Anti-BCMA CAR T cells for the treatment of multiple myeloma

Abstract

The present invention provides a therapeutic agent for treating multiple myeloma. In particular, the present invention provides chimeric antigen receptor (CAR) T cells capable of targeting B cell maturation antigens.

Description

[Technical Field]

[0001] The present invention provides a therapeutic agent for treating multiple myeloma. In particular, the present invention provides chimeric antigen receptor (CAR) T cells capable of targeting B cell maturation antigens. [Background technology]

[0002] Multiple myeloma (MM) remains an incurable hematologic malignancy, accounting for 1% of all cancers and 15%–20% of all hematologic malignancies, with an average annual increase of 0.8% in new cases over the past decade. MM is characterized by the clonal expansion of malignant plasma cells within the bone marrow (BM), resulting in excessive production of monoclonal immunoglobulins (Ig) in the blood and / or urine, leading to osteolytic lesions accompanied by clinical symptoms including hypercalcemia, infections, and organ dysfunction. The natural history of MM is characterized by relapses until refractory disease, with no survival plateau, and fewer than 10% of patients achieve a durable complete remission (CR) beyond 5–10 years after autologous stem cell transplantation (ASCT). Furthermore, patients are rarely cured after high-dose chemotherapy followed by ASCT, and importantly, patients who achieve a CR have a longer survival time than those who do not. Therefore, new strategies are needed to improve the survival of R / R MM patients, especially those with high-risk cytogenetics.

[0003] In recent years, chimeric antigen receptor (CAR) T-cell immunotherapy, which is based on the infusion of autologous T cells genetically modified to recognize antigens expressed on tumor cells, has transformed the treatment of certain hematological malignancies. Specifically, in acute lymphoblastic leukemia (ALL) and lymphoma, this therapy, targeting CD19, has achieved excellent responses, leading to FDA approval of these novel therapies. In MM, B-cell maturation antigen (BCMA), a transmembrane glycoprotein involved in regulating B-cell maturation and survival and with specific and restricted expression on mature B cells and plasma cells, has become the most promising target for CAR T-cell immunotherapy.

[0004] All ongoing clinical trials using CARTBCMA cells in MM patients require higher doses of CART cells (150 × 10) to achieve a response compared with CART19. 6 It has been shown that a lower dose of CART cells is required to achieve a response. Furthermore, it has been shown that the response deepens over time, as the number of very good partial responses progresses to complete responses over time. Furthermore, in order to avoid relapse due to early loss of CART cells, the current trend in CART cell immunotherapy is to use humanized or human CARs instead of mouse CARs (Non-Patent Document 1, Non-Patent Document 2).

[0005] Here, starting from CART19 (ARI1) (Non-Patent Document 3), which has already been used in a multicenter phase II clinical trial for B-cell malignancies, we generated murine CART cells against BCMA (ARI2m) for use by patients in the public healthcare system. After confirming in vitro and in vivo efficacy, we humanized ARI2m to ARI2h. Comparing the efficacy and inflammatory response of both ARI2m and ARI2h, we found that both CARs showed comparable efficacy, but a reduced inflammatory profile was observed with ARI2h. Furthermore, GMP clinical scale-up was successfully achieved for both CARs at our institution. Finally, we analyzed the influence of soluble BCMA (sBCMA) on ARI2 activity and demonstrated how sBCMA can negatively affect CART activity. All these results allow us to conduct a multicenter clinical trial in MM patients in Spain using our ARI2h cells. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Sommermeyer D, Hill T, Shamah SM, et al. Fully human CD19-specific chimeric antigen receptors for T-cell therapy. Leukemia. 2017;31(10):2191-2199

Wood 2

Table 3

Fashion, Water, Life

[0007] [Figure 1]Design and functional characterization of CARTBCMA mice (ARI2m) for multiple myeloma (MM) cells. (A) Design of ARI2m. (B) Transduction efficiency of ARI2 cells and NT T cells before and after cryopreservation. (C) Cytotoxicity assay of ARI2 against two MM cell lines (ARP1 and U266) and one non-myeloma cell line (K562). Limiting dilution cytotoxicity assays against ARP1 and U266 (MM cell lines) and K562 (CML) were performed at ratios of 1:1 to 0.125:1 (T cells:tumor cell line) at 36 hours (D) and 72 hours (E). (F) Cytokine profiles of IFNγ, IL-6, and TNF-α after 24 and 48 hours of coculture (T cells and ARP1 cells). (G-K) In vivo efficacy of ARI2m cells: (G): Diagram of experimental design and quantification of disease progression by weekly bioluminescence (H) and overall survival (I) for mice treated with ARI2m vs. NT T cell group vs. untreated group. (J) Flow cytometry of bone marrow (BM) and spleen of mice at the end of the experiment. (K) Percentage of total T cells and CART cells in BM and spleen of mice treated with ARI2m. (L) Soluble BCMA (sBCMA) ELISA from mouse serum after treatment with ARI2m or NT T cells. [Figure 2]Figure 1 shows the humanization of ARI2m to ARI2h and a comparison of ARI2m vs. ARI2h. (A) Schematic of the amino acid disparity between the scFv heavy and light chains in humanized ARI2 based on the BLAST or Germline algorithms. (B) Limiting dilution cytotoxicity assay of ARI2m vs. both humanized versions (Blast and Germline). (C) Long-term cytotoxicity assay comparing murine and humanized ARI2 against ARP1 (MM) and K562 (CML) and their IFNγ production. (D-I) In vivo results: disease progression by weekly bioluminescence in early-stage (D) and advanced-stage (E) models, respectively, and its quantification (F). (G) Kaplan-Meier curves showing overall survival of untreated, ARI2m, and ARI2h mouse groups in early-stage and advanced-stage disease models. (H) Percentage of total CD3+ T cells and ARI2 cells in the CD3+ T cell population found in the BM and spleen for both early and advanced disease models. (I) ELISA of IFNγ from mouse serum on days 3 and 31 for the early disease model and days 5 and 21 for the advanced disease model. [Figure 3] Figure 1 shows T cell profiles and inflammatory responses of ARI2m versus ARI2h. (A) Schematic of repeated antigen stimulation assay. (B) CD4 / CD8 T cell ratio profiles of ARI2m, ARI2h, and NT T cells during four consecutive challenges, and the percentage of CART cells in the CD4 or CD8 T cell subsets. (C) (D) Schematic of autologous monocyte and T cell isolation from the same buffy coat and their expansion, differentiation, and coculture with MM cell lines. (E) Cytotoxicity of ARI2m against ARP1 cells with or without macrophages, and production of proinflammatory cytokines (IL6, TNFα, and IL1β). (F) (G) Cytokine production of IFNγ, IL6, TNFα, and IL1β over 48 hours after coculture of ARI2m / ARI2h with macrophages and ARP1. [Figure 4]Figure 1 shows the clinical production and activity of ARI2m and ARI2h. Clinical expansion of ARI2m and ARI2h cells (A and B) shows the total number of T cells achieved at the end of expansion (left) and the percentage of CART cells achieved (right). (C) Median of four clinical expansions of ARI2m and ARI2h cells. Results show the percentage and total number of ARI2 cells achieved at the end of expansion. (D) Cytotoxicity assay of both ARI2m and ARI2h cells against U266 MM cell line at the end of expansion. [Figure 5] Soluble BCMA influences ARI2 activity. (A) ELISA of sBCMA from five patients with monoclonal gammopathy of undetermined significance (MGUS), MM at diagnosis (Dx), and at relapse. (B) Confocal fluorescence images of MM cells stained with the cell tracker CMAC and BCMA stained with monoclonal anti-TNRSF17. (C and D) Representative images from two different in vivo time-lapse experiments over 3 hours of ARI2m stained with the cell tracker CMAC and ARP1 MM cells overexpressing BCMA with GFP. (E) Cytotoxicity assay and IFNγ production of ARI2m cocultured with ARP1 MM cells with recombinant BCMA protein (BCMA) added with or without an antibody (Ab) against BCMA (F). (G) MFI of BCMA and its sBCMA quantification (H) in ARP1 MM cell line alone or in coculture with ARI2 / NT T cells, with or without DAPT. (I) Schematic of the cytotoxicity assay with or without transwell (TW). ARI2m / NT T cells were co-cultured with the ARP1 cell line in a well, and additional ARP1 cells were added to the TW as a continuous source of sBCMA release. DAPT was also added in parallel. (J) Cytotoxicity results for the experiment shown in (I). [Figure 6]Further comparison of ARI2m vs. ARI2h in a highly advanced disease model. (A) CFSE assay over 4 days to analyze ARI2 proliferation after encountering ARP1 MM cells. (B) TNFα and IL6 production over 7 days after co-culture of ARP1 MM cells with ARI2m and ARI2h cells at a ratio of 0.125:1 (E:T). (C) Schematic of in vivo experiments in mice receiving ARP1 MM cells and either ARI2m or ARI2h cells. (D) Disease progression of the experiment in (C) followed by weekly bioluminescence. DETAILED DESCRIPTION OF THE INVENTION

