCMA- and CD229-positive cells (U266, MM.1S, and OPM2), BCMA-positive cells (K562 BCMA), and CD229-positive cells (MM.1S BCMAKO and OPM2 BCMAKO). There was no significant increase in cytokine production in the absence of stimulation or in contact with BCMA- and CD229-negative cells (K562).

[0313] In vivo evaluation of the efficacy of GL007 and GL008 To evaluate the efficacy of GL007 and GL008 cells in vivo, we used NOD.Cg-Prkdc scid Il2rd tm1Wjl Xenograft experiments were performed in / SzJ(NSG) mice.

[0314] Mice were randomly assigned to receive MM.1S+UTD cells (A), MM.1S+ARI2h (B), MM.1S+GL007 (C), and MM.1S+GL008 (D). Mice were inoculated with MM.1S-FfLuc+GFP+(CD229+ / BCMA+) cells via the tail vein on day 1. On day 14, mice were injected with either UTD, ARI2h, GL007, or GL008 cells.

[0315] As shown in Figure 15, disease progression was clearly observed after MM.1S cell injection with the UTD cell group. There was no difference in tumor control and survival among the three groups of mice administered any of the three CAR T cells (ARI2h, GL007, and GL008).

[0316] We wanted to analyze the efficacy of GL007 and GL008 against ARI2h and GL002 at lower doses in a model of established tumors, including BCMA-negative disease. To develop the model, we intravenously injected mice with a mixture of 15% MM.1S BCMA-KO tumor cells and 85% wild-type (WT) tumor cells with endogenous BCMA and CD229 expression. Mice were treated with a subtherapeutic medium dose (1 × 10 6 ) dose of CAR T cells. As expected, tumors progressed in control UTD T cell-treated mice. The group receiving ARI2h had progressive disease after two weeks of myeloma control. Nevertheless, there was a survival advantage in the groups receiving GL002, GL007, and GL008 (see Figure 16). Furthermore, GL007 demonstrated superior tumor control and survival compared to GL008. These data suggest that GL002 and both bicistronic CARs (GL007 and GL008) are superior to ARI2h when a population of BCMA-negative tumor cells is present. With regard to bicistronic and GL002, they are comparable; however, GL007 is superior to GL008.

[0317] Finally, MM.1S myeloma cells recovered from the bone marrow of mice at the time of euthanasia confirmed that tumor cells express BCMA + / CD229 + (UTD, GL007, and GL008 groups). However, two mice from each bicistronic group showed loss of both antigens (CD229 and BCMA) (see Figure 17).

[0318] Evaluation of monocistronic and bicistronic CAR T cells in models with reduced BCMA expression. To simulate a model of reduced BCMA expression in tumor cells, but without complete loss of antigen, we derived a cell line with reduced BCMA expression (MM.1S BCMAdim) by FACS of tumor cells with minimal BCMA expression from the MM.1S BCMAKO line (see Figure 18).

[0319] We confirmed that GL002 and GL007 CAR T cells confer specific cytolytic activity against the MM.1S BCMAdim cell line compared to ARI2h. As expected, ARI2h was able to eliminate cell lines that normally express BCMA (MM.1S), no cytotoxicity was observed in cell lines that do not express BCMA (MM.1S BCMA KO), and minimal elimination of cell lines expressing low levels of BCMA (MM.1S BCMAdim). See Figure 19. All these data indicate that CAR GL002 and GL007 T cells exhibit potent and specific cytotoxic effects in vitro against tumor cells that express BCMA at low density.

[0320] We wanted to analyze the efficacy of GL002 and GL007 against ARI2h at lower doses in a model of established tumors with reduced BCMA disease. To develop the model, we intravenously injected mice with a mixture of 50% MM.1S WT tumor cells and 50% MM.1S BCMAdim tumor cells. Mice were treated with a subtherapeutic medium (1 × 10 6) dose of CAR T cells. As expected, tumors progressed in mice treated with control UTD T cells. The group receiving ARI2h developed progressive disease similar to the UTD group. Nevertheless, there was a survival advantage in the groups receiving GL002 and GL007 (see Figure 20). These data suggest that in the presence of a reduced population of BCMA tumor cells, GL002 and GL007 CARs are superior to ARI2h.

