Compositions and methods for treating diseases with chimeric antigen receptors of B cell maturation antigen (BCMA)

Abstract

The present disclosure relates to compositions and methods for treating diseases using chimeric antigen recipient cells and / or antigen binding domains that target BCMA.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 486,391, filed on February 22, 2023, the disclosure of which is incorporated by reference in its entirety.

[0003] References to sequence listings

[0004] This application is filed electronically and includes a sequence listing filed electronically. The sequence listing is named "23-0105-WO_SequenceListing.xml," was created on February 19, 2024, and is 90,152 bytes in size. The sequence listing contained in this .xml file is part of the specification and is incorporated herein by reference in its entirety. Background Art Technical Field

[0005] The present disclosure relates to the use of chimeric antigen receptor cells targeting BCMA to treat cancer and other diseases. Technical Background

[0007] B cell maturation antigen (BCMA) is a member of the tumor necrosis receptor family (TNFR) expressed on cells of the B cell lineage (Laabi et al., Nucleic Acids Research, 22(7): 1147-1154 (1994)). BCMA expression is highest on terminally differentiated B cells, and BCMA is involved in mediating the survival of plasma cells to maintain long-term humoral immunity. BCMA expression has been linked to many cancers, autoimmune disorders, and infectious diseases. Specifically, several researchers have detected BCMA RNA ubiquitously in multiple myeloma cells, and BCMA protein has been detected on the surface of plasma cells from multiple myeloma patients (see, e.g., Novak et al., Blood, 103(2):689-694 (2004); Neri et al., Clinical Cancer Research, 73(19):5903-5909 (2007); Bellucci et al., Blood, 105(10):3945-3950 (2005); and Moreaux et al., Blood, 703(8):3148-3157 (2004)). Consequently, BCMA has been investigated as a possible therapeutic target for multiple myeloma and other diseases.

[0008] Chimeric antigen receptor (CAR)-based cell therapy is a specific form of cell-based immunotherapy that uses engineered immune cells to fight disease. In recent years, such cell therapy has had a transformative effect on patients with hematological malignancies, and in 2017, FDA approved CAR-based therapy for the first time (Larson and Maus, Nat Rev Cancer 21,145–161 (2021); Yu et al., Nature Reviews Drug Discovery 19,583-584 (2020)). In addition, over the past few years, the number of clinical trials studying adoptive cell therapy has grown rapidly.

[0009] Although cell therapy has great potential for curing patients, many factors limit the widespread development and administration of these drugs. Most cell therapies are currently produced in an autologous manner and are accompanied by variable cell product quality, cytokine release syndrome and other toxicities, extended manufacturing time, complex supply chain logistics, high costs, and a limited period in which these therapies can be genetically modified to enhance their efficacy (Larson and Maus, Nat Rev Cancer 21, 145–161 (2021)).

[0010] Specifically, autologous cell therapy with primary human immune cells (e.g., T cells and NK cells) has limited proliferation potential. This significantly limits the window for isolating, amplifying, manufacturing, and gene editing cells while maintaining functionality when reinfused into the patient. Extending the lifespan of these cells by deleting cell cycle-related genes broadens the manufacturing window, allowing for several manipulations that can mitigate cytokine release syndrome and other toxicities, enabling armoring of cells, and most critically, allowing for the generation of large cell libraries of allogeneic edited cells for widespread distribution.

[0011] The BCMA CAR-T cell therapy for multiple myeloma (MM) approved by the current FDA is an autologous product with a transformative effect for all other available treatment-refractory patients, and it is reported that the total response rate is as high as 95%. However, the actual challenge of delivering autologous CAR-T cell products severely limits the recruitment of qualified patients. Moreover, the toxicity associated with BCMA CAR-T cell therapy and the therapy based on BCMA T cell adapters also exceeds those>90% for treatment. Therefore, it is necessary to solve these manufacturing limitations based on the alternative therapy of BCMA CAR cells while minimizing the relevant toxicity. Summary of the Invention

[0012] This disclosure describes compositions and methods for treating cancer and other diseases using CAR-based cell therapies.

[0013] As described below, in a first aspect, the present disclosure provides an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises:

[0014] (a) an antigen-binding domain specific for B-cell maturation antigen (BCMA);

[0015] (b) a transmembrane domain; and

[0016] (c) one or more intracellular domains.

[0017] In some embodiments of the isolated nucleic acid sequence, the antigen binding domain comprises an antibody or antigen binding fragment thereof, Fab, Fab', F(ab')2, Fd, Fv, single chain variable fragment (scFv), single chain antibody, VHH, vNAR, nano antibody (single domain antibody) or any combination thereof. In some embodiments, the antigen binding domain is a single chain variable fragment (scFv). In some embodiments, the antigen binding domain is a scFv comprising an amino acid sequence selected from SEQ ID NO: 9, 36 and 90. In one embodiment of the isolated nucleic acid sequence, the antigen binding domain is a scFv comprising an amino acid sequence of SEQ ID NO: 9.

[0018] In some embodiments of the isolated nucleic acid sequence, the transmembrane domain comprises a transmembrane domain selected from the transmembrane domain of CD4, CD8α, or CD28. In one embodiment, the transmembrane domain comprises the CD28 transmembrane domain.

[0019] In some embodiments of the nucleic acid sequence of separation, the one or more intracellular domains include a costimulatory domain or a portion thereof. In some embodiments, the costimulatory domain includes one or more of the following: CD3z, 4-1BB, CD2, CD27, CD28, OX-40, ICOS, IL-2R β, GITR, MyD88 / CD40a costimulatory domain and / or their variants. In one embodiment of the nucleic acid sequence of separation, the intracellular domain includes CD3z costimulatory domain and CD28 costimulatory domain. In another embodiment of the nucleic acid sequence of separation, the intracellular domain includes CD3z costimulatory domain and 4-1BB costimulatory domain. In another embodiment of the nucleic acid sequence of separation, the intracellular domain includes CD3z costimulatory domain, CD28 costimulatory domain and 4-1BB costimulatory domain.

[0020] In some embodiments of the isolated nucleic acid sequence, the CAR further comprises a hinge / spacer domain, optionally wherein the hinge / spacer domain is located between the antigen binding domain and the transmembrane domain. In some embodiments, the hinge / spacer domain includes an IgG1 hinge domain or a variant thereof, an IgG2 hinge domain or a variant thereof, an IgG3 hinge domain or a variant thereof, an IgG4 hinge domain or a variant thereof, an IgG4P domain, a CD8 hinge domain or a variant thereof, or a CD28 hinge domain or a variant thereof. In one embodiment, the hinge / spacer domain is an IgG4 hinge / spacer domain or a variant thereof, optionally comprising an IgG4P hinge / spacer domain mutated by S241P.

[0021] In one embodiment of the isolated nucleic acid sequence, the nucleic acid sequence encodes a CAR having the amino acid sequence as shown in SEQ ID NO:96.

[0022] In another embodiment of the isolated nucleic acid sequence, the nucleic acid sequence encodes a CAR having the amino acid sequence as shown in SEQ ID NO:97.

[0023] In yet another embodiment of the isolated nucleic acid sequence, the nucleic acid sequence encodes a CAR having the amino acid sequence as shown in SEQ ID NO:98.

[0024] In another aspect, the present disclosure provides an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL);

[0025] wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 29, and 83; and a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 30, and 84;

[0026] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 31, and 85; and

[0027] wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 33, and 87; and a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 34, and 88;

[0028] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 35, and 89.

[0029] In some anti-BCMACAR embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 28, and 82. In some anti-BCMACAR embodiments, the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 32, and 86.

[0030] In one aspect, the present disclosure provides an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL);

[0031] wherein the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and

[0032] wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0033] In some embodiments of the anti-BCMACAR disclosed herein, the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO:5.

[0034] In some embodiments of anti-BCMACAR disclosed herein, the CAR includes a membrane spaning domain and one or more intracellular domains. In some embodiments, the membrane spaning domain includes a membrane spaning domain selected from the membrane spaning domain of CD4, CD8 α or CD28. In some embodiments, the membrane spaning domain includes a CD28 membrane spaning domain.

[0035] In some embodiments of anti-BCMACAR disclosed herein, the one or more intracellular domains include costimulatory domains or parts thereof.In some embodiments, the costimulatory domains include one or more of the following: CD3z, 4-1BB, CD2, CD27, CD28, OX-40, ICOS, IL-2R β, GITR, MyD88 / CD40a costimulatory domains and / or their variants.

[0036] In one embodiment of the anti-BCMACAR disclosed herein, the intracellular domain comprises a CD3z costimulatory domain and a CD28 costimulatory domain.

[0037] In another embodiment of the anti-BCMACAR disclosed herein, the intracellular domain comprises a CD3z costimulatory domain and a 4-1BB costimulatory domain.

[0038] In yet another embodiment of the anti-BCMACAR disclosed herein, the intracellular domain comprises a CD3z costimulatory domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain.

[0039] In some embodiments of anti-BCMACAR disclosed herein, the CAR also includes hinge / spacer domain, optionally, wherein the hinge / spacer domain is between the antigen-binding domain and the membrane spaning domain.In some embodiments, the hinge / spacer domain includes IgG1 hinge domain or its variant, IgG2 hinge domain or its variant, IgG3 hinge domain or its variant, IgG4 hinge domain or its variant, IgG4P domain, CD8a hinge domain or its variant or CD28 hinge domain or its variant.In one embodiment, the hinge / spacer domain is IgG4 hinge / spacer or its variant, optionally including the IgG4P hinge / spacer of S241P mutation.

[0040] In one embodiment of the anti-BCMACAR disclosed herein, the CAR has the amino acid sequence as shown in SEQ ID NO: 96.

[0041] In another embodiment of the anti-BCMACAR disclosed herein, the CAR has the amino acid sequence as shown in SEQ ID NO: 97.

[0042] In yet another embodiment of the anti-BCMACAR disclosed herein, the CAR has the amino acid sequence as shown in SEQ ID NO: 98.

[0043] In another aspect, the present disclosure provides a vector comprising an isolated nucleic acid sequence as disclosed herein or encoding a chimeric antigen receptor as disclosed herein, optionally wherein the vector is a virus, a lentivirus, an adenovirus, a retrovirus, an adeno-associated virus (AAV), a transposon, a DNA vector, mRNA, a lipid nanoparticle (LNP) or a CRISPR-Cas system.

[0044] In yet another aspect, the present disclosure provides a cell comprising a vector as disclosed herein.

[0045] In another aspect, the present disclosure provides a cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) as disclosed herein, further comprising reduced expression or knockout of one or more endogenous regulatory factors.

[0046] In some embodiments of the cells as disclosed herein, the one or more endogenous regulatory factors are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP).

[0047] In some embodiments of the cells as disclosed herein, the cells have reduced expression or knockout of CDKN2A, CDKN2B, and MTAP.

[0048] In some embodiments of the cells as disclosed herein, the cells do not express phosphatase and tensin homolog (PTEN).

[0049] In some embodiments of the cells as disclosed herein, the cells further comprise a transgene encoding B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0050] In some embodiments of the cells as disclosed herein, the cells do not express one or more endogenous immune-related genes. In some embodiments, the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

[0051] In some embodiments of the cells as disclosed herein, the cells do not express cluster of differentiation 38 (CD38).

[0052] In one aspect, the present disclosure provides a cell comprising a BCMA-specific antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL);

[0053] wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 31, and 85; and

[0054] wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 34, and 88; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 35, and 89.

[0055] In some embodiments of the cells as disclosed herein, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 28, and 82. In some embodiments of the cells as disclosed herein, the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 32, and 86.

[0056] In another aspect, the present disclosure provides a cell comprising a BCMA-specific antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL);

[0057] wherein the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and

[0058] wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0059] In some embodiments of the cells as disclosed herein, the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO:5.

[0060] In some embodiments of the cells as disclosed herein, the cells further comprise reduced expression or knockout of one or more endogenous regulatory factors. In some embodiments, the one or more endogenous regulatory factors are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP). In some embodiments, the cells have reduced expression or knockout of CDKN2A, CDKN2B, and MTAP.

[0061] In some embodiments of the cells as disclosed herein, the cells do not express phosphatase and tensin homolog (PTEN).

[0062] In some embodiments of the cells as disclosed herein, the cells further comprise a transgene encoding B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0063] In some embodiments of the cells as disclosed herein, the cells do not express one or more endogenous immune-related genes. In some embodiments, the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

[0064] In some embodiments of the cells as disclosed herein, the cells do not express cluster of differentiation 38 (CD38).

[0065] In another aspect, the present disclosure provides a cell comprising a BCMA-specific antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL);

[0066] wherein the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and

[0067] wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and

[0068] wherein the cells comprise reduced expression or knockout of CDKN2A, CDKN2B, MTAP, B2M, TRAC, and CD38.

[0069] In one embodiment of the cell as disclosed herein, the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO:5.

[0070] In one embodiment of the cell as disclosed herein, the BCMA-specific antigen binding domain comprises the amino acid sequence as shown in SEQ ID NO:96.

[0071] In some embodiments of the cells as disclosed herein, the cells are selected from T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes, regulatory T cells, CD8+ T cells, CD4+ T cells, γδ T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells, and / or combinations thereof.