[0008] definition "Administering" a drug to a patient or "administration" of a drug to a patient (and grammatical equivalents of this phrase) refers to direct administration, which can be administration to the patient by a medical professional or self-administration, and / or indirect administration, which can be the act of prescribing a drug. For example, a patient is administered a drug by a physician who instructs the patient to self-administer the drug or prescribes the drug to the patient.

[0009] The term "affibody" refers to a protein derived from the Z domain of Protein A and engineered to bind to a specific target (see Frejd & Kim, 2017. Exp Mol Med. 49(3): e306).

[0010] 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 or single chain thereof, and combinations thereof. For example, the term "antibody" specifically includes bivalent antibodies and bivalent, bispecific antibodies.

[0011] A typical type of antibody comprises at least two heavy chains (“HC”) and two light chains (“LC”) interconnected by disulfide bonds.

[0012] 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.

[0013] Each "light chain" comprises 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 the kappa or lambda type. The VH and VL domains can be further subdivided into regions of hypervariability, called complementarity determining regions ("CDRs"), interspersed with regions that are more conserved, called "framework regions" ("FW").

[0014] VH and VL each consist of three CDRs and four FWs arranged in the following order from amino terminus to carboxy terminus: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The present disclosure specifically presents VH and VL sequences, as well as subsequences corresponding to CDR1, CDR2, and CDR3.

[0015] The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of known schemes, including those described in 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).

[0016] Therefore, it will be understood by those skilled in the art 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.

[0017] The variable domains of the heavy and light chains contain regions that interact with a binding target, which are also referred to herein as "antigen-binding sites" or "antigen binding sites." The constant domains of the antibody 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 classical antibodies, but also bivalent fragments and variants thereof, such as F(ab')2.

[0018] 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.

[0019] Antibodies can be any of five major classes of immunoglobulins (isotypes): IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, called α, δ, ε, γ, and μ, respectively. Immunoglobulins of different classes have different 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.

[0020] 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.

[0021] 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 in the early stages. As the disease progresses, bone pain, bleeding, frequent infections, and anemia can occur. Complications can include amyloidosis.

[0022] 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.

[0023] The term "BCMA targeting moiety" refers to a substance capable of binding to BCMA. In the context of a CAR, the BCMA targeting moiety targets T cells to BCMA-positive cells, preferably cancer cells. It is understood that in the context of a CAR, the BCMA targeting moiety can be genetically encodable.

[0024] 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 (see Riviere & Sadelain, 2017. Mol Ther. 25(5):1117-1124). Similarly, the term "CART" refers to a T cell containing a CAR.

[0025] "Combination therapy," "in combination with," or "in conjunction with," as used herein, refers to any form of combined, concurrent, simultaneous, sequential, or intermittent treatment with at least two different therapeutic modalities (i.e., compounds, components, targeted agents, or therapeutic agents). As such, these terms refer to the administration of one therapeutic modality to a subject before, during, or after the administration of another therapeutic modality. Modalities in a combination can be administered in any order. Therapeutic 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 administration protocols appropriate for the separate compositions, formulations, or unit dosage forms), in a manner and dosage regimen prescribed by a healthcare professional or in accordance with a regulatory agency. Generally, each therapeutic modality is administered at a dose and / or schedule determined for that therapeutic modality. Optionally, three or more modalities may be used in combination therapy. Additionally, the combination therapies provided herein can be used in combination with other types of treatments, for example, other anti-cancer treatments 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.

[0026] "Complete response" or "complete remission" or "CR" refers to the disappearance of all target lesions as defined in the RECIST v1.1 guidelines. This does not necessarily mean that the cancer has been cured.

[0027] 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 by the CD3ζ chain of the TCR / CD3 complex. In the context of the present invention, the costimulatory domain is 4-1BB. The term "4-1BB" refers to a membrane receptor protein, also known as CD137, a member of the tumor necrosis factor receptor (TNFR) superfamily that is expressed on the surface of activated T cells as an accessory molecule [Kwon et al., Proc. Natl. Acad. Sci. USA 86:1963 (1989); PoUok et al., J. Immunol. 151:771 (1993)]. 4-1BB has a molecular weight of 55 kDa and is found as a homodimer. It has been suggested that 4-1BB mediates a signal transduction pathway from the extracellular to the internal cellular pathway [Kim et al., J. Immunol. 151:1255 (1993)]. The human 4-1BB gene was isolated from a cDNA library made from activated human peripheral T cell mRNA [Goodwin et al., Eur. J. Immunol. 23:2631 (1993)]. The amino acid sequence of human 4-1BB shows 60% homology to that of mouse 4-1BB [Kwon et al., Proc. Natl. Acad. Sci. USA 86:1963 (1989); GenBank No: NM_011612], indicating a high degree of sequence conservation. As mentioned above, 4-1BB belongs to the TNFR superfamily, along with CD40, CD27, TNFR-I, TNFR-II, Fas, and CD30 [Alderson et al., Eur. J. Immunol. 24:2219 (1994)]. Binding of monoclonal antibodies to 4-1BB expressed on the surface of T cells increases anti-CD3 T cell activation many-fold [Pollok et al., J. Immunol. 150:771 (1993)].4-1BB binds to a high-affinity ligand (4-1BBL, also called CD137L) expressed on several antigen-presenting cells, such as macrophages and activated B cells [Pollok et al., J. Immunol. 150:771 (1993) Schwarz et al., Blood 85:1043 (1995)]. 4-1BBL is claimed and described in U.S. Patent No. 5,674,704. The interaction of 4-1BB with its ligand provides a costimulatory signal that leads to T cell activation and growth [Goodwin et al., Eur. J. Immunol. 23:2631 (1993); Alderson et al., Eur. J. Immunol. 24:2219 (1994); Hurtado et al., J. Immunol. 155:3360 (1995); Pollock et al., Eur. J. Immunol. 25:488 (1995); DeBenedette et al., J. Exp. Med. 181:985 (1995)].

[0028] "Disease-free survival" (DFS) refers to the period during and after treatment that a patient remains disease-free.

[0029] As used herein, the term "effective amount" of an agent, e.g., a therapeutic agent such as a CART, is an amount sufficient to bring about a beneficial or desired result, e.g., a clinical result, and thus, an "effective amount" will depend on the context in which it is applied. For example, in the context of administering a therapeutic agent to treat multiple myeloma, an effective amount can reduce the number of cancer cells, reduce tumor size or burden, inhibit (i.e., slow to some extent, and in certain embodiments, stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent, and 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 bring about 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" can be used interchangeably with "effective dose," "therapeutically effective amount," or "therapeutically effective dose."

[0030] The terms "individual," "patient," or "subject" are used interchangeably in this application to designate a human being and are not intended to be limiting in any way. An "individual," "patient," or "subject" may be of any age, sex, and physical condition.

[0031] "Infusion" or "infuse" refers to the introduction of a solution containing a therapeutic agent into the body via a vein for therapeutic purposes. Typically, this is accomplished by an intravenous bag.