Claims

1. 1. A CD229 targeting moiety that is an scFV comprising a VL domain and a VH domain, - the VL domain comprises a sequence having at least 85% sequence identity with SEQ ID NO: 1, and the VL domain comprises CDR regions consisting only of LCDR1 as set forth in SEQ ID NO: 4, LCDR2 as set forth in SEQ ID NO: 5, and LCDR3 as set forth in SEQ ID NO: 6; - a CD229 targeting moiety, wherein said VH domain comprises a sequence having at least 85% sequence identity to SEQ ID NO:2, and said VH domain comprises CDR regions consisting only of HCDR1 set forth in SEQ ID NO:7, HCDR2 set forth in SEQ ID NO:8, and HCDR3 set forth in SEQ ID NO:

9.

2. 2. The CD229 targeting moiety of claim 1, wherein the VL domain consists solely of SEQ ID NO: 1 and the VH domain consists solely of SEQ ID NO:

2.

3. 3. The CD229 targeting moiety of claim 1, wherein the VL domain is positioned upstream of the VH domain (GL002).

4. The CD229 targeting moiety of any one of claims 1 to 3, wherein the CD229 targeting moiety is an scFv consisting solely of SEQ ID NO: 17 (GL002) or SEQ ID NO: 3 (GL001).

5. a. an extracellular domain comprising a CD229 targeting moiety, wherein said CD229 targeting moiety is as defined in any one of claims 1 to 4; b. a transmembrane domain; and c. Intracellular signaling domain A chimeric antigen receptor (CAR) comprising:

6. The CAR of claim 5, wherein the transmembrane domain comprises the transmembrane domain of CD8.

7. The CAR of claim 5 or 6, wherein the intracellular signaling domain comprises the intracellular domain of CD3ζ.

8. The CAR according to any one of claims 5 to 7, wherein the costimulatory signaling domain comprises an intracellular domain of CD137.

9. The CAR according to any one of claims 5 to 8, consisting solely of SEQ ID NO: 18 (CAR GL002) or 13 (CAR GL001).

10. A nucleic acid encoding the CAR according to any one of claims 5 to 9.

11. A cell comprising the nucleic acid of claim 10.

12. 12. The CD229 targeting moiety of any one of claims 1 to 4, the CAR of any one of claims 5 to 9, the nucleic acid of claim 10, or the cell of claim 11 for use in a method of treating a CD229-positive cancer, said method comprising administering said CD229 targeting moiety, said CAR, said nucleic acid, or said cell to a patient in need thereof, preferably wherein said cancer is multiple myeloma.

13. 13. The CD229 targeting moiety, the CAR, the nucleic acid, or the cell for use according to claim 12, wherein the use is in combination with anti-BCMA therapy.

14. 14. The CD229 targeting moiety, the CAR, the nucleic acid, or the cell for use according to claim 13, wherein the anti-BCMA therapy comprises administering a CAR against BCMA.

15. The CAR against BCMA is a) an extracellular domain comprising said BCMA targeting moiety, said BCMA targeting moiety comprising a VL domain and a VH domain; - the VL domain comprises a sequence having at least 85% sequence identity with SEQ ID NO: 25, and the VL domain comprises CDR regions consisting only of LCDR1 as set forth in SEQ ID NO: 36, LCDR2 as set forth in SEQ ID NO: 20, and LCDR3 as set forth in SEQ ID NO: 21; an extracellular domain, wherein the VH domain comprises a sequence having at least 85% sequence identity with SEQ ID NO: 26, and wherein the VH domain comprises CDR regions consisting only of HCDR1 as set forth in SEQ ID NO: 22, HCDR2 as set forth in SEQ ID NO: 23, and HCDR3 as set forth in SEQ ID NO: 24; b) a transmembrane domain, preferably the transmembrane domain of CD8; c) an intracellular signaling domain, preferably the intracellular domain of CD3ζ, and d) optionally at least a costimulatory signaling domain, preferably the intracellular domain of CD137 15. The CD229 targeting moiety, the CAR, the nucleic acid, or the cell for use according to claim 14, comprising:

16. 16. The CD229-targeting moiety, the CAR, the nucleic acid, or the cell for use according to claim 15, wherein the CAR against BCMA consists solely of SEQ ID NO: 29 (CAR ARI2h).