[0072] In one aspect, the present disclosure provides a method for treating a disease, the method comprising: administering to a subject in need thereof an effective amount of cells comprising an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL),

[0073] wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 29, and 83; and a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 30, and 84;

[0074] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 31, and 85; and

[0075] wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 33, and 87; and a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 34, and 88;

[0076] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 35, and 89.

[0077] In some embodiments of the methods as disclosed herein, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 28, and 82. In some embodiments of the methods as disclosed herein, the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 32, and 86.

[0078] In another aspect, the present disclosure provides a method for treating a disease, comprising: administering to a subject in need thereof an effective amount of cells comprising an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL),

[0079] wherein the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and

[0080] wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0081] In some embodiments of the methods as disclosed herein, the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO:5.

[0082] In some embodiments of the methods as disclosed herein, the method further comprises inhibiting cancer growth, inducing cancer regression, and / or prolonging the survival of the subject.

[0083] In some embodiments of the methods disclosed herein, the cell further comprises reduced expression or knockout of one or more endogenous regulatory factors. In some embodiments, the one or more endogenous regulatory factors are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP). In one embodiment, the cell has reduced expression or knockout of CDKN2A, CDKN2B, and MTAP.

[0084] In some embodiments of the methods as disclosed herein, the cell does not express phosphatase and tensin homolog (PTEN).

[0085] Methods In some embodiments of the methods disclosed herein, the cell further comprises a transgene encoding B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0086] In some embodiments of the methods disclosed herein, the cell does not express one or more endogenous immune-related genes. In some embodiments, the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

[0087] In some embodiments of the methods as disclosed herein, the cells do not express cluster of differentiation 38 (CD38).

[0088] In some embodiments of the methods as disclosed herein, the cell is an autologous cell.

[0089] In some embodiments of the methods as disclosed herein, the cell is an allogeneic cell.

[0090] In some embodiments of the methods as disclosed herein, the cell is selected from T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), tumor-infiltrating lymphocytes, regulatory T cells, CD8+ T cells, CD4+ T cells, γδ T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells, and / or combinations thereof.

[0091] In some embodiments of the methods as disclosed herein, the disease is a cancer selected from multiple myeloma (MM), chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). In one embodiment, the cancer is multiple myeloma.

[0092] In some embodiments of the methods as disclosed herein, the disease is an autoimmune disease. In one embodiment, the autoimmune disease is lupus.

[0093] In one aspect, the present disclosure provides a pharmaceutical composition comprising an isolated nucleic acid as disclosed herein, an anti-BCMACAR as disclosed herein, a vector as disclosed herein, or a cell as disclosed herein, and a pharmaceutically acceptable excipient.

[0094] In some embodiments of the method as disclosed herein, the method includes administering to the subject an isolated nucleic acid as disclosed herein, an anti-BCMACAR as described in any one of the claims disclosed herein, a carrier as disclosed herein, a cell as disclosed herein, or a pharmaceutical composition as disclosed herein. In some embodiments of the method as disclosed herein, the disease is cancer or an autoimmune disease. In some embodiments, the cancer is selected from multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphoblastic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). In one embodiment, the cancer is multiple myeloma. In one embodiment, the autoimmune disease is lupus.

[0095] In one aspect, the present disclosure provides use of an isolated nucleic acid as disclosed herein, an anti-BCMACAR as disclosed herein, a vector as disclosed herein, a cell as disclosed herein, or a pharmaceutical composition as disclosed herein for treating a disease in a subject in need thereof.

[0096] In some embodiments, the disease is cancer or an autoimmune disease. In some embodiments, the cancer is selected from multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphoblastic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). In one embodiment, the cancer is multiple myeloma. In one embodiment, the autoimmune disease is lupus.

[0097] In one aspect, the present disclosure provides the use of an engineered cell for the manufacture of a medicament for treating a disease in a patient, wherein the engineered cell comprises an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL),

[0098] wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 29, and 83; and a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 30, and 84;

[0099] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 31, and 85; and

[0100] wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 33, and 87; and a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 34, and 88;

[0101] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 35, and 89.

[0102] In some embodiments of the uses as disclosed herein, the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0103] In some embodiments of the uses as disclosed herein, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 28, and 82. In some embodiments of the uses as disclosed herein, the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 32, and 86.

[0104] In some embodiments of the uses as disclosed herein, the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO:5.

[0105] In one embodiment of the use as disclosed herein, the CAR has the amino acid sequence as shown in SEQ ID NO: 96.

[0106] In one embodiment of the use as disclosed herein, the CAR has the amino acid sequence as shown in SEQ ID NO: 97.

[0107] In one embodiment of the use as disclosed herein, the CAR has the amino acid sequence as shown in SEQ ID NO: 98.

[0108] In some embodiments of the uses as disclosed herein, the uses further comprise inhibiting cancer growth, inducing cancer regression, and / or prolonging the survival of the subject.

[0109] In some embodiments of the uses as disclosed herein, the engineered cells further comprise reduced expression or knockout of one or more endogenous regulatory factors. In some embodiments of the uses as disclosed herein, the one or more endogenous regulatory factors are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP). In one embodiment, the engineered cells have reduced expression or knockout of CDKN2A, CDKN2B, and MTAP.

[0110] In some embodiments of the uses as disclosed herein, the engineered cell does not express phosphatase and tensin homolog (PTEN).

[0111] In some embodiments of the uses as disclosed herein, the engineered cell further comprises a transgene encoding B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0112] In some embodiments of the uses as disclosed herein, the engineered cells do not express one or more endogenous immune-related genes. In some embodiments of the uses as disclosed herein, the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

[0113] In some embodiments of the uses as disclosed herein, the engineered cells do not express cluster of differentiation 38 (CD38).

[0114] In some embodiments of the uses as disclosed herein, the engineered cells are autologous cells.

[0115] In some embodiments of the uses as disclosed herein, the engineered cell is an allogeneic cell.

[0116] In some embodiments of the uses as disclosed herein, the engineered cells are selected from T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes, regulatory T cells, CD8+ T cells, CD4+ T cells, γδ T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells and / or combinations thereof.

[0117] In some embodiments of the uses as disclosed herein, the disease is a cancer selected from multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphoblastic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). In some embodiments of the uses as disclosed herein, the cancer is multiple myeloma. In some embodiments of the uses as disclosed herein, the disease is an autoimmune disease. In some embodiments of the uses as disclosed herein, the autoimmune disease is lupus.

[0118] In one aspect, the present disclosure provides an engineered cell for use in the manufacture of a medicament for treating a disease in a patient, wherein the engineered cell comprises an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL),

[0119] wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 29, and 83; and a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 30, and 84;

[0120] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 31, and 85; and

[0121] wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 33, and 87; and a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 34, and 88;

[0122] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 35, and 89.

[0123] In some embodiments of the engineered cells as disclosed herein, the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0124] In some embodiments of the engineered cells as disclosed herein, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 28, and 82. In some embodiments of the engineered cells as disclosed herein, the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 32, and 86.

[0125] In one embodiment of the engineered cell as disclosed herein, the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO:5.

[0126] In one embodiment of the engineered cell as disclosed herein, the CAR has the amino acid sequence as shown in SEQ ID NO: 96.

[0127] In one embodiment of the engineered cell as disclosed herein, the CAR has the amino acid sequence as shown in SEQ ID NO: 97.

[0128] In one embodiment of the engineered cell as disclosed herein, the CAR has the amino acid sequence as shown in SEQ ID NO: 98.

[0129] In some embodiments of the engineered cells as disclosed herein, the cells further comprise inhibiting cancer growth, inducing cancer regression, and / or prolonging survival of a subject.

[0130] In some embodiments of the engineered cells as disclosed herein, the engineered cells further comprise reduced expression or knockout of one or more endogenous regulatory factors. In some embodiments of the engineered cells as disclosed herein, the one or more endogenous regulatory factors are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP). In one embodiment, the engineered cells have reduced expression or knockout of CDKN2A, CDKN2B, and MTAP.

[0131] In some embodiments of the engineered cells as disclosed herein, the engineered cells do not express phosphatase and tensin homolog (PTEN).

[0132] In some embodiments of the engineered cells as disclosed herein, the engineered cells further comprise a transgene encoding B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0133] In some embodiments of the engineered cells as disclosed herein, the engineered cells do not express one or more endogenous immune-related genes. In some embodiments of the engineered cells as disclosed herein, the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

[0134] In some embodiments of the engineered cells as disclosed herein, the engineered cells do not express cluster of differentiation 38 (CD38).

[0135] In some embodiments of the engineered cells as disclosed herein, the engineered cells are autologous cells.

[0136] In some embodiments of the engineered cells as disclosed herein, the engineered cells are allogeneic cells.

[0137] In some embodiments of the engineered cells as disclosed herein, the engineered cells are selected from T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes, regulatory T cells, CD8+ T cells, CD4+ T cells, γδ T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells, and / or combinations thereof.

[0138] In some embodiments of the engineered cells as disclosed herein, the disease is a cancer selected from multiple myeloma (MM), chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). In one embodiment, the cancer is multiple myeloma. In some embodiments, the disease is an autoimmune disease. In one embodiment, the autoimmune disease is lupus.

[0139] These and other features and advantages of the present disclosure will be more fully understood from the following detailed description taken in conjunction with the appended claims.It is noted that the scope of the claims is defined by the recitation therein rather than by the specific discussion of the features and advantages set forth in this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0140] The accompanying drawings are included to provide a further understanding of the methods and compositions of the present disclosure. The drawings illustrate one or more embodiments of the present disclosure, and together with the description serve to explain the principles and operations of the disclosure.

[0141] Figure 1 Surface plasmon resonance binding of anti-BCMA scFv-Fc to soluble BCMA protein is shown. Average values ​​of ka, kd, and KD are reported.

[0142] Figure 2 Membrane proteome array showing the specificity of anti-BCMA scFv-Fc for BCMA. Anti-BCMA scFv-Fc was used in a cell-based membrane proteome array of >6000 membrane proteins to examine off-target binding. Figure 2 The scFv exemplified for anti-BCMACAR-T is specific for BCMA.

[0143] Figure 3 Anti-BCMACAR staining of primary T cells is shown. Figure 3 Demonstrates successful expression of CAR on the surface of T cells. Staining is representative of 5 different donors.

[0144] Figure 4 BCMACAR-T binding curves are shown. Figure 4 The binding properties of each anti-BCMA CAR-T clone to soluble BCMA antigen are exemplified.

[0145] Figure 5 Anti-BCMACAR-T amplification is shown. Figure 5 Illustrated are the differences in growth kinetics and expansion length of anti-BCMACAR-T clones in primary T cells using IL-2-containing medium.

[0146] Figure 6A and Figure 6B In vitro cytotoxicity against multiple myeloma cell lines is shown. Figure 6A and Figure 6B The cytotoxicity of BCMACAR-T clones across the spectrum of multiple myeloma (MM) cell lineages is demonstrated and compared to two clinical benchmarks. Figure 6A Figure 2 is a heatmap of the average cytotoxicity percentages of anti-BCMACAR-T clones from four donors across different MM cell lines at an E:T ratio of 1:2. Figure 6B Shown are bar graphs showing the mean percentage cytotoxicity and standard deviation from four donors.

[0147] Figure 7 Effector cytokine production by anti-BCMACAR-T clones is shown. Figure 7Representative cytokine production by anti-BCMA CAR-T cells when challenged with the huh7 engineered cell line expressing BCMA is illustrated.

[0148] Figure 8 Resistance to soluble BCMA is shown. Figure 8 Illustrated are the differences in percentage of cell lysis observed 40 hours after co-culture with BCMA-expressing huh7 cells in the presence or absence of soluble BCMA protein (sBCMA).

[0149] Figure 9 Effector cytokine production in the presence of soluble BCMA is shown. Figure 9 The effect of soluble BCMA on CAR-T production of IFNγ and IL-2 effector cytokines is exemplified.

[0150] Figure 10A and Figure 10B Shown are the persistence and expansion of CAR-T cells after repeated antigen stimulation in the presence or absence of soluble BCMA. Figure 10A Schematic diagram of the expansion of anti-BCMACAR-T cells after repeated antigen stimulation (JJN3 co-culture) over the course of 12 days. During this period, the percentage of tumor cell control was used as a measure of T cell function and persistence. Figure 10B Similarly, antigen-dependent CAR-T cell expansion in the presence of soluble antigen (JJN3 co-culture) is demonstrated. The percentage of target cell lysis highlights the impact of soluble BCMA protein on the function and persistence of anti-BCMA CAR-T clones.

[0151] Figure 11 In vivo tumor control of anti-BCMACAR-T clones and clinical benchmarks is shown. Figure 11 Demonstrates in vivo CAR-T functional efficacy at high and low doses of CAR-T cells compared to clinical benchmarks in a disseminated MM model (MM1.S).

[0152] Figure 12 Serum cytokine levels of the in vivo tumor challenge model are shown. Figure 12 We demonstrated CAR-T function via effector cytokine production at two doses following in vivo MM1.S tumor challenge.

[0153] Figure 13 BCMACAR-T REX BCMA binding curve. Figure 13 Illustrated in CAR-T REX BCMA binding properties of the 7A8.11 CAR when expressed in-frame and benchmarked against primary CAR-T and clinical products.

[0154] Figure 14 In vitro cytotoxicity against multiple myeloma cell lines is shown. Figure 14 BCMACAR-T across the multiple myeloma cell lineage spectrum is exemplified REX Cytotoxicity was assessed and compared with primary T cells and two clinical benchmarks.