[0032] "Intracellular signaling domain," as used herein, refers to all or a portion of one or more domains of a molecule (here, a chimeric receptor molecule) that leads to lymphocyte activation. The intracellular domain of such a molecule mediates signals by interacting with cellular mediators, leading to 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 ligation, as well as any derivatives or variants of these sequences, and any synthetic sequence that has the same function.

[0033] The term "monobody" refers to a protein derived from the fibronectin type III domain and engineered to bind to a specific target (see Koide et al., 2013. J Mol Biol. 415(2):393-405).

[0034] 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).

[0035] "Overall survival" (OS) refers to the time from patient enrollment until death or censoring at the last known survival date. OS includes an increase in life expectancy compared to untreated or untreated individuals or patients. Overall survival refers to the survival of a patient for a specified period of time, e.g., 1 year, 5 years, etc., from the time of diagnosis or treatment.

[0036] "Partial response" or "PR" refers to a reduction in the sum of diameters of target lesions by at least 30% referenced to the baseline sum of diameters in response to treatment, as defined in the RECIST v1.1 guidelines.

[0037] The term "peptide aptamer" refers to a short sequence of 5-20 amino acid residues that can bind to a specific target. Peptide aptamers are typically inserted into loop regions of stable protein scaffolds (see Reverdatto et al., 2015. Curr Top Med Chem. 15(12):1082-101).

[0038] 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 pharmaceutical 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, 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, mannose, Examples of suitable pharmaceutically acceptable carriers include organic sugars or sugar alcohols such as sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), and polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; 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 also be included in the pharmaceutical compositions described herein, provided they do not adversely affect the desired properties of the pharmaceutical composition.

[0039] "Progressive disease" or "advanced disease" refers to the appearance of another new lesion or tumor and / or overt progression of an existing non-target lesion as defined in the RECIST v1.1 guidelines. Progressive or advanced disease may also refer to tumor growth of more than 20 percent from the start of treatment due to an increase in either tumor mass or spread.

[0040] "Progression-free survival" (PFS) refers to the time from enrollment to disease progression or death. PFS is generally measured using the Kaplan-Meier method and Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 standard. Generally, progression-free survival refers to the situation in which a patient continues to live without their cancer getting worse.

[0041] The term "RECIST" refers to Response Evaluation Criteria in Solid Tumors. RECIST guidelines, criteria, or standards describe a standard approach to measuring and defining solid tumors for the objective assessment of changes in tumor size used in clinical trials of adult and pediatric cancers. RECIST v1.1 refers to the revised RECIST guidelines, version 1.1, published in European Journal of Cancers 45 (2009) 228-247.

[0042] The term "repebody" refers to proteins derived from leucine-rich repeat modules and engineered to bind to specific targets (see Lee et al., 2012. PNAS. 109(9): 3299-3304).

[0043] The term "respond favorably" generally refers to producing a beneficial state in a subject. In the context of cancer treatment, this term refers to producing a therapeutic effect in a subject. A favorable therapeutic effect in cancer can be measured in many ways (see Weber, 2009. J Nucl Med. 50 Suppl 1:1S-10S). For example, tumor growth inhibition, molecular marker expression, serum marker expression, and molecular imaging techniques can all be used to assess the therapeutic efficacy of anti-cancer therapies. Regarding tumor growth inhibition, according to the NCI standard, T / C≦42% is the minimum level of anti-tumor activity. T / C<10% is considered a high level of anti-tumor activity, where T / C (%)=median tumor volume with treatment / median tumor volume of control×100. A favorable response can be assessed, for example, by 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.

[0044] The term "sequence identity" refers to the percentage value obtained when two sequences are compared using a pairwise sequence alignment tool. In this case, sequence identity is obtained using the global alignment tool "EMBOSS Needle" with default settings (Rice et al., 2000. Trends Genet. 16(6):276-7; Li et al., 2015. Nucleic Acids Res. 43(W1):W580-4). The global alignment tool is available at https: / / www.ebi.ac.uk / Tools / psa / .

[0045] The term "single-chain antigen-binding fragment" or "scFab" refers to a fusion protein comprising one variable domain and one constant domain of an antibody light chain attached to one variable domain and one constant domain of an antibody heavy chain, the heavy and light chains being linked by a short peptide.

[0046] The term "single-chain variable fragment" or "scFv" refers to a fusion protein comprising the variable domains of the heavy (VH) and light (VL) chains of an antibody linked together by a peptide linker. This term also includes disulfide-stabilized Fvs (dsFvs). Methods for stabilizing scFvs by disulfide bonds are disclosed in Reiter et al., 1996. Nat Biotechnol. 14(10):1239-45.

[0047] "Stable disease" refers to disease without progression or relapse, as defined by RECIST v1.1 guidelines. Stable disease is neither sufficient tumor shrinkage to be considered a partial response nor sufficient tumor growth to be considered progressive disease.

[0048] "Time to progression" (TTP) is defined as the time from enrollment to disease progression. TTP is generally measured using RECIST v1.1 criteria.

[0049] The terms "treatment" and "therapy" as used in this application refer to a range of hygienic, pharmacological, surgical, and / or physical measures used with the goal of ameliorating a health problem, with the intent of curing and / or alleviating a disease and / or symptom. The terms "treatment" and "therapy" include prophylactic and curative methods, as both are directed to maintaining and / or restoring the health of an individual or animal. The administration of suitable medications to alleviate and / or cure a health problem, regardless of the cause of the symptom, disease, or disorder, should be construed as a form of treatment or therapy within the context of this application.

[0050] Detailed Description of the Invention The present inventors have successfully developed BCMA-directed CART cells (ARI2m cells) with 4-1BB as the costimulatory domain, which are being administered in a multicenter Phase I clinical trial to treat MM patients who have relapsed or become refractory (R / R) to at least two prior therapies, including a proteasome inhibitor, an immunomodulatory agent, and an anti-CD38 monoclonal antibody. Our ARI2m cells were humanized to ARI2h, demonstrating that anti-MM activity was retained in the humanized version, and furthermore, a lower cytotoxicity profile was observed with the ARI2h cells.

[0051] BCMA emerged as a promising antigen for the treatment of MM using CART cells in 2013 (Carpenter RO, Evbuomwan MO, Pittaluga S, et al. B-cell maturation antigen is a promising target for adoptive T-cell therapy of multiple myeloma. Clin Cancer Res. 2013;19(8):2048-2060), leading to the first clinical study in MM patients in 2016 who received CART BCMA cells with CD28 as their costimulatory domain (Ali SA, Shi V, Maric I, et al. T cells expressing an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma. Blood. 2016;128(13):1688-1700). These CART cells demonstrated efficacy but displayed a high cytotoxicity profile, as all patients treated with active doses developed severe CRS. Thus, CD28 was replaced with 4-1BB, a new CAR called bb2121, which showed manageable toxicity and required 150 × 10 6showed that a minimum dose of CAR T cells is required (Raje N, Berdeja J, Lin Y, et al. Anti-BCMA CAR T-Cell Therapy bb2121 in Relapsed or Refractory Multiple Myeloma. N Engl J Med. 2019;380(18):1726-1737). In parallel, two additional studies in MM patients (Cohen AD, Garfall AL, Stadtmauer EA, et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J Clin Invest. 2019;130 and Zhao WH, Liu J, Wang BY, et al. A phase 1, open-label study of LCAR-B38M, a chimeric antigen receptor T cell therapy directed against B cell maturation antigen, in patients with relapsed or refractory multiple myeloma. J Hematol Oncol. 2018;11(1):141) demonstrated that fewer prior therapies were associated with better responses and that, although lymphodepletion was not absolutely required for CAR T cell expansion and activity in vivo, short-term expansion after lymphodepletion was more consistent. Because studies have shown that most MM patients eventually relapse (a finding not observed in ALL patients treated with CART19), various factors influence the growth and persistence of CART, which enhances long-term disease control, factors that need to be improved with CARTBCMA therapy. In this regard, the persistence of CART cells may be improved by the use of human or humanized CARs, as the murine component of the CAR's scFv initiates an immune response by the human immune system, leading to the early disappearance of CART cells.Based on these previous studies, and supported by results showing that both ARI2m and ARI2h equally prevented disease progression, we selected ARI2h cells, humanized CARTBCMA cells containing the 4-1BB costimulatory domain.