17. i) a CAR against CD229 according to any one of claims 5 to 9, or a nucleic acid molecule encoding the same; and ii) a CAR against BCMA, or a nucleic acid molecule encoding the same 20. A combination therapy comprising administering

18. ii) the CAR against BCMA is a) an extracellular domain comprising said BCMA targeting moiety, said BCMA targeting moiety comprising a VL domain and a VH domain; - the VL domain comprises a sequence having at least 85% sequence identity with SEQ ID NO: 25, and the VL domain comprises CDR regions consisting only of LCDR1 as set forth in SEQ ID NO: 36, LCDR2 as set forth in SEQ ID NO: 20, and LCDR3 as set forth in SEQ ID NO: 21; an extracellular domain, wherein the VH domain comprises a sequence having at least 85% sequence identity with SEQ ID NO: 26, and wherein the VH domain comprises CDR regions consisting only of HCDR1 as set forth in SEQ ID NO: 22, HCDR2 as set forth in SEQ ID NO: 23, and HCDR3 as set forth in SEQ ID NO: 24; b) a transmembrane domain, preferably the transmembrane domain of CD8; c) an intracellular signaling domain, preferably the intracellular domain of CD3ζ, and d) optionally at least a costimulatory signaling domain, preferably the intracellular domain of CD137 18. The combination therapy of claim 17, comprising:

19. i) the CAR against CD229 consists solely of SEQ ID NO: 18 (GL002) or a nucleic acid molecule encoding same; ii) the CAR against BCMA consists solely of SEQ ID NO: 29 (ARI2h) or a nucleic acid molecule encoding same; 19. The combination therapy of claim 18.

20. i) a nucleic acid sequence encoding a CD229 targeting moiety comprising a VL domain and a VH domain, in any order, - the VL domain comprises a sequence having at least 85% sequence identity with SEQ ID NO: 1, and the VL domain comprises CDR regions consisting only of LCDR1 [SSVSY] (SEQ ID NO: 4), LCDR2 [DTS] (SEQ ID NO: 5), LCDR3 [CQQWSSYPPTF] (SEQ ID NO: 6); - a nucleic acid sequence in which the VH domain comprises a sequence having at least 85% sequence identity with SEQ ID NO: 2, and the VH domain comprises CDR regions consisting only of HCDR1 [GYTFTHYY] (SEQ ID NO: 7), HCDR2 [IFPESGST] (SEQ ID NO: 8) and HCDR3 [CARGTGFFDYW] (SEQ ID NO: 9), and ii) a nucleic acid sequence encoding a BCMA targeting moiety comprising a VL domain and a VH domain in any order, - the VL domain comprises a sequence having at least 85% sequence identity with SEQ ID NO: 25, and the VL domain comprises CDR regions consisting only of LCDR1 of SEQ ID NO: 36, LCDR2 of SEQ ID NO: 20, and LCDR3 of SEQ ID NO: 21; - a nucleic acid sequence in which the VH domain comprises a sequence having at least 85% sequence identity with SEQ ID NO: 26, and the VH domain comprises CDR regions consisting only of HCDR1 of SEQ ID NO: 22, HCDR2 of SEQ ID NO: 23, and HCDR3 of SEQ ID NO:

24. in any order.

21. i) a nucleic acid sequence encoding a CD229-targeting moiety which is an ScFV and consists solely of SEQ ID NO: 17 (GL002); ii) a nucleic acid sequence encoding a BCMA-targeting portion that is an ScFV and consists solely of SEQ ID NO: 28 (ARI2h); 21. The bicistronic nucleic acid of claim 20, comprising in the 5' to 3' direction:

22. i) a nucleic acid sequence encoding SEQ ID NO: 18 (CAR GL002), and ii) a nucleic acid sequence encoding SEQ ID NO: 29 (CAR ARI2h) 22. The bicistronic nucleic acid of claim 20 or 21, comprising in the 5' to 3' direction:

23. 23. The bicistronic nucleic acid of claim 22, comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:

38.

24. A cell comprising the bicistronic nucleic acid of any one of claims 20 to 23.

25. 25. The combination therapy of any one of claims 17 to 19, the bicistronic nucleic acid of any one of claims 20 to 23, or the cell of claim 24, for use in a method for treating a CD229-positive / BCMA-positive cancer, preferably multiple myeloma.

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