[0155] Figure 15 Compared with primary T cells, BCMACAR-T REX Effector cytokine profile of cells. Figure 15 exemplifies the expression of BCMACAR-T when encountering the multiple myeloma target cell line JJN3 at an E:T ratio of 1:1. REX Representative effector cytokine production by cells. BCMACAR-T REX They demonstrated 2%-15% effector cytokine production compared to primary T cells with the same CAR and less than 5% of clinical comparators, suggesting a potentially safer cytokine profile.

[0156] Figure 16 Compared with clinical benchmarks, BCMACAR-T REX In vivo tumor control of MM1.S cells. Figure 16 BCMACAR-T REX The cells demonstrated comparable or improved tumor clearance kinetics relative to primary T cells expressing the same CAR and clinical benchmarks.

[0157] Figures 17A-17C Daratumumab (Dara) treatment protects against BCMA-T REX The number of cells and the remaining T REX After 5 h, the co-cultures were evaluated by flow cytometry to quantify NK cells and anti-BCMA-T cells. REX Cell number, demonstrating Dara-mediated anti-BCMA-T REX Protection of cell number ( Figure 17A Cells were further progressed by two rounds of serial killing of JJN3 target cells at the specified E:T ratios, where the percentage of tumor cell lysis was measured by luciferase assay ( Figure 17B ); or further progression of cells by a single round of cell killing with the SNU-182 adherent cell line ectopically expressing BCMA, where tumor cell killing was assessed by Xcelligence ( Figure 17C ).

[0158] Figure 18 showed that even when administered immediately after cryopreservation, BCMACAR-T REXThe cells also effectively cleared tumors from the bone marrow. REX The cells showed comparable tumor clearance and mouse bone marrow recovery to other groups.

[0159] Figure 19 It was shown that when administered immediately after cryopreservation, BCMACAR-T REX NSG mice were inoculated with MM1S-luciferase tumor cells and 3 days later, primary BCMACAR-T cells or BCMACAR-T cells from two donors were administered at the indicated doses immediately after cryopreservation. REX cells (7A8.11).

[0160] Figure 20 Figure 2 shows the expression of BCMA and the characterization of B cell subsets in blood from healthy donors and SLE donors. Healthy donor PBMC and SLE donor PBMC were isolated from fresh whole blood and stained with antibodies. The cells shown are lymphocytes defined by FSC / SSC, viable cells, negative for dump gate, CD20+ and CD19+ / -. DN2 cells (IgD-, CD27-), memory B cells (CD27+IgD-), naive B cells (IgD+CD27-), NCSM (non-class switched memory cells, IgD+CD27+), plasma cells (CD19 low / negative CD138+CD38 low) and plasmablasts (CD19 低 / 阴性 CD138-CD38+CD27+).

[0161] Figure 21 BCMACAR-T cell consumption healthy human plasma cell is shown to the degree similar to MM1S (BCMA+) tumor cell.By primary plasmablast, plasma cell and multiple myeloma MM1S cell and BCMACAR-T cell or the T cell of not transducing with 1:1,1:2,1:4 effect: target ratio coculture 24 hours, then dye and pass through flow cytometry assessment.T cell is gated as live, exclude gate positive.Plasma cell is gated as live, exclude gate negative, CD19+, CD20 is low, CD38+, CD27+ (plasma cell+plasmablast) by pre-gating.

[0162] Figure 22Figure 5 shows the dose-dependent consumption (E:T) of BCMA CART cells shown in vitro differentiation plasmablasts from SLE donors and healthy donors. From freezing healthy donors and SLE patients, separate the original B cells, and utilize the proprietary mixture plate inoculation of cytokines to drive B cell differentiation. Differentiation after 5 days, by the B cells of differentiation and BCMA CART cells or the T cells of non-transduction with 1:1,1:2,1:4,1:8,1:16 and 1:32 effect: target ratio co-culture 24 hours, then dye and pass through flow cytometry assessment. Consumption percentage is calculated by 1-(plasmablast percentage in experimental well / average plasmablast percentage in only stimulation well)*100.

[0163] Figure 23 CAR-T cells targeting BCMA are shown to reduce BCMA+ cells in an allogeneic model of graft-versus-host disease. Whole blood was collected for FACS analysis at the end of the study on the 12th day after transplantation. The upper figure shows a representative FACS histogram of spleen CD27+ memory B cells expressing BCMA. The lower figure shows the BCMA+ percentage of CD27+ memory B cells in spleen, blood and bone marrow. The mean + / - standard error of each treatment group is shown. In order to determine statistically significant differences, one-way ANOVA with Tukey's multiple comparison test was performed; *P<0.05, ****P<0.0001.

[0164] Figure 24 Serum cytokines / cytolytic granzymes are shown to increase with BCMACAR-T treatment. Serum collected from mice treated with PBS, UTT, and BCMACAR-T was collected on day 12 after transplantation, and serum levels of IFN-γ, GM-CSF, TNF-α, IL-2, granzyme A, and granzyme B were assessed via ELISA. Mean values ​​+ / - standard errors of each cytokine from each treatment group are shown. To determine statistically significant differences, one-way ANOVA and Multiple comparison test; *P<0.05, **P<0.01.

[0165] It will be appreciated by those skilled in the art that the elements in the accompanying drawings are illustrated for simplicity and clarity and are not necessarily drawn to scale. For example, the sizes of some elements in the drawings may be magnified relative to other elements to help improve understanding of the embodiments of the present disclosure. DETAILED DESCRIPTION

[0166] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this disclosure pertains. The following references provide general definitions of many of the terms used in this disclosure to those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed. 1988); The Glossary of Genetics, 5th ed., R. Rieger et al. (eds.), Springer-Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise indicated, the following terms have the meanings assigned to them below.

[0167] As used herein, the terms "comprise" and "include" and variations thereof (e.g., "comprises," "comprising," "includes," and "including") will be understood to indicate the inclusion of stated components, features, elements, or steps, or groups of components, features, elements, or steps, but not the exclusion of any other components, features, elements, or steps, or groups of components, features, elements, or steps. Any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms while retaining their ordinary meaning.

[0168] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0169] The percentages disclosed herein may vary in amount from the disclosed values ​​by ±10%, 20%, or 30% and remain within the range contemplated for disclosure.

[0170] Unless otherwise indicated or otherwise apparent from the context and understanding of one of ordinary skill in the art, values ​​expressed herein as ranges can be assumed to represent any specific value or sub-range within the stated range in different embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0171] As used herein, ranges and amounts can be expressed as "about" a particular value or range. The term "about" also includes an exact amount. For example, "about 5%" means "about 5%" and also means "5%." The term "about" can also refer to ±10% of a given value or range of values. Thus, about 5% also means, for example, 4.5%-5.5%. In addition, "about" or "substantially comprising" can mean a range of up to ±10%. Furthermore, particularly with respect to biological systems or processes, these terms can mean a value of up to one order of magnitude or up to 5 times. When a specific value or composition is provided in the present application and claims, unless otherwise stated, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value or composition. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0172] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the recited range, and where appropriate, fractions thereof (such as tenths and hundredths of integers).

[0173] Units, prefixes and symbols are expressed in their international system of units (SI) recognized form. Numerical ranges include numerical values ​​that limit the range. Unless otherwise indicated, nucleotide sequences are written from left to right in a 5' to 3' orientation. Amino acid sequences are written from left to right in an amino to carboxyl orientation. The headings provided herein are not limitations on the various aspects of the present disclosure, which can be obtained by reference to the entire specification. Therefore, the terms defined immediately below are more fully defined by reference to the entire description.

[0174] As used herein, the terms "or" and "and / or" can describe multiple components that are in combination with each other or that are exclusive of each other. For example, "x, y, and / or z" can mean "x alone," "y alone," "z alone," "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z."

[0175] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other terms used to refer to one or more chains of two or more amino acids are included in the definition of "polypeptide," and the term "polypeptide" may be used instead of or interchangeably with any of these terms.

[0176] As used herein, "protein" may refer to a single polypeptide, ie, a single amino acid chain as defined above, but may also refer to two or more polypeptides associated, for example, by disulfide bonds, hydrogen bonds, or hydrophobic interactions to produce a multimeric protein.

[0177] "Isolated" material, e.g., an isolated nucleic acid, is material that is not in its natural environment, but it is not necessarily purified. For example, an isolated nucleic acid is a nucleic acid that is not produced or located in its native or natural environment (such as a cell). The isolated material can be separated, fractionated, or at least partially purified by any suitable technique.

[0178] As used herein, the terms "antibody" and "antigen-binding fragment thereof" refer to at least the minimum portion of an antibody that is capable of binding to a designated antigen targeted by the antibody, for example, in the context of a typical antibody produced by a B cell, at least some of the complementarity determining regions (CDRs) of the heavy chain variable domain (VH) and the light chain variable domain (VL). In some antibodies, such as naturally occurring IgG antibodies, the heavy chain constant region consists of a hinge and three domains, CH1, CH2, and CH3. In some antibodies, such as naturally occurring IgG antibodies, each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate immunoglobulin binding 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. The heavy chain may or may not have a C-terminal lysine. Unless otherwise indicated herein, the amino acids in the variable region are numbered using the Kabat numbering system, and those in the constant region are numbered using the EU system.

[0179] The antibody or antigen-binding fragment thereof can be or can be derived from a polyclonal antibody, a monoclonal antibody, a human antibody, a humanized antibody or a chimeric antibody, a single chain antibody, an epitope-binding fragment (e.g., Fab, Fab' and F(ab')2), a Fd, a Fv, a single chain fragment variable fragment (scFv), a single chain antibody, a V HH, vNAR, nanobody, (single domain antibody), disulfide bonded Fv (sdFv), comprising a separate VL domain or VH domain or a fragment combined with a portion of an opposing domain (e.g., a whole VL domain and a partial VH domain with one, two or three CDRs) and a fragment produced by a Fab expression library. scFv molecules are known in the art and are described in, for example, U.S. Patent No. 5,892,019. The antibody molecules encompassed by the present disclosure can be or be derived from any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY) of immunoglobulin molecules, classes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses of immunoglobulin molecules.

[0180] In certain aspects, the present disclosure provides an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 11, 20, 29, 38, 47, 56, 65, 74 and 83; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 12, 21, 30, 39, 48, 57, 66, 75 and 84; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 13, 22, 31, 40, 49, 58, 67, 76 and 85; and wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 15, 24, 33, 42, 51, 60, 69, 78 and 87; a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: NO: 7, 16, 25, 34, 43, 52, 61, 70, 79 and 88; and a CDR3 comprising an amino acid sequence selected from SEQ ID NO: 8, 17, 26, 35, 44, 53, 62, 71, 80 and 89. In certain embodiments, the present disclosure provides an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence selected from SEQ ID NO: 1, 10, 19, 28, 37, 46, 55, 64, 73 and 82; and wherein the VL comprises an amino acid sequence selected from SEQ ID NO: 5, 14, 23, 32, 41, 50, 59, 68, 77 and 86.

[0181] In one aspect, the present disclosure provides an anti-BCMACAR comprising or consisting of the amino acid sequence shown in SEQ ID NO: 96. In another aspect, the present disclosure provides an anti-BCMACAR comprising or consisting of the amino acid sequence shown in SEQ ID NO: 97. In yet another aspect, the present disclosure provides an anti-BCMACAR comprising or consisting of the amino acid sequence shown in SEQ ID NO: 98.

[0182] As used herein, antibodies or antigen-binding fragments thereof also include "single domain antibodies," which are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples of single domain antibodies include heavy chain antibodies, antibodies naturally lacking light chains, single domain antibodies derived from conventional four-chain antibodies, and engineered or recombinant single domain antibodies. Single domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, and cattle. Single domain antibodies can be naturally occurring single domain antibodies, referred to as heavy chain antibodies lacking light chains. Specifically, camelid species, such as camels, dromedaries, llamas, alpacas, and guanacos, produce heavy chain antibodies naturally lacking light chains. The variable heavy chain of single domain antibodies lacking light chains is referred to as "VHH" or "nanoantibodies." Similar to conventional VH domains, VHH contains four FRs and three CDRs. Nanoantibodies have advantages over conventional antibodies: they are smaller than IgG molecules, so correctly folded functional nanoantibodies can be produced by in vitro expression while achieving high yields. For example, VHH domains, nanobodies, and proteins / polypeptides containing them can be produced using microbial fermentation and do not require the use of mammalian expression systems; VHH domains and nanobodies are relatively small (approximately 15 kDa, or 1 / 10 of conventional IgG), and therefore exhibit high (higher) permeability to tissues (including but not limited to solid tumors and other dense tissues) compared to such conventional 4-chain antibodies and antigen-binding fragments thereof; VHH domains and nanobodies can exhibit so-called cavity binding properties (especially due to their extended CDR3 loops compared to conventional VH domains), and therefore can also access targets and epitopes that are inaccessible to conventional 4-chain antibodies and antigen-binding fragments thereof. In addition, nanobodies are very stable and resistant to the action of proteases.