[0052] Other factors that influence CART cell persistence include the exhaustion profile of CART cells and the CD4 / CD8 ratio in leukapheresis products, which correlates with in vivo CART expansion in CARTBCMA cells. In our study, regardless of the initial CD4 / CD8 ratio, all in vitro expansions achieved CD4 / CD8 >1, and normalized to approximately equal CD4 and CD8 levels after tumor cell exposure due to preferential CD8 T cell proliferation. Furthermore, continuous exposure to tumor cells demonstrated that ARI2h cells achieved higher proliferation than ARI2m, suggesting less exhaustion in ARI2h cells. In this regard, studies with CART19 using CD28 and 4-1BB costimulatory domains have shown that strong activation of CART cells with high affinity or high expression of target antigens leads to an effector T cell phenotype with increased exhaustion, whereas weaker activation with lower affinity leads to a T cell memory phenotype with reduced exhaustion. Here, the humanization process involves amino acid sequence changes that may reduce CART affinity in ARI2h cells, explaining slower in vitro activity in assays after a single challenge; on the contrary, after successive challenges with tumor cells, sustained and higher CART cell proliferation occurs, allowing longer in vivo disease control in highly advanced tumor models.

[0053] Although the high incidence of CRS and neurotoxicity are common events following CART cell administration and are effectively managed according to international guidelines, ideal CART treatment should strive to minimize the onset of CRS. Here, the use of ARI2h cells instead of ARI2m cells is further supported by the observation of a lower in vivo toxicity profile and lower in vitro TNFα production of ARI2h cells compared to ARI2m cells. IL6 is an effector cytokine of CRS produced by monocytes and macrophages and increases exponentially as CRS develops, whereas other cytokines, such as TNFα and IL1β, are major initiators of CRS and are produced early by monocytes and macrophages upon activation by IFNs produced by CART cells. Indeed, TNFα acts as an initiator cytokine orchestrating cytokine cascades in many inflammatory diseases, emerging as a therapeutic target for various inflammatory diseases. Here, our in vitro model using macrophages, which mimics a model more similar to the in vivo scenario, showed that ARI2h cells led to a decrease in TNFα production by macrophages, a finding relevant to CRS in MM patients after CARTBCMA, which is associated with a high peak of TNFα.

[0054] Finally, this study carefully analyzed the effect of sBCMA on CART activity, confirming that sBCMA entertains CART cells from targets and potentially causing fratricide between CARTBCMA cells. Although preclinical and clinical studies using CARTBCMA in MM have not shown any correlation between sBCMA and CART activity, we observed that the high in vitro CART activity, which rapidly eliminates MM cells, precluded proper analysis of the role of sBCMA in preclinical studies. Furthermore, the higher dose of CARTBCMA cells required to induce a response in MM compared with CART19 in ALL patients led us to hypothesize that sBCMA may be involved in this higher dose of CART. Therefore, our in vitro model was performed at a low CARTBCMA:MM ratio to create an environment with sustained release of sBCMA, and the addition of a γ-secretase inhibitor confirmed the negative effect of sBCMA on CARTBCMA activity.

[0055] In conclusion, we present herein a humanized CART BCMA (wherein the CAR corresponds to ARI2h of SEQ ID NO: 13) with 4-1BB as the costimulatory domain, which retains high efficacy and exhibits a lower toxicity profile than its murine counterpart (ARI2m). This CAR (ARI2h) is efficiently grown under GMP conditions for use in clinical trials. Therefore, the main objective of the present invention is to identify chimeric antigen receptors for ARI2h and their variants, as described in the following description.

[0056] ARI2h chimeric antigen receptor and its mutants In one aspect, the present invention provides a chimeric antigen receptor (CAR) of ARI2h or a variant thereof, comprising an extracellular domain comprising a BCMA targeting moiety, a transmembrane domain, and an intracellular signaling domain, such domains being described in more detail below.

[0057] BCMA targeting moiety In some embodiments, the BCMA targeting moiety is an antibody, anticalin, lipibody, monobody, scFv, Fab, scFab, affibody, fynomer, DARPin, nanobody, or peptide aptamer that specifically binds to BCMA.

[0058] Binding molecules that specifically bind to BCMA may be very useful in the diagnosis and treatment of MM. Several mouse monoclonal antibodies against BCMA are known in the art. However, the in vivo use of mouse antibodies is limited by problems associated with their administration to humans, such as short serum half-life, inability to elicit certain human effector functions, and generation of undesirable immune responses against mouse antibodies. To overcome these previously mentioned drawbacks, new human antibodies have been developed.

[0059] Phage display and combinatorial methods for generating antibodies are known in the art (e.g., U.S. Pat. No. 5,223,409 to Ladner et al., WO 92 / 18619 to Kang et al., WO 91 / 17271 to Dower et al., WO 92 / 20791 to Winter et al., WO 92 / 15679 to Markland et al., WO 93 / 01288 to Breitling et al., WO 92 / 01047 to McCafferty et al., WO 92 / 09690 to Garrard et al., WO 90 / 02809 to Ladner et al., Fuchs et al. (1991) Bio / Technology 9:1370-1372, Hay et al. (1992) Hum Antibod Hybridomas 3:81-85, Huse et al. (1989) Science 246:1275-1281, Griffiths et al. (1993) EMBO J 12:725-734, Hawkins et al. (1992) J Mol Biol 226:889-896, Clackson et al. (1991) Nature 352:624-628, Gram et al. (1992) PNAS 89:3576-3580, Garrad et al. (1991) Bio / Technology 9:1373-1377, Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137 and Barbas et al. (1991) PNAS 88:7978-7982, the contents of both of which are incorporated herein by reference).

[0060] Additionally, methods for generating and selecting non-immunoglobulin scaffolds that bind to specific targets are known in the art (see, e.g., Skrlec, et al., 2015. Trends Biotechnol. 33(7):408-18).

[0061] In some embodiments, the BCMA targeting moiety, preferably an antibody, scFv, Fab, or scFab, comprises a VH domain, wherein the VH domain is set forth in SEQ ID NO:1: TIFF0007729561000001.tif21170, or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

[0062] In some embodiments, the BCMA targeting moiety, preferably an antibody, scFv, Fab, or scFab, comprises a VH domain as described above and further comprises a VL domain, wherein the VL domain is set forth in SEQ ID NO:2: TIFF0007729561000002.tif21170, or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2.

[0063] In some embodiments, the BCMA targeting moiety, preferably an antibody, scFv, Fab, or scFab, comprises a VL domain and a VH domain, wherein the VL domain and the VH domain comprise or consist of SEQ ID NOs: 1 and 2 or a variant of any of these sequences as defined above. Preferably, the VL domain and the VH domain comprise or consist of SEQ ID NO: 3: TIFF0007729561000003.tif34170, or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3.

[0064] As used herein, the VL domain and VH domain of SEQ ID NO: 3 as described above may be linked with a linker sequence, in particular SEQ ID NO: 4: Note that this contains the linker sequence TIFF0007729561000004.tif6170, although other linker sequences may be used.

[0065] Furthermore, the VL and VH domains, in particular SEQ ID NO: 3, may further comprise a peptide signal, preferably the signal peptide is SEQ ID NO: 5: TIFF0007729561000005.tif7170, or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:5.

[0066] In some embodiments, the BCMA targeting moiety, preferably an antibody, scFv, Fab, or scFab, has the sequence of SEQ ID NO:6: TIFF0007729561000006.tif45170, or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6.

[0067] Transmembrane domain The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane region may comprise at least one or more transmembrane regions 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. Preferably, the transmembrane region comprises at least one or more transmembrane regions of CD8a.

[0068] The transmembrane domain may be synthetic 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.

[0069] 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 at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. Preferably, the transmembrane domain comprises or consists of at least the transmembrane domain of CD8a.

[0070] In some embodiments, the transmembrane domain comprises or consists of the transmembrane domain of CD8a or a variant thereof, wherein the variant has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0071] In particular, in some embodiments, the transmembrane domain comprises or consists of SEQ ID NO:7 or a variant having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:7.