[0183] As used herein, "VHH domain" refers to the variable domain present in naturally occurring heavy chain antibodies, so as to distinguish them from the heavy chain variable domain present in conventional four-chain antibodies (referred to herein as "VH domain") and the light chain variable domain present in conventional four-chain antibodies (referred to herein as "VL domain"). In some embodiments, the recombinant polypeptides of the present disclosure correspond to the amino acid sequence of a naturally occurring VHH domain, but have been "humanized", i.e., by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence with one or more amino acid residues that appear at the corresponding position in the VH domain of a conventional four-chain antibody from a human. This can be done in a manner known in the art.

[0184] In one embodiment, the present disclosure provides a recombinant polypeptide sequence, such as an immunoglobulin sequence (in some embodiments, a VHH antibody sequence) capable of binding to an envelope epitope of BCMA, wherein the immunoglobulin sequence comprises four framework regions (FR1, FR2, FR3, and FR4) and three complementarity determining regions (CDR1, CDR2, and CDR3), wherein:

[0185] a) CDR1 is an amino acid sequence of SEQ ID NO: 2, 11, 20, 29, 38, 47, 56, 65, 74 and 83; or is selected from the group consisting of SEQ ID NO: 2, 11, 20, 29, 38, 47,

[0186] 56, 65, 74 and 83 have at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity; or selected from the group consisting of amino acid sequences that have 2 or only 1 amino acid difference compared to the amino acid sequence of SEQ ID NO: 2, 11, 20, 29, 38, 47, 56, 65, 74 and 83;

[0187] b) CDR2 is an amino acid sequence of SEQ ID NO: 3, 12, 21, 30, 39, 48, 57, 66, 75 and 84; or is selected from the group consisting of SEQ ID NO: 3, 12, 21, 30, 39, 48,

[0188] 57, 66, 75 and 84 have at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity; or selected from the group consisting of amino acid sequences that have 2 or only 1 amino acid difference compared to the amino acid sequence of SEQ ID NO: 3, 12, 21, 30, 39, 48, 57, 66, 75 and 84;

[0189] c) CDR3 is an amino acid sequence of SEQ ID NO: 4, 13, 22, 31, 40, 49, 58, 67, 76 and 85; or is selected from the group consisting of SEQ ID NO: 4, 13, 22, 31, 40, 49,

[0190] 58, 67, 76 and 85 have at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity; or selected from the group consisting of amino acid sequences that have 2 or only 1 amino acid difference compared to the amino acid sequence of SEQ ID NO: 4, 13, 22, 31, 40, 49, 58, 67, 76 and 85;

[0191] And wherein the framework sequence can be any suitable framework sequence, such as the framework sequence of a single domain antibody, in particular the framework sequence of a VHH antibody.

[0192] B-cell maturation antigen (BCMA; also known as BCM; CD269; and TNFRSF13A) is a member of the TNF receptor superfamily. This receptor is expressed in mature B lymphocytes and may be important for B-cell development and autoimmune responses. BCMA is also known as TNF receptor superfamily member 17 and has been shown to bind to tumor necrosis factor superfamily member 13b, leading to NF-κB and MAPK8 / JNK activation. This receptor also binds to various TRAF family members and, as such, can transduce signals for cell survival and proliferation.

[0193] As used herein, the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human BCMA). The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody (e.g., an anti-BCMA antibody described herein) include (i) a Fab fragment (fragment from papain cleavage) or a fragment consisting of a V L 、V H (ii) a F(ab')2 fragment (fragment resulting from pepsin cleavage) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a fragment consisting of V H and CH1 domains; (iv) a V fragment consisting of a single-arm antibody L and V H Fv fragment composed of structural domains, (v) consists of V H(vi) an isolated complementarity determining region (CDR), and (vii) a combination of two or more isolated CDRs optionally linked by a synthetic linker. In addition, although the two domains V and V of the Fv fragment are L and V H Encoded by separate genes, they can be joined using recombinant methods via a synthetic linker that enables them to become a single protein chain, where V L Area and V H The antibody fragments are paired with the corresponding regions to form monovalent molecules (called single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0194] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered antigen-binding polypeptide comprising an antigen-binding domain, a transmembrane domain, and one or more intracellular domains (e.g., costimulatory domains). In some embodiments, CAR may optionally include a spacer domain and / or a flexible hinge domain to provide conformational freedom to facilitate binding to a target antigen on a target cell. In some embodiments, CAR may optionally include an armor domain comprising a nucleic acid sequence encoding an armor molecule. CAR expression on the surface of a cell (e.g., an immune cell) allows the cell to target and bind to a specific antigen. In some embodiments, CAR is expressed by an immune cell, such as a T cell. In some embodiments, the antigen-binding domain includes Fab, Fab', F(ab')2, Fd, Fv, a single-chain variable fragment (scFv), a single-chain antibody, VHH, vNAR, a nanobody (single domain antibody), or any combination thereof. In some embodiments, the transmembrane domain includes a transmembrane domain selected from the transmembrane domain of CD4, CD8α, or CD28. In some embodiments, the one or more intracellular domains include a costimulatory domain or a portion thereof. In some embodiments, the intracellular domain includes a costimulatory domain or a portion thereof. In some embodiments, the intracellular domain includes a costimulatory domain or a variant thereof of CD3z. For example, CD3z costimulatory domain variants may contain only 1 or 2 functional ITAMs in three immunoreceptor tyrosine-based activation motifs (ITAMs) present in wild-type CD3z. In some embodiments, the intracellular domain includes a costimulatory domain selected from the group consisting of: CD3zeta costimulatory domain, CD28 costimulatory domain, CD27 costimulatory domain, 4-1BB costimulatory domain, ICOS costimulatory domain, OX-40 costimulatory domain, GITR costimulatory domain, CD2 costimulatory domain, IL-2R β costimulatory domain, MyD88 / CD40 costimulatory domain and any combination thereof. CAR may also include a "hinge" or "spacer" domain. Non-limiting examples of hinge / spacer domains include immunoglobulin hinge / spacer domains, such as IgG1 hinge domains, IgG2 hinge domains, IgG3 hinge domains, IgG4 hinge domains, IgG4P hinge domains (IgG4 hinge domains comprising S241P mutations), CD8a hinge domains, or CD28 hinge domains. In one embodiment, the CAR comprises a hinge comprising a sequence of SEQ ID NO: 93.

[0195] As used herein, the term "polynucleotide" includes single nucleic acids as well as multiple nucleic acids, and refers to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA) or plasmid DNA (pDNA). The term "nucleic acid" includes any nucleic acid type, such as DNA or RNA. "Conservative amino acid substitution" refers to the replacement of an amino acid residue by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, the predicted nonessential amino acid residue in the BCMA-binding portion (e.g., anti-BCMA CAR or antibody) is replaced with another amino acid residue from the same side chain family.

[0196] Percent identity between two sequences is a function of the number of identical positions that the sequences have (i.e., the total number of homology percentage=number of identical positions / positions × 100), taking into account the number of spaces that need to be introduced and the length of each space for optimal alignment of the two sequences. As described in the following non-limiting examples, a mathematical algorithm can be used to compare the sequences and determine the percent identity between the two sequences.

[0197] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (freely available) using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm incorporated into the GAP program in the GCG software package, using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0198] The nucleic acid and protein sequences described herein can further be used as a "query sequence" to search against public databases, for example, to identify related sequences. Such searches can be performed using the NBLAST program and the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized, as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0199] As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which an additional DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to a viral genome. Some vectors can replicate autonomously in a host cell (e.g., a bacterial vector and a mammalian episomal vector) into which they are introduced. Other vectors (e.g., non-episodic mammalian vectors) can be integrated into the host cell genome after being introduced into the host cell, thereby replicating together with the host genome. Moreover, some vectors can guide the expression of genes operatively connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally speaking, in recombinant DNA technology, there are practical expression vectors that are often in the form of plasmids. In this specification sheets, "plasmids" and "vectors" can be used interchangeably because plasmids are the most commonly used vector forms. However, other forms of expression vectors that play an equivalent function are also included, such as viral vectors (for example, lentiviral vectors, replication-defective retroviruses, adenoviruses and adeno-associated viruses) or transposons (for example, DNA transposons or retrotransposons). In certain embodiments, CAR and / or antibodies or their antigen-binding fragments are covered and / or delivered to cells and / or patients by viruses, lentiviruses, adenoviruses, retroviruses, adeno-associated viruses (AAV), transposons, DNA vectors, mRNA, lipid nanoparticles (LNPs) or CRISPR-Cas systems. In one embodiment, lentiviral vectors are used.

[0200] As used herein, the term "vector" may refer to a nucleic acid molecule that is introduced into a host cell to produce a transformed host cell. A vector may include a nucleic acid sequence that permits its replication in the host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. Specific types of vectors contemplated herein may be associated with or incorporated into viruses to promote cell transformation.

[0201] "Transformed" cells or "host" cells are cells into which nucleic acid molecules have been introduced by molecular biology techniques. It is contemplated herein that all techniques by which nucleic acid molecules can be introduced into such cells include transfection with viral vectors, transformation with plasmid vectors, and the introduction of naked DNA accelerated by electroporation, lipofection, and particle guns. In certain embodiments, cells are transformed using one or more techniques, including viral, lentivirus, adenovirus, retrovirus, adeno-associated virus (AAV), transposon, DNA vector, mRNA, lipid nanoparticles (LNP), or CRISPR-Cas systems.

[0202] As used herein, the term "affinity" refers to a measure of the strength of binding of an antigen or target (such as an epitope) to its cognate binding domain (such as a paratope). As used herein, the term "avidity" refers to the overall stability of the complex between a population of epitopes and paratopes (i.e., antigens and antigen-binding domains).

[0203] The term "epitope" refers to a site on an antigen (e.g., BCMA) to which a chimeric antigen receptor, immunoglobulin, or antibody specifically binds, e.g., as defined by a specific method for identifying the site. An epitope can be formed by continuous amino acids (usually a linear epitope) or by non-continuous amino acids juxtaposed by the tertiary folding of the protein (usually a conformational epitope). Epitopes formed by continuous amino acids are usually, but not always, retained when exposed to a denaturing solvent, while epitopes formed by tertiary folding are typically lost when treated with a denaturing solvent. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation.

[0204] "Immunotherapy" refers to the treatment of a subject having a disease or at risk of contracting a disease or suffering recurrence of a disease by methods that include inducing, enhancing, suppressing, or otherwise altering the immune system or immune response.

[0205] " immune response " is as understood in the art, and generally refers to the biological response for exogenous factors or abnormal cells (for example, cancer cells) in vertebrates, and the reaction protects organisms against these factors and the disease caused by them.Immune response is mediated by the effect of one or more cells (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells or neutrophils) of immune system and the soluble macromolecules (including antibodies, cytokines and complement) produced by any cell in these cells or liver, which causes selective targeting, combination, damage, destruction and / or elimination of invading pathogens, cells or tissues of infected pathogens, cancer cells or other abnormal cells or normal human cells or tissues in the case of autoimmunity or pathological inflammation in vertebrates.Immune response includes activation or suppression of such as T cells (for example, effector T cells, Th cells, CD4+ cells, CD8+ T cells or Treg cells), or activation or suppression of any other cell (for example, NK cells) of immune system.

[0206] As used herein, the term "treating," when used in the context of treating cancer, refers to reducing disease pathology, reducing or eliminating disease symptoms, promoting increased survival, and / or reducing discomfort. For example, treating can refer to the ability of a therapy to reduce disease symptoms, signs, or causes when administered to a subject. Treating also refers to alleviating or reducing at least one clinical symptom and / or inhibiting or delaying the progression of a condition and / or preventing or delaying the onset of a disease or illness.

[0207] As used herein, the term "subject," "individual," or "patient" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, and the like.

[0208] As used herein, the term "effective amount" or "therapeutically effective amount" of an administered therapeutic substance (such as CAR T cells) is an amount sufficient to carry out a specifically described or intended purpose (such as treating cancer or cancer therapy). An "effective amount" can be determined empirically with respect to the described purpose. In certain embodiments, a therapeutically effective amount may refer to the number of cells administered to a subject in need of treatment. The number of cells per dose, the number of doses, and the frequency of administration will depend on various parameters, such as the patient's age, weight, clinical assessment, disease type, cancer type, tumor type, tumor load, and / or other factors, including the judgment of the attending physician.

[0209] The terms "T cell" or "T lymphocyte" are art-recognized and are intended to include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cell can be a T helper (Th) cell, such as a T helper 1 (Th1) or T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4 + T cells)CD4 + T cells, cytotoxic T cells (CTL; ​​CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells or any other subpopulation of T cells. Other illustrative T cell populations suitable for use in certain embodiments include naive T cells and memory T cells.

[0210] As used herein, the term "proliferation" refers to an increase in cell division (symmetrical or asymmetrical cell division). In certain embodiments, "proliferation" refers to symmetrical or asymmetrical division of T cells. "Increased proliferation" occurs when the number of cells in a treated sample increases compared to cells in an untreated sample.

[0211] The term "expand" in the disclosed methods refers to the process of increasing the number of cells in a cell culture. During the expansion step, in one embodiment, the cells are fed and the culture medium is changed regularly according to a feeding schedule. The specific timing and amount of culture medium added in a particular feeding schedule will depend on the number of cells and metabolite levels in the culture.

[0212] As used herein, the term "differentiation" refers to a process of reducing the potential or proliferation of a cell or causing the cell to move to a more developmentally restricted state. In certain embodiments, the differentiated T cells acquire immune effector cell function.

[0213] An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). Illustrative immune effector cells contemplated herein are NK cells or T lymphocytes, particularly cytotoxic T cells (CTLs; CD8 + T cells), TILs, and helper T cells (HTL; CD4 + T cells).

[0214] "Modified T cells" refer to T cells that have been modified by introducing polynucleotides encoding the engineered CARs contemplated herein. Modified T cells include both genetic modifications and non-genetic modifications (e.g., episomal or extrachromosomal modifications).

[0215] As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material of a cell.

[0216] The terms "genetically modified cells," "modified cells," and "redirected cells" are used interchangeably.

[0217] The acronym "SMART" (Short-Term Manipulated Autoreplicating T Cells) refers to a short-term T cell production and expansion process in which cells are cultured in the presence of IL-21 (and optionally IL-2).

[0218] The acronym "TNT" (traditionally cultured T cells) refers to traditional T cell expansion methods that do not employ IL-21 and typically involve culturing cells for more than 7 days and / or typically involve the use of IL-2.

[0219] The term "stimulation" refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, including but not limited to signal transduction via the TCR / CD3 complex.

[0220] "Stimulatory molecule" refers to a molecule on a T cell that specifically binds to its cognate stimulatory ligand.

[0221] As used herein, "stimulatory ligand" means a ligand that, when present on an antigen presenting cell (e.g., APC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner on a T cell (referred to herein as a "stimulatory molecule"), thereby mediating a primary response of the T cell, including but not limited to activation, initiation of an immune response, proliferation, etc. Stimulatory ligands include but are not limited to CD3 ligands (e.g., anti-CD3 antibodies) and CD2 ligands (e.g., anti-CD2 antibodies) as well as peptides (e.g., CMV, HPV, EBV peptides).

[0222] The term "activated" refers to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. In a specific embodiment, activation can also be associated with induced cytokine production and detectable effector functions. The term "activated T cells" refers in particular to T cells that are proliferating. The signal generated by a single TCR is not sufficient to fully activate the T cells, and one or more secondary or costimulatory signals are also required. Therefore, T cell activation includes a primary stimulation signal and one or more secondary costimulatory signals through a TCR / CD3 complex. Costimulation can be demonstrated by the proliferation and / or cytokine production of T cells that have received primary activation signals, such as stimulation by a CD3 / TCR complex or by CD2.

[0223] A "co-stimulatory signal" refers to a signal that, in combination with a primary signal (such as TCR / CD3 ligation), results in T cell proliferation, cytokine production, and / or up- or down-regulation of specific molecules (eg, CD28).

[0224] "Costimulatory ligand" refers to a molecule that binds to a costimulatory molecule. A costimulatory ligand can be soluble or provided on the surface. A "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand (e.g., an anti-CD28 antibody).

[0225] As used herein, "autologous" refers to cells from the same subject. In some embodiments, the cells of the present disclosure are autologous.

[0226] As used herein, "allogeneic" refers to a cell of the same species that is genetically different from the cell being compared. In some embodiments, the cells of the disclosure are allogeneic.

[0227] As used herein, "isogenic" refers to a cell from a different subject that is genetically identical to the cell being compared. In some embodiments, the cells of the present disclosure are isogenic.

[0228] As used herein, "allogeneic" refers to a cell of a different species than the cell being compared. In some embodiments, the cells of the disclosure are allogeneic.

[0229] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that exhibits symptoms of a disease that can be treated with the gene therapy vectors, cell-based therapeutic agents, and methods disclosed elsewhere herein. Suitable subjects (e.g., patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and livestock or pets (such as cats or dogs). Non-human primates are included, and preferably human patients are included. Typical subjects include human patients who have cancer, have been diagnosed with cancer, are at risk for cancer, or are currently suffering from cancer.

[0230] "Enhancement" or "promotion" or "increase" or "amplification" generally refers to the ability of the compositions encompassed herein to produce, induce or cause a greater physiological response (i.e., downstream effect) than the response caused by a vehicle or control molecule / composition. Measurable physiological responses may include an increase in T cell expansion, activation, persistence, and / or an increase in cancer cell death and killing ability, as well as other factors apparent from the understanding of the art and the description herein. An "increased" or "enhanced" amount is generally a "statistically significant" amount and may include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500 times, 1000 times) (including all integers and decimal points between and greater than 1, such as 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by a vehicle or control composition.

[0231] "Reduced" or "diminished" or "lessened" or "reduced" or "mitigated" generally refers to the ability of the compositions encompassed herein to produce, elicit, or cause less of a response (i.e., a physiological response) than a response elicited by a vehicle or control molecule / composition. A "reduced" or "lowered" amount is generally a "statistically significant" amount and can include a reduction of 10-11, 5-6, 2-3, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, or less (e.g., 1-500, 1-1000) (including all integers and decimal points between and less than 1, e.g., 2-3, 5-8, 1-17, 5-9, etc.) of the response produced by a vehicle, a control composition (reference response), or in a particular cell lineage.

[0232] "Maintain" or "maintain" or "no change" or "no substantial change" or "no substantial reduction" generally refers to the ability of the compositions contemplated herein to produce, elicit or cause a lower physiological response (i.e., downstream effect) in a cell compared to the response elicited by vehicle, a control molecule / composition, or a response in a particular cell lineage. An equivalent response is one that is not significantly different or measurably different from a reference response.

[0233] Overview

[0234] In some aspects, the present disclosure relates to compositions and methods for treating diseases using chimeric antigen receptor (CAR) cell therapy. More particularly, the present disclosure relates to CAR cell therapy, in which transformed cells (such as T cells or NK cells) express CAR targeting BCMA. Furthermore, the CAR constructs disclosed herein, the transformed cells expressing these constructs, and the therapy utilizing transformed cells can provide robust treatment for cancer, autoimmune diseases, or other diseases expressing BCMA.

[0235] Without wishing to be bound by theory, BCMA is considered a viable disease target across multiple modalities and is believed to be a promising target for CAR cell therapy.

[0236] CAR construct design

[0237] The CAR construct of the present disclosure may have several components, many of which may be selected based on the desired or refined functions of the resulting CAR construct. In addition to the antigen binding domain, the CAR construct may also have a spacer domain, a hinge domain, a signal peptide domain, a transmembrane domain, and one or more intracellular domains (e.g., one or more costimulatory domains). In some embodiments, CAR may optionally include an armor domain comprising a nucleic acid sequence encoding an armor molecule. The selection of one component relative to another component (i.e., the selection of a specific costimulatory domain from a receptor relative to a costimulatory domain from different receptors) may affect clinical efficacy and safety characteristics.

[0238] Antigen binding domain

[0239] The antigen binding domains considered herein may include an antibody or one or more antigen binding fragments thereof. A contemplated CAR construct for targeting BCMA includes a single-chain variable fragment (scFv), which contains light chain variable regions and heavy chain variable regions from one or more antibodies specific for BCMA, and these light chain variable regions and heavy chain variable regions are directly connected together or connected together via a flexible linker (e.g., with 1,2,3 or more G4S repeats of repeated sequences). In one embodiment, the linker includes SEQ ID NO:92 sequence.

[0240] As disclosed herein, the antigen binding domain of the CAR targeting BCMA can be changed in terms of its binding affinity to the BCMA protein. Compared to antibodies that generally expect higher affinity, in the case of CAR, the relationship between binding affinity and efficacy may be more nuanced. For example, preclinical studies of receptor tyrosine kinase-like orphan receptor 1 (ROR1)-CAR derived from high-affinity scFv (dissociation constant of 0.56nM) produced an increased therapeutic index compared to lower affinity variants. On the contrary, other examples have been reported, namely, improved differentiation between cells with different antigen densities for lower affinity engineered scFv. This can be used to improve the therapeutic specificity for antigens that are differentially expressed on tumors and normal tissues.

[0241] A variety of methods can be used to determine the binding affinity of an antigen binding domain. In some embodiments, methods that exclude avidity effects can be used. Avidity effects involve multiple antigen binding sites interacting with multiple target epitopes simultaneously, typically in a multimeric structure. Therefore, avidity functionally represents the cumulative strength of multiple interactions. An example of a method that excludes avidity effects is any method in which one or both of the interacting proteins are monomeric / monovalent, because if one or both partners contain only a single interaction site, multiple simultaneous interactions are impossible.

[0242] spacer domain

[0243] The CAR construct of the present disclosure may have a spacer domain to provide conformational freedom to promote binding to the target antigen on the target cell. The optimal length of the spacer domain may depend on the proximity of the binding epitope to the target cell surface. For example, a proximal epitope may require a longer spacer, while a distal epitope may require a shorter spacer. In addition to promoting the binding of CAR to the target antigen, achieving an optimal distance between CAR cells and cancer cells can also help to spatially block the immune synapse formed between large inhibitory molecules and the CAR cells and target cancer cells. The CAR targeting BCMA may have a long spacer, an intermediate spacer, or a short spacer. The long spacer may include the CH2CH3 domain (about 220 amino acids) of immunoglobulin G1 (IgG1) or IgG4 (natural or with common modifications in therapeutic antibodies, such as S228P mutations), and the CH3 region may be used alone to construct an intermediate spacer (about 120 amino acids). Shorter spacers may be derived from segments (<60 amino acids) of CD28, CD8α, CD3, or CD4. The short spacer region can also be derived from the hinge region of an IgG molecule. These hinge regions can be derived from any IgG isotype and may or may not contain mutations commonly found in therapeutic antibodies, such as the S228P mutation mentioned above. For example, the hinge domain can comprise an IgG1 hinge domain or a variant thereof, an IgG2 hinge domain or a variant thereof, an IgG3 hinge domain or a variant thereof, an IgG4 hinge domain or a variant thereof, a CD8 hinge domain or a variant thereof, or a CD28 hinge domain or a variant thereof.

[0244] Hinge domain

[0245] CAR targeting BCMA can also have a hinge domain. A flexible hinge domain is a short peptide fragment that provides conformational freedom to facilitate binding to a target antigen on a tumor cell. It can be used alone or in combination with a spacer sequence. The terms "hinge" and "spacer" are generally used interchangeably - for example, an IgG4 sequence can be considered to be both a "hinge" sequence and a "spacer" sequence (i.e., a hinge / spacer sequence). In some embodiments, the hinge domain may include an IgG1 hinge domain or a variant thereof, an IgG2 hinge domain or a variant thereof, an IgG3 hinge domain or a variant thereof, an IgG4 hinge domain or a variant thereof (especially an IgG4P hinge domain), a CD8 hinge domain or a variant thereof, or a CD28 hinge domain or a variant thereof. In one embodiment, the hinge domain includes SEQ ID NO:93 sequence.

[0246] signal peptide

[0247] CAR targeting BCMA may also include a sequence comprising a signal peptide. The signal peptide plays a role in prompting cells to translocate CAR to the cell membrane. Examples include IgG1 heavy chain signal polypeptide, Igκ or λ light chain signal peptide, granulocyte-macrophage colony stimulating factor receptor 2 (GM-CSFR2 or CSFR2) signal peptide, CD8a signal polypeptide or CD33 signal peptide. In one embodiment, the signal peptide comprises the sequence of SEQ ID NO:91.

[0248] transmembrane domain

[0249] The CAR targeting BCMA may also include a sequence comprising a transmembrane domain. The transmembrane domain may include a hydrophobic alpha helix that spans the cell membrane. The properties of the transmembrane domain have not been studied as carefully as other aspects of the CAR construct, but they may potentially affect the expression of CAR and the association with endogenous membrane proteins. The transmembrane domain may be derived from, for example, CD3, CD4, CD8α or CD28. Any transmembrane domain may be used in the compositions disclosed herein. In some embodiments, the transmembrane domain includes a transmembrane domain selected from a transmembrane domain of CD3, CD4, CD8α or CD28. In some embodiments, the transmembrane domain includes a CD28 transmembrane domain. In one embodiment, the transmembrane domain includes a sequence of SEQ ID NO:94.

[0250] Intracellular domain / costimulatory domain

[0251] The CAR targeting BCMA can also include one or more sequences forming an intracellular domain and / or a costimulatory domain (sometimes also referred to as a signaling domain).Costimulatory domain is a domain that can strengthen or regulate the response of immune effector cells (i.e., the response of immune effector cells can be started). In some embodiments, costimulatory domain and / or signaling domain include the primary activation signal derived from the cytoplasmic domain of CD3 ζ, which contains a sequence motif called an activation motif (ITAM) based on immunoreceptor tyrosine. In certain embodiments, the intracellular domain refers to a costimulatory domain (e.g., a costimulatory domain from 4-1BB or CD28) plus the primary activation signal of CD3 ζ (CD3z or CD3zeta).Costimulatory domains may include sequences, such as costimulatory domains from one or more of CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2R β, and MyD88 / CD40. In certain embodiments, the co-stimulatory domain selected from CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2R β and MyD88 / CD40 is combined with the primary activation signal of CD3 ζ (CD3z or CD3zeta). In some embodiments, the co-stimulatory domain may include a variant of the co-stimulatory domain of one or more of CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2R β and MyD88 / CD40. In certain embodiments, the co-stimulatory domain variant is selected from the co-stimulatory domain variant of CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2R β and MyD88 / CD40, and is combined with the primary activation signal of CD3 ζ (CD3z or CD3zeta). In one embodiment, the CAR co-stimulatory domain may also include the modification of the CD3z domain. For example, CD3z signaling domain variants may contain one or two functional ITAMs in three immunoreceptor tyrosine-based activation motifs (ITAMs) present in wild-type CD3z. The selection of costimulatory domains affects the phenotype and metabolic characteristics of CAR cells. For example, CD28 costimulation produces an effective but transient effector-like phenotype with high levels of cytolytic capacity, interleukin-2 (IL-2) secretion and glycolysis. In contrast, T cells modified with CARs carrying 4-1BB costimulatory domains tend to expand and last longer in vivo, have increased oxidative metabolism, are not easily exhausted, and have increased ability to produce central memory T cells. In some embodiments, the intracellular signaling domain includes a costimulatory domain or a portion thereof. In one embodiment, the intracellular domain includes SEQ ID NO:95 sequence.