[0072] The transmembrane domain from CD8a (SEQ ID NO: 7: TIFF0007729561000007.tif6170).

[0073] In some embodiments, the domain derived from CD8a is the CD8 hinge, preferably SEQ ID NO: 8: TIFF0007729561000008.tif6170, or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:8.

[0074] Thus, in some further embodiments, the transmembrane domain further comprises a CD8 hinge and is set forth in SEQ ID NO:9: TIFF0007729561000009.tif14170, or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 9.

[0075] Intracellular signaling domains The intracellular signaling domain results in activation of at least one function of a cell expressing the CAR after binding to a ligand expressed on a tumor cell. 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 activation of at least one function of a cell containing the CAR.

[0076] In some embodiments, the intracellular signaling domain comprises or consists of the intracellular domain of CD3zeta, FcRgamma, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, CD66b, or a variant thereof, which variant has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0077] In some embodiments, the intracellular signaling domain comprises or consists of the intracellular domain of CD3ζ or a variant thereof, wherein the variant has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0078] In some embodiments, the intracellular signaling domain comprises SEQ ID NO: 10, or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10. Note that SEQ ID NO: 10 is represented by the following sequence: TIFF0007729561000010.tif21170

[0079] Costimulatory signaling domain It should be noted that the CAR of the present invention, an ARI2h CAR, or variant thereof, must further comprise a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain comprises the intracellular domain of 4-1BB or a variant thereof, wherein the variant has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0080] In some embodiments, the costimulatory signaling domain comprises or consists of the intracellular domain of 4-1BB or a variant thereof, wherein the variant has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0081] In some embodiments, the costimulatory signaling domain comprises or consists of SEQ ID NO: 11 or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 11. As used herein, a costimulatory signaling domain derived from 4-1BB is represented by SEQ ID NO: 11: Note that the image is represented as TIFF0007729561000011.tif7170.

[0082] Full-sequence CARs according to the present invention The full amino acid sequence of the ARI2h CAR according to the present invention is SEQ ID NO: 13: TIFF0007729561000012.tif72170 (where, the signal peptide consists of SEQ ID NO: 5, the VH domain consists of SEQ ID NO: 1; The linker sequence consists of SEQ ID NO: 4, the VL domain consists of SEQ ID NO:2; CD8 hinge consists of SEQ ID NO: 8, the transmembrane domain consists of SEQ ID NO: 7; the 4-1BB domain consists of SEQ ID NO: 11; Note that the CD3z domain comprises or consists solely of SEQ ID NO: 10).

[0083] In some embodiments, a CAR according to the invention may be characterized by comprising: (i) a BCMA targeting moiety, preferably an antibody, scFv, Fab, or scFab, comprising or consisting of a VL domain and a VH domain, wherein the VH domain and the VL domain comprise or consist of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, or a variant of either thereof, which variant has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to either SEQ ID NO: 1 and / or SEQ ID NO: 2; (ii) a transmembrane domain linked to a hinge domain comprising or consisting solely of SEQ ID NO:9 or a variant thereof, wherein the variant has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9; (iii) a costimulatory signaling domain comprising or consisting of SEQ ID NO:11 or a variant thereof, wherein the variant has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:11; and (iv) An intracellular signaling domain comprising or consisting of SEQ ID NO:10 or a variant thereof, wherein the variant has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:10.

[0084] In some embodiments, the CAR is SEQ ID NO: 13: TIFF0007729561000013.tif72170, or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13.

[0085] nucleic acid In one aspect, the invention provides a nucleic acid encoding any one of the CARs of the invention, including any one of the CARs disclosed above. The nucleic acid sequence encoding the chimeric receptor connects multiple modular components that can be excised and replaced with other components to customize the chimeric receptor for efficient T cell activation and recognition of BCMA.

[0086] 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.

[0087] In some embodiments, the nucleic acid is codon-optimized for expression in mammalian cells. Codon optimization methods are known in the art (see, e.g., Parret et al., 2016. Curr Opin Struct Biol. 39: 155-162).

[0088] The nucleic acids of the invention can be included in lentiviral vectors that can be used to transduce or transform T cells (see Riviere & Sadelain, 2017. Mol Ther. 25(5):1117-1124). Currently, transduction of T cells with lentiviral vectors is a more widely used technique in humans. Nucleic acids can also be inserted into cells using DNA transposons, RNA transfection, or genome editing technologies such as TALEN, ZFN, and CRISPR / Cas9 (see Riviere & Sadelain, 2017. Mol Ther. 25(5):1117-1124).

[0089] The complete nucleotide sequence of CARS according to the present invention is SEQ ID NO: 12: Preferably, it comprises or consists solely of TIFF0007729561000014.tif153170.

[0090] cell In one 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).

[0091] In some embodiments, the cells are naive T cells, memory stem T cells, or central memory T cells, which are currently believed to be better suited for adaptive immunotherapy (see Riviere & Sadelain, 2017. Mol Ther. 25(5):1117-1124).

[0092] In some embodiments, the cells are autologous T cells. The term "autologous cells" refers to cells obtained from the same patient being treated with any one of the methods of the present invention.

[0093] In some embodiments, the cells are allo-tolerant T cells. The term "allo-tolerant cells" refers to cells that have been modified to reduce the risk of graft-versus-host disease responses. In some embodiments, this is achieved by genome editing-mediated deletion of the TCR and / or β2-microglobulin. 15、19 Allogeneic tolerant cells are known in the art (see the chapter on allogeneic T cells in Riviere & Sadelain, 2017. Mol Ther. 25(5):1117-1124).

[0094] In some embodiments, the cells are lymphoid precursors, embryonic stem cells, or induced pluripotent stem cells that have the capacity to differentiate into mature T cells (see Riviere & Sadelain, 2017. Mol Ther. 25(5):1117-1124).

[0095] Pharmaceutical Composition In one aspect, the invention provides a pharmaceutical composition comprising a plurality of cells of the invention and a pharmaceutically acceptable carrier or diluent.

[0096] 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 confer stability to 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 with low toxicity in biological systems are generally used.

[0097] In some embodiments, the cells are formulated by first harvesting them from their culture medium, then washing the cells and concentrating them in a therapeutically effective amount in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier). A suitable infusion medium can be any isotonic medium formulation, typically saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter); 5% dextrose in water or lactated Ringer's solution can also be used. The infusion medium may also be supplemented with human serum albumin, fetal bovine serum, or other human serum components.

[0098] In one aspect, the present invention provides a cell according to the present invention or a pharmaceutical composition according to the present invention for use as a medicament.

[0099] Treatment method In one aspect, the present invention provides a method for treating multiple myeloma, comprising administering to a patient in need thereof a cell of the present invention or a pharmaceutical composition of the present invention.

[0100] In some embodiments, the patient is administered a therapeutically effective amount of cells. In some embodiments, the patient is administered at least 10 2 pieces, 10 3 pieces, 10 4 pieces, 105 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 Pieces or 10 10 The number of cells will depend on the intended end use of the composition, as will the type of cells included in the composition.

[0101] In some embodiments, the cells or pharmaceutical composition are administered intravenously, intraperitoneally, into the bone marrow, into the lymph nodes, and / or into the cerebrospinal fluid.

[0102] In some embodiments, the method comprises combination therapy. In some embodiments, the method comprises further administering an immune checkpoint inhibitor (see Lim & June, 2017. Cell. 168(4):724-740). In further embodiments, the method comprises further administering an immune checkpoint inhibitor and / or an IAP inhibitor (see WO 2016 / 054555).

[0103] In some embodiments, the cells or pharmaceutical compositions described herein are administered in combination with chemotherapeutic agents and / or immunosuppressants. In one embodiment, a patient is first treated with a chemotherapeutic agent that inhibits or destroys other immune cells, followed by the cells or pharmaceutical compositions described herein. In some cases, chemotherapy can be avoided entirely.