[0252] In some embodiments, the intracellular domain comprises a costimulatory domain selected from the group consisting of the following intracellular domains: CD28 costimulatory domain, CD27 costimulatory domain, 4-1BB costimulatory domain, ICOS costimulatory domain, OX-40 costimulatory domain, GITR costimulatory domain, CD2 costimulatory domain, IL-2Rβ costimulatory domain, MyD88 / CD40 costimulatory domain, and any combination thereof. In some embodiments, the intracellular domain comprises a CD28 costimulatory domain. In some embodiments, the intracellular domain comprises a 4-1BB costimulatory domain. In some embodiments, the intracellular domain comprises a CD28 costimulatory domain in combination with CD3zeta. In some embodiments, the intracellular domain comprises a 4-1BB costimulatory domain in combination with CD3zeta. In one embodiment, the intracellular domain comprises the sequence of SEQ ID NO:95.

[0253] In certain embodiments, the intracellular domain comprises a costimulatory domain comprising a portion of the intracellular T cell receptor (TCR) signaling domain CD3zeta (or CD3z; the CD3z signaling domain is also referred to herein as a "CD3z costimulatory domain"). In some embodiments, CD3zeta comprises one or more modifications to the CD3z format. For example, a CD3z signaling domain variant may contain one or two functional ITAMs of the three immunoreceptor tyrosine-based activation motifs (ITAMs) present in wild-type CD3z (e.g., 1XX, XX, or X2X).

[0254] Exemplary CARs

[0255] According to all aspects of the invention, the CAR may comprise or consist of the amino acid sequence shown in SEQ ID NO: 96. According to all aspects of the invention, the CAR may comprise or consist of the amino acid sequence shown in SEQ ID NO: 97. According to all aspects of the invention, the CAR may comprise or consist of the amino acid sequence shown in SEQ ID NO: 98.

[0256] In certain aspects, the present disclosure provides an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 11, 20, 29, 38, 47, 56, 65, 74 and 83; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 12, 21, 30, 39, 48, 57, 66, 75 and 84; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 13, 22, 31, 40, 49, 58, 67, 76 and 85; and wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 15, 24, 33, 42, 51, 60, 69, 78 and 87; a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: NO:7, 16, 25, 34, 43, 52, 61, 70, 79 and 88; and a CDR3 comprising an amino acid sequence selected from SEQ ID NO:8, 17, 26, 35, 44, 53, 62, 71, 80 and 89.

[0257] In certain embodiments, the present disclosure provides an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 10, 19, 28, 37, 46, 55, 64, 73 and 82; and wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 14, 23, 32, 41, 50, 59, 68, 77 and 86.

[0258] The CAR constructs of the present disclosure may include a certain combination of modular components as described herein. For example, in some embodiments of the present disclosure, the CAR constructs include BCMA scFv antigen binding domains. In some embodiments of the present disclosure, the CAR constructs include CD33 signal peptides. In one embodiment, CAR includes a signal peptide containing SEQ ID NO:91 sequence. In some embodiments, the CAR constructs include IgG4 hinge / spacer domains (IgG4P) carrying S241P mutations. In one embodiment, CAR includes a hinge domain containing SEQ ID NO:93 sequence. In some embodiments, the CAR constructs include CD28 transmembrane domains. In one embodiment, CAR includes a transmembrane domain containing SEQ ID NO:94 sequence.

[0259] Different costimulatory domains can be utilized in the CAR constructs of the present disclosure. In some embodiments, the CAR construct includes a costimulatory domain, which includes a signaling domain (for example, a part of an intracellular T cell receptor (TCR) signaling domain, CD3zeta (or CD3z) or its variant) from the intracellular domain of CD3z. In some embodiments, the CAR construct includes a CD28 costimulatory domain. In some embodiments, the CAR construct includes a 4-1BB costimulatory domain. In some embodiments, the CAR construct includes a costimulatory domain from CD3z and CD28, as described herein. In some embodiments, the CAR construct includes a costimulatory domain from CD3z and 4-1BB, as described herein. In some embodiments, the CAR construct includes a costimulatory domain from all CD3z, CD28 and 4-1BB, as described herein. In some embodiments, the CAR construct includes a costimulatory domain from ICOS, OX-40 and / or GITR. In one embodiment, CAR includes an intracellular domain containing a sequence of SEQ ID NO:95.

[0260] cell

[0261] CAR-based cell therapy can be used for a variety of cell types, such as lymphocytes. The specific types of cells that can be used include T cells, natural killer (NK) cells, natural killer T (NKT) cells, constant natural killer T (iNKT) cells, αβT cells, γδT cells, virus-specific T (VST) cells, cytotoxic T lymphocytes (CTL), tumor infiltrating lymphocytes and regulatory T cells (Treg). In some embodiments, cells are autologous. In certain embodiments, cells are allogeneic. In other embodiments, cells can be from genetically similar but not identical donors (allogeneics).

[0262] In some embodiments, the cell population may also include amplified populations and / or engineered T cells. In some embodiments, the cell population may include total T cells, CD4 positive T cells, CD8 positive T cells, regulatory T cells, gamma-delta T cells, mucosal associated constant T (MAIT) T cells, natural killer (NK) cells, or natural killer T (NKT) cells. T cells are broadly divided into cells expressing CD4 on the surface (also referred to as CD4 positive cells) and cells expressing CD8 on the surface (also referred to as CD8 positive cells).

[0263] In some embodiments, T cells suitable for use according to the methods provided herein are mononuclear lymphocytes derived from bone marrow (BM), peripheral blood (PB) or cord blood (CB) of a human donor. These cells can be collected directly from BM, PB or CB, or collected after mobilization or stimulation via administration of growth factors and / or cytokines (such as granulocyte colony stimulating factor (G-CSF) or granulocyte-macrophage colony stimulating factor (GM-CSF)) to an allogeneic donor or an autologous donor. It will be appreciated by those skilled in the art that there are many established protocols for isolating peripheral blood mononuclear cells (PBMC) from peripheral blood. The separation of PBMC can be assisted by a density gradient separation protocol, typically employing density gradient centrifugation techniques, using or For separating lymphocytes from other components in the blood. Preferably, PBMC separation is performed under sterile conditions. PBMC separation can also be performed using a negative selection kit. Alternatively, cell elutriation can be used to isolate mononuclear cell populations. In some embodiments, the cell population is human cells. In certain embodiments, the cell population is human primary immune cells.

[0264] In some embodiments, the cell compositions and methods of the present disclosure may include cells that are genetically engineered to resist replicative senescence (RRS). In some embodiments, the cells that resist replicative senescence may include a transgene encoding B-cell lymphoma-extra large (Bcl-xL). In specific embodiments, the cells that resist replicative senescence may include a transgene encoding B-cell lymphoma-extra large (Bcl-xL) and / or B-cell lymphoma 2 (Bcl-2). In some embodiments, the cells that resist replicative senescence may include knockout of one or more endogenous regulatory factors or have suppressed expression, and the one or more endogenous regulatory factors are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B) and S-methyl-5'-thioadenosine phosphorylase (MTAP). In some embodiments, the cells that resist replicative senescence may include knockout of one or more endogenous immune-related genes in primary immune cells or suppressed expression. In certain embodiments, the endogenous immune-related gene is beta-2 microglobulin (B2M) or T cell receptor alpha constant region (TRAC).In some embodiments, the cells resistant to replicative senescence may comprise a knockout or suppressed expression of CD38.

[0265] The term "genetically engineered" refers to alterations to the genetic material of a cell. Gene editing includes adding, removing, or altering genetic material in a genetically engineered cell. In certain embodiments, gene editing includes introducing a transgene into the cell and / or inhibiting gene expression in the cell. In certain embodiments, introducing one or more gene edits includes introducing one or more transgenes encoding anti-apoptotic factors or virus-derived factors into the cell.

[0266] The term "transgenic" refers to any nucleic acid sequence introduced into a cell by experimental manipulation. Transgenic can be an "endogenous DNA sequence" or a "heterologous DNA sequence". The term "endogenous" refers to development or origin within a cell, tissue, or organism or within a part of a cell, tissue, or organism. Transgenic can be isolated and obtained in a suitable amount using one or more methods well known in the art. These methods and other methods that can be used to isolate transgenics are described, for example, in Sambrook et al. (ibid.) and in Berger and Kimmel (Methods in Enzymology: Guide to Molecular Cloning Techniques, Vol. 152, Academic Press, Inc., San Diego, CA (1987)). Transgenic can be incorporated into a "transgenic construct," which comprises a gene of interest and other regulatory DNA sequences required for transient expression or cell-specific expression or enhanced expression of the transgenic of interest. Transgenic can be introduced into cells by any suitable method or technique known in the art. In some embodiments, transgenic is introduced using a plasmid-based DNA transposon, a lentiviral platform, or site-specific integration via CRISPR. Transgene expression in cells can be constitutive or inducible.

[0267] In certain embodiments, the transgene encodes a virus-derived factor. A "virus-derived factor" refers to either a naturally occurring viral peptide, polypeptide, or protein, or a peptide, polypeptide, or protein that exhibits a degree of sequence identity and / or similarity to a viral protein and / or maintains one or more structural, mechanical, or antigenic properties of a viral protein. In specific embodiments, the virus-derived factor is from Saimiriine gammaherpesvirus type 2 StpAA11, Herpesvirus saimiri StpC, Herpesvirus Tip, or a modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1.

[0268] In some embodiments, the cells as described herein further comprise suppressed expression of one or more endogenous regulatory factors in the cell, such that the activity of the endogenous regulatory factors is eliminated or reduced. As used herein, “regulatory factor” refers to a gene encoding a protein involved in regulating cell cycle arrest, cell death, or signal suppression. Endogenous regulatory factors can be downregulated or blocked by any suitable method or technique known in the art. Known methods for downregulating gene expression of factors or reducing the activity of factors include, but are not limited to, CRISPR / Cas (including cytosine and adenine base editors), microRNA, shRNA, RNAi, TALEN, zinc finger nucleases, large-range nucleases, neutralizing antibodies, small molecule inhibitors, chemical inhibitors that block downstream signal transduction pathways, and the like. The inhibition of endogenous regulatory factors can be complete inhibition of gene expression, partial inhibition, downregulation, or reduction of the activity of the factor. In some embodiments, the activity or gene expression of an endogenous regulatory factor is reduced by 1%-100% (i.e., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%). Regulatory factors include genes encoding proteins involved in regulating cell cycle arrest, cell death, or signal repression. In specific aspects, the one or more endogenous regulatory factors are cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP). In certain embodiments, the one or more endogenous regulatory factors are RB transcriptional corepressor 1 (RB1), TP53, autophagy and Beclin 1 regulatory factor 1 (AMBRA1), neurofibromatosis type 1 (NF1), tyrosine-protein phosphatase non-receptor type 2 (PTPN2), or suppressor of cytokine signaling 1 (SOCS1).

[0269] In some embodiments, cells as disclosed herein include suppressed expression of one or more endogenous immune-related genes in the cell, so that the activity of the immune-related genes is eliminated or reduced. As used herein, "immune-related genes" refer to genes encoding proteins involved in achieving an immune response. In some aspects, immune-related genes encode proteins involved in host-versus-graft (HvG) and graft-versus-host (GvH) allogeneic immune responses. Immune-related genes can be lowered or blocked by any suitable method or technology known in the art. Known methods for lowering the gene expression of immune-related genes or reducing the activity of immune-related genes include but are not limited to CRISPR / Cas (including cytosine and adenine base editors), microRNA, shRNA, RNAi, TALEN, zinc finger nucleases, large-range nucleases, neutralizing antibodies, small molecule inhibitors, chemical inhibitors that block downstream signal transduction pathways, etc. The inhibition of endogenous immune-related genes can be complete inhibition of gene expression, partial inhibition, lowering, or reducing the activity of factors. In some embodiments, the activity or gene expression of endogenous immune-related genes is reduced by 1%-100% (i.e., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%). Immune-related genes include genes that refer to proteins that encode proteins involved in achieving an immune response. Immune-related genes can encode proteins involved in host-versus-graft (HvG) and graft-versus-host (GvH) allogeneic immune responses. In specific embodiments, the one or more endogenous immune-related genes are beta-2 microglobulin (B2M) or T cell receptor alpha constant region (TRAC). In a specific embodiment, the one or more endogenous immune-related genes are genes of the major histocompatibility complex (MHC), human leukocyte antigen class I genes (e.g., HLA-A, HLA-B, HLA-C), human leukocyte antigen class II genes (HLA-DR, HLA-DQ and HLA-DP), T cell receptors (e.g., αβ T cell receptors), interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 23 (IL-23), interferon-γ (IFNγ), CCL2, CCL3, CCL4, CCL5, CXCL2, CXCL9-11, CCL17, CCL27, programmed death protein-1 (PD-1), TIM3 or TIGIT.