[0104] The following examples serve to illustrate the invention without, however, limiting it. [Example]

[0105] Materials and Methods Ethics statement: Studies involving human material were approved by the Clinical Research Ethics Committee (Hospital Clinic, Barcelona). Peripheral blood (PB) T cells were obtained from healthy donors with informed consent. All work involving animals was performed under the Animal Research Ethics Committee (Hospital Clinic, Barcelona).

[0106] Cell culture: RPMI8226, U266, and K562 were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA). The ARP1 cell line was kindly provided by the Multiple Myeloma Research Center (Little Rock, AK, USA). Cell lines (K562, RPMI8226, and ARP1) were cultured in RPMI containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Pen / Strep), and U266 containing 15% FBS. 293-T cells were cultured in DMEM containing 10% FBS and 1% Pen / Strep. Lymphocytes were obtained from healthy donors by magnetic depletion with Ficoll and a T cell isolation kit (Miltenyi Biotec). T cells were expanded in Click's medium (50% RPMI, 50% Click's medium, 5% human serum, and 1% Pen / Strep, Irvine Scientific) and activated every other day with Dynabeads Human T-Activator CD3 / CD28 (Thermo Fisher Scientific) and IL-2 (100 IU). Experiments were performed 8–10 days after T cell expansion. Macrophages were differentiated from monocytes after one week of expansion in RPMI, 10% FBS, and 0.1 mg / ml M-CSF (Thermo Fisher Scientific).

[0107] Cloning and humanization: The ARI2m transmembrane domain, costimulatory domain (4-1BB), and CD3ζ were obtained from a lentiviral vector containing CART19 used in our laboratory (pCCL-EF1α-CD19-CD8a-41BB-CD3ζ). The scFv-CD19 was derived from the anti-BCMA antibody J22.9 previously published by Oden F. et al. and used instead of the scFv BCMA, which is freely available in NCBI-Protein Genbank. The complete amino acid sequence corresponding to the signal peptide, VH, linker, VL, CD8 hinge, CD8 TM, 4-1BB, and CD3ζ of mouse ARI2m is shown below.

[0108] Mouse (ARI2m) (SEQ ID NO: 14): TIFF0007729561000015.tif45170 Signal peptide (SEQ ID NO: 5): TIFF0007729561000016.tif5170VH (SEQ ID NO: 15): TIFF0007729561000017.tif8170 Linker (SEQ ID NO: 4): TIFF0007729561000018.tif4170VL (SEQ ID NO: 16): TIFF0007729561000019.tif8170CD8 Hinge (SEQ ID NO: 8): TIFF0007729561000020.tif4170CD8 TM (SEQ ID NO: 7): TIFF0007729561000021.tif41704-1BB (SEQ ID NO: 11): TIFF0007729561000022.tif4170CD3ζ (SEQ ID NO: 10): TIFF0007729561000023.tif9170

[0109] To obtain ARI2h, the scFv of the ARI2m sequence was humanized using two prediction models (Blast and Germline) to replace murine amino acids with their human homologs and remove both the complementarity-determining regions (CDRs) and the Vernier Zone. Once the sequence was ready, it was cloned into the pCCL vector using the same procedure as for ARI2m. The amino acid sequence differences between ARI2h and ARI2m, along with the humanized sequence, are shown below.

[0110] Humanized (ARI2h (Germline variant) corresponding to SEQ ID NO: 13): TIFF0007729561000024.tif41170VH (SEQ ID NO: 1): TIFF0007729561000025.tif9170 Linker (SEQ ID NO: 4): TIFF0007729561000026.tif4170VL (SEQ ID NO: 2): TIFF0007729561000027.tif9170CD8 Hinge (SEQ ID NO: 8): TIFF0007729561000028.tif4170CD8 TM (SEQ ID NO: 7): TIFF0007729561000029.tif41704-1BB (SEQ ID NO: 11): TIFF0007729561000030.tif4170CD3ζ (SEQ ID NO: 10): TIFF0007729561000031.tif9170

[0111] Amino acid differences introduced during the humanization process (ARI2m vs. ARI2h): Bold: CDR Italics: Vernier zone Underlined: AA changes VH: Mouse (AR2m): TIFF0007729561000032.tif11170 Humanized (AR2h): TIFF0007729561000033.tif10170VL: Mouse (AR2m): TIFF0007729561000034.tif10170 Humanized (AR2h): TIFF0007729561000035.tif10170

[0112] Virus production and CAR expression: 293-T cells were transfected with lentiviral vectors (pCCL-EF1α-BCMA, pREV-REV, pMDLg / pRRE, and pCMV-VSV-G) to produce lentivirus. After 48 hours, supernatants were collected and concentrated using a LentiX concentrator (Clontech, Takara) according to the manufacturer's protocol. The concentrated lentivirus was kept at -80°C until use. T cells from healthy donors were activated with Dynabeads on day 0 and transduced with the concentrated lentivirus on day 2 by adding polybrene (Merck Millipore) and centrifuging at 2000 rpm for 1 hour.

[0113] Flow cytometry: For CAR-BCMA detection, cells were incubated with recombinant BCMA-Fc protein (Enzo Life Sciences) followed by a secondary antibody, anti-human IgG Fc, conjugated to Brilliant Violet (BV)-421 (Biolegend). Antibodies used for T cell staining and exhaustion were CD3-APC and CD8-PE (Becton Dickinson), PD1-APC, TIM3-APC, and LAG3-APC (Thermo Fisher Scientific). Multiple myeloma cells were stained with CD138-BV421 (Becton Dickinson) and BCMA-APC (Biolegend). Flow cytometry analysis for all experiments was performed using FlowJo software.

[0114] Proliferation assay: CAR-T cells were stained with the CellTrace™ CFSE Cell Proliferation Kit (Invitrogen, Thermo Fisher Scientific) and then co-cultured with different conditions and cell lines for 96 hours. Proliferation was analyzed by flow cytometry.

[0115] Cytokine production and sBCMA: IFN-γ, TNF-α, IL-6, IL-1β cytokines were quantified by ELISA (ELISA MAX™ Deluxe Set, Biolegend) according to the manufacturer's protocol. Soluble BCMA was detected by ELISA (Human BCMA / TNFRSF17 Duo Set ELISA, R&D Systems) according to the manufacturer's protocol.

[0116] Confocal microscopy: RPMI cell lines were transduced with lentiviral particles to overexpress BCMA fused to green fluorescent protein (GFP) and then cocultured with CART cells stained with CellTracker™ Blue CMAC dye (Thermo Fisher Scientific). BCMA was also detected by confocal fluorescence microscopy using a monoclonal anti-TNRSF17 mouse antibody (Sigma-Aldrich) and secondary anti-mouse IgG Alexa 647 (Cell Signaling Technologies). Images were acquired using a Leica SP5 microscope. Lasers 405, 488, and 633 were used for excitation, and Z-stack acquisition images were generated with corresponding filters applied. For in vivo time-lapse imaging, images were acquired every 20 seconds.

[0117] Cytotoxicity: Assays were performed by co-culturing T cells with tumor cells modified with a lentiviral vector (pLV) to overexpress GFP-firefly luciferase (GFP-ffLuc) for 24 to 96 hours at various effector:target ratios ranging from 1:1 to 0.125:1. The percentage of remaining viable GFP+ tumor cells was determined by flow cytometry using the following formula: % viable cells = % GFP+ cells at time point x / % GFP+ cells at 0 h.

[0118] In vivo myeloma mouse model: 8- to 12-week-old NOD / SCID IL-2Rc null (NSG) mice were irradiated with 2G on days -1 and 0, and inoculated with GFP-ffLuc-ARP-1 cells at 1 or 1.5 × 10 cells per mouse, depending on whether the mouse was female or male. 6Mice received either 100 ARP1 cells / mouse. Tumor cells were allowed to grow for 6–14 days, and then either NT T cells or CART cells were inoculated into the mice. Mice were subjected to weekly bioluminescence imaging (BLI). BLI was performed using a Hamamatsu color CDD camera (Hamamatsu Photonics Systems, Bridgewater, NJ) following an IP injection of 100 μL of D-luciferin (20 mg / mL PBS). Signal quantification was performed using ImageJ software.