[0270] In a further embodiment, the cells as disclosed herein include suppressed expression of cluster of differentiation 38 (CD38) in the cells, such that the activity of CD38 is eliminated or reduced. CD38 can be downregulated or blocked by any suitable method or technique known in the art. Known methods for downregulating the gene expression of CD38 or reducing the activity of CD38 include, but are not limited to, CRISPR / Cas (including cytosine and adenine base editors), microRNA, shRNA, RNAi, TALEN, zinc finger nucleases, large-range nucleases, neutralizing antibodies, small molecule inhibitors, chemical inhibitors that block downstream signaling pathways, and the like. In some embodiments, the activity or gene expression of CD38 is reduced by 1%-100% (i.e., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%).

[0271] In a further embodiment, cells as disclosed herein include suppressed expression of phosphatase and tensin homologue (PTEN) in primary immune cells, such that the activity of PTEN is eliminated or reduced. PTEN can be down-regulated or blocked by any suitable method or technique known in the art. Known methods for down-regulating the gene expression of PTEN or reducing the activity of PTEN include but are not limited to CRISPR / Cas (including cytosine and adenine base editors), microRNA, shRNA, RNAi, TALEN, zinc finger nucleases, large range nucleases, neutralizing antibodies, small molecule inhibitors, chemical inhibitors that block downstream signal transduction pathways, etc. In some embodiments, the activity or gene expression of PTEN is reduced by 1%-100% (i.e., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%).

[0272] The term "T REX " refers to "regeneratively expandable T cells" using, for example, the techniques and genetic modifications provided herein. More specifically, T REX The cells refer to cells in which the expression of some or all of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) is reduced or eliminated.

[0273] In some embodiments, the inhibition of expression of one or more endogenous regulatory factors occurs after the one or more transgenes are introduced into the cells. In some aspects, the cells into which the one or more transgenes have been introduced are cultured for at least 2 days, at least 5 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days prior to the inhibition of one or more endogenous regulatory factors. In further embodiments, the inhibition of expression of PTEN occurs after the one or more transgenes are introduced into the cells. In some embodiments, the method comprises the following sequential steps: i) introducing one or more transgenes into immune cells and then culturing the cells for at least 2 days, 5 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days; ii) inhibiting one or more endogenous regulatory factors by culturing the cells for at least 2 days, 5 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days; and iii) inhibiting PTEN expression.

[0274] T cells / T REX SMART / activation and expansion of cells

[0275] The present disclosure also relates to methods for culturing T cells transduced with chimeric antigen receptors (CARs) that produce persistent T cell populations exhibiting increased antigen-independent activation. The acronym "SMART" (short-term manipulated, autonomously replicating T cells) refers to a short-term T cell manufacturing and expansion process in which cells are cultured in the presence of IL-21 (and optionally IL-2).

[0276] Some aspects of the present disclosure relate to cells comprising polynucleotides or polypeptides disclosed herein. Some aspects of the present disclosure relate to a cell comprising (i) a polynucleotide encoding a chimeric antigen receptor (CAR) that binds to human BCMA. In some embodiments, the cell further comprises (ii) a polynucleotide encoding an armor molecule. In some embodiments, the cell is an immune cell. In some embodiments, the cell is an autologous cell of the recipient. In some embodiments, the cell is selected from the group consisting of: T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, γδT cells, TSCM cells, CMV+T cells, tumor infiltrating lymphocytes, and any combination thereof. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0277] Before T cell amplification and gene modification of the present disclosure, T cell sources are obtained from the subject. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infection site, ascites, pleural effusion, spleen tissue and tumor. In certain embodiments of the present disclosure, many T cell lines available in the art can be used. In certain embodiments of the present disclosure, many techniques known to the skilled person can be used, such as FicollTM separation, to obtain T cells from a unit of blood collected from the subject. In one embodiment, cells from individual circulating blood are obtained by apheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma portion, and the cells are placed in an appropriate buffer or medium for subsequent processing steps. In some embodiments, cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the washing solution lacks calcium ions and may lack magnesium ions or may lack many (if not all) divalent cations. Similarly, the initial activation step in the absence of calcium ions results in amplified activation. As will be readily appreciated by those skilled in the art, the washing step can be accomplished by methods known to those skilled in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, unwanted components in the apheresis can be removed and the cells can be directly resuspended in culture medium.

[0278] In other embodiments, by lysing red blood cells and depleting monocytes, for example, by PERCOLL TMGradient centrifugation or countercurrent centrifugal elutriation is used to separate T cells from peripheral blood lymphocytes. Specific subpopulations of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further separated by positive or negative selection techniques. In some embodiments, T cells are separated by positive selection for CD4 and CD8 expression. For example, in one embodiment, T cells are separated by incubating with anti-CD4 / anti-CD8 conjugated microbeads for a period of time sufficient to positively select the desired T cells. In one embodiment, the period is about 30 minutes. In another embodiment, the period ranges from 30 minutes to 36 hours or longer, and all integer values ​​therebetween. In another embodiment, the period is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. In yet another embodiment, the period is 10 hours to 24 hours. In any case where there are only a few T cells compared to other cell types, such as when separating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals, longer incubation times can be used to separate T cells. In addition, using longer incubation times can increase the capture efficiency of CD8+ T cells. Therefore, by simply shortening or extending the time allowing T cells to bind to CD4 / CD8 microbeads and / or by increasing or decreasing the ratio of microbeads to T cells (as further described herein), T cell subsets can be preferentially selected at the beginning of culture or at other time points during the process. In addition, by increasing or decreasing the ratio of anti-CD4 and / or anti-CD8 antibodies on microbeads or other surfaces, T cell subsets can be preferentially selected at the beginning of culture or at other desired time points. Those skilled in the art will recognize that multiple rounds of selection can also be used in the context of the present disclosure. In certain embodiments, it may be desirable to perform a selection procedure and use "unselected" cells in the activation and amplification process. "Unselected" cells can also be selected in other rounds.

[0279] Enrichment of T cell populations by negative selection can be accomplished with a combination of antibodies for surface markers specific to the cells selected negatively. One method is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry, which uses a mixture of monoclonal antibodies for cell surface markers present on the cells selected negatively. For example, in order to enrich CD4+ cells by negative selection, the monoclonal antibody mixture typically includes antibodies for CD14, CD20, CD11b, CD16, and HLA-DR. In certain embodiments, it may be desirable to enrich or positively select regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain embodiments, regulatory T cells are consumed by anti-C25-conjugated microbeads or other similar selection methods.

[0280] In order to separate the desired cell mass by positive selection or negative selection, the concentration of cells and surfaces (for example, particles such as microbeads) can be changed. In certain embodiments, it may be desirable to significantly reduce the volume (that is, increase the concentration of cells) that microbeads and cells mix together to ensure the maximum contact of cells and microbeads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In another embodiment, greater than 100 million cells / ml is used. In another embodiment, a cell concentration of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75 million, 80 million, 85 million, 90 million, 95 million or 100 million cells / ml is used. In other embodiments, a concentration of 125 million or 150 million cells / ml can be used. Use of high concentrations can result in increased cell yield, cell activation, and cell expansion.

[0281] In a related embodiment, it may be desirable to use a lower concentration of cells. By significantly diluting the mixture of T cells and a surface (e.g., particles such as microbeads), interactions between the particles and the cells are minimized. This selects cells that express a large amount of the desired antigen to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than a dilute concentration of CD8+ T cells. In one embodiment, the cell concentration used is 5×10 6 In other embodiments, the concentration used may be about 1×10 5 cells / ml to 1×10 6 per ml, and any integer value in between.

[0282] In other embodiments, cells can be incubated at 2°C-10°C or at room temperature on a rotator at different speeds for different lengths of time.

[0283] The T cells used for stimulation can also be frozen after the washing steps. In some embodiments, the freezing and subsequent thawing steps can provide a more uniform product by removing granulocytes and, to a certain extent, monocytes from the cell population. After the washing steps to remove plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and would be useful in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, followed by freezing the cells to -80°C at a rate of 1° / minute and storing in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods can be used, as well as immediate uncontrolled freezing at -20°C or in liquid nitrogen.

[0284] In certain embodiments, cryopreserved cells are thawed and washed and allowed to stand at room temperature for one hour prior to activation using the methods of the disclosure.

[0285] In the context of the present disclosure, it is also contemplated that a period of time before the cells of amplification as described herein may be needed, blood samples are collected from the subject or products are collected separately. Therefore, the source of the cell to be amplified can be collected at any necessary time point, and the desired cells (such as T cells) are separated and frozen for use later in the T cell therapy of many diseases or conditions (such as those described herein) that will benefit from T cell therapy. In one embodiment, a blood sample or a single blood component is taken from a subject in overall health. In certain embodiments, a blood sample or a single blood component is taken from a subject in the overall health of the risk of developing a disease but not yet developing a disease, and the cells of interest are separated and frozen for use later. In certain embodiments, T cells can be amplified, frozen and used later. In certain embodiments, samples are collected from a patient soon after diagnosing a specific disease as described herein but before any treatment. In another embodiment, cells are isolated from a blood sample or apheresis of a subject prior to a number of relevant treatment modalities including, but not limited to, treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506), antibodies or other immunoablative agents (such as CAMPATH, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228), and radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signaling (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In another embodiment, the cells are isolated for use in the patient and frozen for later use in combination with (e.g., before, simultaneously with, or after) bone marrow or stem cell transplantation, T cell ablative therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cells are isolated before B cell ablative therapy (such as an agent that reacts with CD20, e.g., Rituxan), and the cells can be frozen for later use in treatment.

[0286] In another embodiment of the present disclosure, T cells are directly obtained from the patient after treatment. In this regard, it has been observed that after certain cancer treatments, especially after treatment with drugs that damage the immune system, soon after treatment, during normal recovery from treatment in the patient, the quality of the T cells obtained may be optimal or improved for the ability of their ex vivo expansion. Similarly, after ex vivo manipulation using the methods described herein, these cells can be in a preferred state for enhancing implantation and in vivo expansion. Therefore, in the context of the present disclosure, it is considered that blood cells are collected during the convalescent period, including other cells of T cells, dendritic cells or hematopoietic lineages. In addition, in certain embodiments, mobilization (for example, mobilization with GM-CSF or G-CSF) and conditioning regimens can be used for producing in the subject conditions that are wherein conducive to the regeneration, recirculation, regeneration and / or expansion of specific cell types, especially during the limited time window after treatment. Illustrative cell types include T cells, B cells, dendritic cells and other cells of the immune system.

[0287] Whether before or after genetically modifying T cells to express the desired CAR, T cells can generally be activated and expanded using methods such as those described in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0288] Typically, the T cells of the present disclosure are amplified by contacting the surface of the medicament to which the CD3 / TCR complex-related signal is attached and the ligand for stimulating the co-stimulatory molecules on the T cell surface. Specifically, T cell groups can be stimulated as described herein, such as by contacting with anti-CD3 antibodies or their antigen-binding fragments or anti-CD2 antibodies fixed on the surface, or by contacting with protein kinase C activators (e.g., bryostatin) together with calcium ion carriers. In order to costimulate the auxiliary molecules on the T cell surface, a ligand for binding auxiliary molecules is used. For example, under conditions suitable for stimulating T cell proliferation, T cell groups are contacted with anti-CD3 antibodies and anti-CD28 antibodies. In order to stimulate the proliferation of CD4+T cells or CD8+T cells, anti-CD3 antibodies and anti-CD28 antibodies are used. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besangon, France). Other methods known in the art may also be used (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):1319-328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0289] In certain embodiments, the primary stimulation signal and the costimulatory signal of T cell can be provided by different schemes.For example, the medicament providing each signal can be in solution or coupled to the surface.When coupled to the surface, the medicament can be coupled to the same surface (i.e., in " cis " form) or to a separate surface (i.e., in " trans " form). Alternatively, a kind of medicament can be coupled to the surface, and another kind of medicament is in solution. In one embodiment, the medicament providing the costimulatory signal is combined with the cell surface, and the medicament providing the primary activation signal is in solution or coupled to the surface. In certain embodiments, both medicaments can be in solution. In another embodiment, the medicament can be a soluble form, which is then cross-linked to the surface, such as cells or antibodies expressing Fc receptors or other binding agents that will be combined with the medicament. In this regard, referring to, for example, artificial antigen presenting cells (aAPCs) in U.S. Patent Application Publication No. 20040101519 and No. 20060034810, it is contemplated that these cells are used in the present invention for activation and amplification of T cells.

[0290] In one embodiment, two agents are immobilized on microbeads, either on the same microbead, i.e., "cis", or on separate microbeads, i.e., "trans". For example, the agent that provides the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent that provides the co-stimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof, and the two agents are co-immobilized on the same microbead with equal molecular weight. In one embodiment, a 1:1 ratio of each antibody bound to the microbeads is used for CD4+ T cell expansion and T cell growth. In certain embodiments of the present disclosure, a ratio of anti-CD3:CD28 antibodies bound to the microbeads is used such that an increase in T cell expansion is observed compared to the expansion observed using a 1:1 ratio.