[0119] result Design and functional characterization of murine CARTBCMA (ARI2m) The design of ARI2m was based on our CART19 (ARI1) gene, which contains CD8a, 4-1BB, and CD3ζ as the hinge, transmembrane domain, costimulatory domain, and signaling domain, respectively. A single-chain variable fragment (scFv) encoding anti-CD19 (A3B1 antibody) was replaced with the sequence of the anti-BCMA antibody J22.9, which has been successfully tested against MM. This entire sequence was cloned into a third-generation pCCL lentiviral vector (Figure 1A). Transfection efficiency of CART cells exceeded 30% and varied between 30% and 60% in all in vitro and in vivo experiments where CART cells were cryopreserved and maintained after thawing (Figure 1B). The efficacy of ARI2 against different MM cell lines (ARP1 and U266) was confirmed after co-culture of T cells and MM cells at an E:T ratio of 1:1 for 4 days, demonstrating efficient elimination of MM cells compared to untransduced T cells (Figure 1C). Furthermore, K562 non-MM cells, which do not express BCMA, were not eliminated by ARI2m cells, demonstrating their specificity (Figure 1C). Furthermore, limiting dilution cytotoxicity assays at E:T ratios ranging from 1:1 to 0.125:1 demonstrated the high efficacy of ARI2m cells in eliminating MM cells at low E:T ratios within 36 hours (Figure 1D), and efficacy continued to increase after 72 hours (Figure 1E). As expected, no toxicity to K562 cells was detected (Figure 1D and Figure 1E).

[0120] Proinflammatory cytokine production by ARI2m cells was further analyzed by co-culture of ARI2 cells with MM cells at different E:T ratios for 24 and 48 hours. High IFNγ production was observed in ARI2m cells at 24 hours and continued to increase after 48 hours (Figure 1F). As expected, some IFNγ production was detected in non-transduced (NT) T cells, as NT T cells are activated by in vitro expansion. Minimal levels of IL-6 were also detected at 24 hours and increased after 48 hours of co-culture. Furthermore, as expected, some IL-6 secretion was observed only in MM cells (Figure 1F). TNFα production decreased at 48 hours compared to 24 hours, indicating that TNFα is produced early in CART activation (Figure 1F). Unfortunately, IL-1β production could not be detected in this in vitro system.

[0121] The in vivo efficacy of ARI2m cells was analyzed in our mouse model, with NSG mice receiving 1 × 10 6 ARP1 MM cells and 6 days later 10 x 10 6 NT cells or 2 x 10 6 10 x 10 containing ARI2m cells 6 Mice were treated with either NT T cells or ARI2m cells (Figure 1G). Disease progression and subsequent bioluminescence analysis showed that ARI2m cells prevented disease progression compared to untreated mice and mice treated with NT T cells (Figure 1H), leading to higher survival rates (Figure 1I). Furthermore, analysis of mouse tissues at the end of the experiment showed the absence of MM cells in the BM and spleen (Figure 1J). T cells were found primarily in the spleen (Figure 1K), whereas CART cells expanded primarily in the BM, and the percentage of CART cells in the total T cell population was higher in the BM than in the spleen (Figure 1K), a highly relevant finding since MM is a BM disease. Furthermore, as an additional marker of MM progression, we analyzed the amount of sBCMA in mouse serum and confirmed high levels of sBCMA in mice treated with NT T cells and a complete absence of sBCMA in mice treated with ARI2m cells (Figure 1L).

[0122] Humanization of ARI2m to ARI2h and comparison of ARI2m versus ARI2h The early loss of CAR cells in patients can lead to non-sustained responses and is associated with xenorecognition by the human immune system of the mouse scFv components within the CAR. Therefore, we performed scFv humanization of ARI2m. Two different variants of ARI2 (Blast and Germline) were generated based on two different prediction algorithms by substituting mouse amino acids (aa) with aa more frequently found in the human code. In the heavy chain, both variants showed the same number of substituted aa compared to the mouse sequence, whereas in the light chain, the Germline variant had fewer substituted aa than the Blast variant (Figure 2A). When the efficacy of both variants was compared in vitro, the Germline variant showed slightly higher anti-MM activity (Figure 2B), and neither variant cleared K562 cells, indicating specificity for MM cells (Figure 2B). Therefore, to compare ARI2m with ARI2h, we selected the Germline variant for all additional assays (see Materials and Methods for the complete sequences of both CARs). Because the in vitro efficacy of ARI2h versus ARI2m was slightly lower (Figure 2B), we performed a long-term cytotoxicity assay in which tumor cells and CAR T cells were co-cultured at a lower E:T ratio (0.125:1). This assay demonstrated that ARI2h, while slower than ARI2m, achieved its goal by eliminating all MM cells (Figure 2C). Furthermore, proliferation assays confirmed the slower proliferation rate of ARI2h cells. Furthermore, while the same in vitro IFNγ production was observed for both CARs in the long-term cytotoxicity assay (Figure 2C), ARI2h versus ARI2m showed lower TNFα and IL-6 production, suggesting a lower toxicity profile for ARI2h.

[0123] ARI2h and ARI2m were compared in vivo in two different models of MM disease (early and advanced). Mice received MM cells on day 0 and 5 × 10 cells on either day 6 or day 14. 6We generated early and advanced disease models by treating mice with 100 CART cells each (Figure 2D and Figure 2E). In the early disease model, both ARI2h and ARI2m equally prevented MM disease progression (Figure 2D and Figure 2F). As expected, mice began to show signs of xenograft-versus-host disease (GVHD) at approximately 50 days, which was more severe in the ARI2m group and resulted in reduced survival in this group (Figure 2G). In the advanced disease model, ARI2m did not result in disease progression, while some disease signals were detected in the ARI2h group at the designated time points (Figure 2E), although not significantly (Figure 2F). Furthermore, mice treated with ARI2h had a longer survival time due to much less toxicity compared to ARI2m (Figure 2G). Analysis of mouse tissues again demonstrated higher T cell homing in the spleen compared to the BM (Figure 2H). In both models, T cell proliferation was higher with ARI2m than with ARI2h, potentially explaining the higher toxicity of ARI2m (Figure 2H). Importantly, in both CARs and both disease models, the majority of T cells in the BM were CART cells (Figure 2H). Finally, serum analysis of mice showed that both CARs secreted large amounts of IFNγ. However, consistent with previous observations that ARI2h had a lower toxicity profile, IFNγ production by ARI2h was slower than that by ARI2m. Thus, in the early model, 3 days after CART infusion, no IFNγ production was detectable in the ARI2h group, whereas 31 days after CART infusion, both ARI2m and ARI2h showed high levels of IFNγ production (Figure 2I). In the advanced model, the same pattern was observed: no IFNγ production by ARI2h after 5 days of CART administration, and at 21 days, high IFNγ production was observed with both CARs but low with ARI2h (Figure 2I).

[0124] These results suggest that ARI2m versus ARI2h activity is faster, which may lead to faster CART cell exhaustion when tumor burden is high. Therefore, a third in vivo experiment using a lower CART cell dose (3 × 10 6) was performed. In this case, bioluminescence imaging showed that when mice received CART cells on day 14, the disease progressed more rapidly compared to the previous progression model. In this model, neither ARI2m nor ARI2h were able to prevent disease progression. However, ARI2h performed better than ARI2m with slower disease progression, suggesting that its slower activity may lead to reduced CART cell exhaustion when tumor burden is high.

[0125] Response to sequential tumor cell challenge and inflammatory response of ARI2m versus ARI2h Previous results have shown that ARI2h exhibits slower in vivo activity and is less toxic with respect to xenograft-versus-host disease (GVHD). Furthermore, we hypothesize that a high tumor burden reduces the efficacy of ARI2m, which may be due to faster exhaustion of ARI2m versus ARI2h. To confirm this hypothesis, we exposed CART cells to repeated in vitro challenges with tumor cells (Figure 3A). These experiments first demonstrated that in vitro expansion of T cells achieved a higher number of CD4 T cells compared with CD8 T cells, a finding observed with both CART and NT T cells. However, exposure to tumor cells enhanced CD8 T cell proliferation, leading to a normalization of the CD4 / CD8 ratio (Figure 3B).

[0126] Furthermore, continuous challenge of CART cells with tumor cells showed increased CD4 and CD8 CART cell proliferation in ARI2h, but this proliferation was not sustained in ARI2m (Figure 3C), suggesting that ARI2m cells were exhausted or dead after continuous challenge with MM cells.

[0127] Furthermore, we compared the pro-inflammatory profiles of both CARs, establishing a model more similar to the cytokine release syndrome (CRS) observed in patients, in which macrophages are the primary producers of IL6, IL1β, and TNFα after activation by CART cells. Therefore, monocytes and T cells isolated from the same individual were differentiated into macrophages and CART cells, respectively, and both were added to in vitro coculture with MM cells to assess cytotoxicity and cytokine production (Figure 3E). The addition of macrophages did not adversely affect CART anti-MM activity (Figure 3F), but induced a slight increase in IFNγ production (Figure 3G), and a significant increase in IL6 and TNFα production (Figure 3G). Furthermore, IL1β, which was undetectable in the absence of macrophages, was detected in large amounts after macrophage addition (Figure 3G). Therefore, in this context, we compared the pro-inflammatory activity of ARI2m and ARI2h over a 2-day period, demonstrating similar IFNγ, IL6, and IL1β production in both CARs (Figure 3G), with lower TNF production in ARI2h (Figure 3H), suggesting lower pro-inflammatory and toxic activity of ARI2h, as previously shown in in vivo studies (Figures 2D-G).

[0128] Efficient clinical production and activity of ARI2m and ARI2h These previous data, starting with ARI2 cells generated at our institution in 2019 and provided to all participating centers, supported the development of a Phase I multicenter clinical trial in MM patients (EudraCT code: 2019-001472-11). Therefore, ARI2m and ARI2h were expanded in our institution's GMP facility following the same protocol used at our institution for a multicenter Phase II clinical trial of B-cell malignancies with ARI1 cells. 17 Both ARI2m (Figure 4A and 4C) and ARI2h (Figure 4B and 4C) were efficiently increased, demonstrating the minimal required dose (150 × 10) to achieve a response in MM patients. 6ARI2h achieved more CART cells than ARI2m cells (>100 CART cells). Comparison of the four clinical expansions showed that comparable numbers of ARI2h and ARI2m cells were achieved at the end of the expansion; despite the higher proportion of ARI2h cells, this difference was not significant, likely due to the higher viral titer of ARI2h (Figure 4C). Furthermore, these CART cells demonstrated high efficacy in eliminating MM cells at low E:T ratios (Figure 4D).

[0129] Soluble BCMA influences ARI2 activity Clinical studies using CART19 in ALL have shown that CARTBCMA (150 × 10 6 Compared with studies using 100x10 CART cell doses, a lower dose (100x10 CART cell doses) was required to achieve a complete response (CR). 6) has been shown to be effective. In this regard, BCMA expression on the surface of MM cells is not stable because it is continuously released into the extracellular environment as sBCMA. Therefore, we hypothesize that sBCMA binds to CARTBCMA cells and transiently inhibits their activity, explaining the high doses required to achieve CR in MM patients. Therefore, we first measured the amount of sBCMA in the serum of patients with monoclonal gammopathy of undetermined significance (MGUS), newly diagnosed MM patients, and at the time of relapse, and confirmed higher amounts of sBCMA in MM patients (Figure 5A). Furthermore, confocal fluorescence microscopy confirmed that BCMA is released from MM cells in vesicles (Figure 5B). Therefore, to confirm that sBCMA can transiently affect CARTBCMA activity, we co-cultured MM cells overexpressing BCMA fused to GFP (MM-BCMA-GFP) with ARI2 cells for 3 hours and performed in vivo time-lapse imaging. We confirmed that vesicularly released sBCMA bound to ARI2 cells and recruited ARI2 cells from their target MM cells (Figure 5C). Furthermore, we also observed that after contact with MM cells, ARI2 cells were able to acquire a portion of BCMA from the surface of MM cells into their membranes, resulting in fratricide between ARI2 cells (Figure 5D).

[0130] To further confirm that sBCMA inhibits CART activity, MM cells were co-cultured with ARI2m cells in the presence of recombinant BCMA protein, with or without anti-BCMA antibodies. The results confirmed that recombinant BCMA protein inhibited ARI2m activity in terms of cytotoxicity and IFNγ production (Figures 5E and 5F). Addition of anti-BCMA partially restored this inhibition in terms of IFNγ production only, but not cytotoxicity (Figures 5E and 5F). Furthermore, sBCMA released from MM cells led to a decrease in BCMA expression in MM cells, an effect mediated by γ-secretase, which directly cleaves soluble BCMA, releasing BCMA, and this can be circumvented by using a γ-secretase inhibitor. Therefore, we first analyzed the effect of a γ-secretase inhibitor (DAPT) on BCMA expression on MM cells and the amount of released sBCMA. As expected, DAPT treatment increased BCMA expression and reduced sBCMA release in MM cells (Figures 5G and 5H). This DAPT-associated increase in BCMA expression was also detected after coculture of MM cells with NT T cells (Figure 5G). Addition of DAPT to ARI2 / MM cell cocultures also reduced the amount of sBCMA. However, due to the high in vitro activity of ARI2, which eliminated MM cells, the effect on BCMA expression was barely detectable in standard in vitro cocultures (Figures 5G and 5H). To confirm the possible role of DAPT in enhancing ARI2 activity by reducing the amount of sBCMA, we analyzed its effect in an in vitro setting involving the sustained release of sBCMA by MM cells not exposed to ARI2 cells. Therefore, we performed the same in vitro experiment in parallel in transwell plates, where MM cells not exposed to ARI2 cells could sustainably release sBCMA (Figure 5I). In this situation, sustained release of sBCMA by untouched MM cells reduced ARI2 activity ( Figure 5J ), confirming that the addition of DAPT partially circumvented the adverse effects of sBCMA released from untouched MM cells ( Figure 5J ).

Claims

1. A chimeric antigen receptor (CAR) comprising an extracellular domain comprising a BCMA targeting moiety, a transmembrane domain, and an intracellular signaling domain, the BCMA targeting moiety comprises a VH domain and a VL domain, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 1; The VL domain comprises the amino acid sequence of SEQ ID NO:

2. Chimeric antigen receptor.

2. The chimeric antigen receptor of claim 1 , wherein the VH domain and VL domain consist of the amino acid sequences of SEQ ID NOs: 1 and 2, respectively.

3. The chimeric antigen receptor of claim 2, wherein the BCMA targeting moiety comprises the amino acid sequence of SEQ ID NO:

3.

4. 3. The chimeric antigen receptor of claim 2, wherein the BCMA targeting moiety is a single-chain variable fragment (scFv) BCMA targeting moiety and comprises a VL domain and a VH domain, wherein the VH domain and the VL domain comprise the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

5. a. a single chain variable fragment (scFv) BCMA targeting moiety comprising a VL domain and a VH domain, wherein the VH domain and the VL domain comprise the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively; b) a transmembrane domain connected to a hinge domain comprising the amino acid sequence of SEQ ID NO: 9; and c) a costimulatory signaling domain comprising the amino acid sequence of SEQ ID NO:

11. d. an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 10; The chimeric antigen receptor (CAR) of claim 4, comprising:

6. The chimeric antigen receptor (CAR) according to claim 5, wherein the CAR consists only of the amino acid sequence of SEQ ID NO:

13.

7. A nucleic acid encoding the CAR according to claim 5 or 6.

8. A cell comprising the nucleic acid of claim 7.

9. The cell of claim 8 , wherein the cell is a T cell.

10. A pharmaceutical composition comprising a plurality of cells according to claim 8 and a pharmaceutically acceptable carrier or diluent.

11. The cell of claim 9 or the pharmaceutical composition of claim 10, which is a pharmaceutical.

12. 11. The cell of claim 8 or the pharmaceutical composition of claim 10 for use in a method for treating multiple myeloma, the method comprising administering the cell or composition to a patient in need thereof.

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