[0291] In other embodiments of the present disclosure, cells (such as T cells) are combined with agent-coated microbeads, the microbeads and cells are subsequently separated, and the cells are then cultured. In an alternative embodiment, the agent-coated microbeads and cells are not separated before culture, but are cultured together. In another embodiment, the microbeads and cells are first concentrated by applying a force (such as a magnetic force), causing an increase in the connection of cell surface markers, thereby inducing cell stimulation.

[0292] Suitable conditions for T cell culture include an appropriate culture medium (e.g., minimum essential medium or RPMI medium 1640 or X-vivo 15 (Lonza)), which may contain factors necessary for proliferation and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), IL-21, insulin, IFN-7, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α or any other additives known to those skilled in the art for cell growth. Other additives for cell growth include, but are not limited to, surfactants, human plasma protein powder (plasmanate) and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Culture medium can comprise RPMI1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer added with amino acids, sodium pyruvate and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a set of defined hormones, and / or cytokines sufficient for the growth and expansion of T cells. Antibiotics (e.g., penicillin and streptomycin) are included only in experimental cultures and are not included in cultures of cells to be infused into a subject. Target cells are maintained under conditions necessary to support growth, e.g., appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO 2). In one embodiment, culture medium is an X-VIVO 15 serum-free culture medium containing 1% (v / v) recombinant serum replacement (ITSE-A).

[0293] In one embodiment, T cells are cultured in a culture medium containing 10IU / mL to 300IU / mL recombinant human IL-2. In one embodiment, T cells are cultured in a culture medium containing 10IU / mL, 15IU / mL, 20IU / mL, 25IU / mL, 30IU / mL, 35IU / mL, 40IU / mL, 45IU / mL, 50IU / mL, 55IU / mL, 60IU / mL, 65IU / mL, 70IU / mL, 75IU / mL, 80IU / mL, 85IU / mL, 90IU / mL, 95IU / mL, 100IU / mL, 200IU / mL or 300IU / mL recombinant human IL-2. In another embodiment, T cells are cultured in a culture medium also containing 0.1U / mL to 0.3U / mL recombinant IL-21. In another embodiment, T cells are cultured in medium containing IL-2 and 0.1 U / mL, 0.2 U / mL, 0.5 U / mL, 1 U / mL, 2 U / mL, 5 U / mL, 10 U / mL, 15 U / mL, 20 U / mL, 25 U / mL, 30 U / mL, 40 U / mL, 50 U / mL, 75 U / mL, or 100 U / mL of recombinant human IL-21. In another embodiment, T cells are cultured in a culture medium containing IL-2 and 0.10U / mL, 0.11U / mL, 0.12U / mL, 0.13U / mL, 0.14U / mL, 0.15U / mL, 0.16U / mL, 0.17U / mL, 0.18U / mL, 0.19U / mL, 0.20U / mL, 0.21U / mL, 0.22U / mL, 0.23U / mL, 0.24U / mL, 0.25U / mL, 0.26U / mL, 0.27U / mL, 0.28U / mL, 0.29U / mL or 0.30U / mL recombinant human IL-21. In one embodiment, T cells are cultured in a culture medium containing 40IU / mL recombinant human IL-2 and 0.24U / mL recombinant human IL-21.

[0294] In one embodiment of the present disclosure, the cells are cultured for up to 14 days. In another embodiment, the mixture can be cultured for 4 days. The T cells can be stirred at any stage of the culture. In one embodiment, the cells are stirred in a culture medium containing IL-2 and IL-21 during the cell culture. In certain embodiments, the T cells harvested on day 4 exhibit higher target-independent killing activity compared to the CAR-T cells harvested on day 6.

[0295] In one embodiment of the present disclosure, CD8 +T cells. The cells were cryopreserved or activated using CD3 / CD28 stimulation immediately after isolation. After 3 days of activation, CRISPR knockout of CDKN2A, CDKN2B, and MTAP (called REX editing) was performed to confer resistance to replicative aging. The cells were further manipulated using site-specific CRISPR knock-in of BCMA-CAR. In some embodiments, B2M knockout was performed to limit the patient's CD8 + In some embodiments, CD38 is knocked out to achieve resistance to daratumumab. In some embodiments, cells are edited at the TRAC locus to eliminate TCRαβ expression and eliminate the risk of graft-versus-host disease.

[0296] Vectors, host cells and pharmaceutical compositions disclosed herein

[0297] In some embodiments, the polynucleotides of the present disclosure are present in a vector. Therefore, provided herein are vectors comprising the polynucleotides of the present disclosure. In some embodiments, the disclosure relates to vectors or vector groups comprising polynucleotides encoding CAR as described herein.

[0298] Any carrier known in the art may be suitable for the present disclosure. In some embodiments, the carrier is a viral vector. In some embodiments, the carrier is a retroviral vector, a DNA vector, a murine leukemia virus vector, a SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, a transposon or any combination thereof. In certain embodiments, CAR and / or antibody or its antigen-binding fragment is covered and / or delivered to cells and / or patients by viruses, lentivirus, adenovirus, retrovirus, adeno-associated virus (AAV), transposon, DNA vector, mRNA, lipid nanoparticles (LNP) or CRISPR-Cas systems.

[0299] In other embodiments, provided herein are host cells comprising polynucleotides or vectors of the present disclosure. In some embodiments, the disclosure relates to host cells, such as in vitro cells, which contain polynucleotides encoding CAR or TCR, as described herein. In other embodiments, the disclosure relates to in vitro cells, which contain polypeptides encoded by polynucleotides encoding CARs that specifically bind to BCMA. In other embodiments, the disclosure relates to cells, such as in vitro cells, which contain polypeptides encoded by polynucleotides encoding antibodies or antigen binding molecules thereof that specifically bind to BCMA, as disclosed herein.

[0300] Any cell can be used as a host cell for the polynucleotides, vectors or polypeptides of the present disclosure. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell or a higher eukaryotic cell, such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as gram-negative organisms or gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, for example Escherichia coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example Salmonella typhimurium; Serratia, for example Serratia marcescens. Marcescens and Shigella; Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell.

[0301] Other embodiments of the present disclosure relate to compositions comprising polynucleotides as described herein, carriers as described herein, polypeptides as described herein, or cells as described herein. In some embodiments, the composition includes a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and / or adjuvant. In some embodiments, the composition includes an excipient. In one embodiment, the composition includes polynucleotides encoding CAR, wherein the CAR includes antigen binding molecules specifically bound to BCMA. In another embodiment, the composition includes CAR encoded by the polynucleotides of the present disclosure, wherein the CAR includes antigen binding molecules specifically bound to BCMA. In another embodiment, the composition includes T cells containing polynucleotides encoding CAR, wherein the CAR includes antigen binding molecules specifically bound to BCMA. In another embodiment, the composition includes cells (e.g., T cells, such as CAR-T cells) containing polynucleotides encoding CAR, and the CAR includes antigen binding domains specifically bound to BCMA, as disclosed herein.

[0302] In other embodiments, the composition is configured to be delivered parenterally, for suction or for delivery by the digestive tract, such as oral.The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art. In certain embodiments, the composition is maintained at physiological pH or slightly lower pH using buffer, typically maintained in the pH range of about 5 to about 8. In certain embodiments, when considering parenteral administration, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution with or without an additional therapeutic agent in a pharmaceutically acceptable vehicle. In certain embodiments, the vehicle for parenteral injection is a sterile distilled water with or without at least one additional therapeutic agent, which is formulated as a sterile isotonic solution, suitably preserved. In certain embodiments, preparation is directed to preparing the desired molecule together with a polymeric compound (such as polylactic acid or polyglycolic acid), microbeads or liposomes, which provide the controlled or sustained release of the product, are then delivered via reservoir injection. In certain embodiments, an implantable drug delivery device is used to introduce the desired molecule.

[0303] Treating Disease with CARs

[0304] In some embodiments, the present disclosure provides CAR cells for treating diseases. In certain embodiments, the present disclosure provides CAR cells for treating cancer and / or hematological malignancies. In one embodiment, the present disclosure provides CAR cells for treating cancer and / or hematological malignancies expressing BCMA. Compositions described herein (e.g., CAR constructs and CAR cells) and methods of use thereof are particularly useful for inhibiting neoplastic cell growth or spread; particularly neoplastic cell growth in which BCMA plays a role.

[0305] In one embodiment, it is contemplated that the cancer treated herein includes any cancer expressing BCMA on the cell surface of cancer cells.It is contemplated herein that the cancer treated may include but is not limited to multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphoblastic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).In one embodiment, the present disclosure provides CAR cells for the treatment of multiple myeloma.

[0306] In some embodiments, the present disclosure provides CAR cells for treating autoimmune diseases. In certain embodiments, the present disclosure provides CAR cells for treating autoimmune diseases involving BCMA. Compositions described herein (e.g., CAR constructs and CAR cells) and methods of use thereof are particularly useful for treating autoimmune diseases in which BCMA plays a role. In certain embodiments, the present disclosure provides CAR cells for treating lupus.

[0307] Treatment

[0308] CAR modified cells of the present disclosure, such as CAR T cells, can be administered alone or as a pharmaceutical composition with a diluent and / or other components associated with cytokines or cell groups. In short, the pharmaceutical composition of the present disclosure may include, for example, CAR cells as described herein, and one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers, such as neutral buffered saline, buffered saline, etc.; sulfates; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins, polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The pharmaceutical composition of the present disclosure may be suitable for treatment (or prevention).

[0309] In some embodiments, the present disclosure provides a method for treating a disease by administering an effective amount of cells comprising an anti-BCMA chimeric antigen receptor (CAR) to a subject in need thereof, the anti-BCMA chimeric antigen receptor comprising an antigen binding domain. The antigen binding domain can be an antibody, Fab or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). In certain embodiments, the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 11, 20, 29, 38, 47, 56, 65, 74, and 83; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 12, 21, 30, 39, 48, 57, 66, 75, and 84; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 13, 22, 31, 40, 49, 58, 67, 76, and 85; and the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 15, 24, 33, 42, 51, 60, 69, 78, and 87; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 16, 25, 34, 43, 52, 61, 70, 79, and 88; and a CDR4 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: NO:8,17,26,35,44,53,62,71,80 and 89 amino acid sequence CDR3. In certain embodiments, the VH comprises an amino acid sequence selected from SEQ ID NO:1,10,19,28,37,46,55,64,73 and 82. In some embodiments, the VL comprises an amino acid sequence selected from SEQ ID NO:5,14,23,32,41,50,59,68,77 and 86. In one aspect, the present disclosure provides an anti-BCMACAR comprising the amino acid sequence as shown in SEQ ID NO:96 or consisting of the amino acid sequence as shown in SEQ ID NO:96. In another aspect, the present disclosure provides an anti-BCMACAR comprising the amino acid sequence as shown in SEQ ID NO:97 or consisting of the amino acid sequence as shown in SEQ ID NO:97. In yet another aspect, the present disclosure provides an anti-BCMACAR comprising the amino acid sequence as shown in SEQ ID NO:98 or consisting of the amino acid sequence as shown in SEQ ID NO:98.

[0310] As used herein, the term "effective amount" or "therapeutically effective amount" of an administered therapeutic substance (such as CAR T cells) is an amount sufficient to carry out a specifically described or intended purpose (such as treating a disease or disease treatment). An "effective amount" can be determined empirically with respect to the described purpose. In certain embodiments, a therapeutically effective amount may refer to the number of cells administered to a subject in need of treatment. The number of cells per dose, the number of doses, and the frequency of administration will depend on various parameters, such as the patient's age, weight, clinical assessment, disease type, cancer type, tumor type, tumor load, and / or other factors, including the judgment of the attending physician.

[0311] In some embodiments, the cancer treated by the method is multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphoblastic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). In one embodiment, the present disclosure provides a CAR cell for treating multiple myeloma.

[0312] In some embodiments, the present disclosure provides CAR cells for treating autoimmune diseases. In certain embodiments, the method of the present disclosure provides CAR cells for treating autoimmune diseases involving BCMA. Compositions described herein (e.g., CAR constructs and CAR cells) and methods of use thereof are particularly useful for treating autoimmune diseases in which BCMA plays a role. In certain embodiments, the present disclosure provides CAR cells for treating lupus.

[0313] It should be understood that the specific aspects of the description described herein are not limited to the specific embodiments presented and may vary. It should also be understood that the terminology used herein is only for the purpose of describing specific aspects and is not intended to be limiting unless expressly defined herein. In addition, as will be appreciated by those skilled in the art, the specific embodiments disclosed herein may be combined with other embodiments disclosed herein without limitation.

[0314] Implementation Plan :

[0315] Embodiment 1. An isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises:

[0316] (a) an antigen-binding domain specific for B-cell maturation antigen (BCMA);

[0317] (b) a transmembrane domain; and

[0318] (c) one or more intracellular domains.

[0319] Embodiment 2. The isolated nucleic acid sequence of embodiment 1, wherein the antigen binding domain comprises an antibody or antigen binding fragment thereof, Fab, Fab', F(ab')2, Fd, Fv, a single-chain variable fragment (scFv), a single-chain antibody, VHH, vNAR, a nanobody (single domain antibody), or any combination thereof.

[0320] Embodiment 3. The isolated nucleic acid sequence of embodiment 2, wherein the antigen binding domain is a single-chain variable fragment (scFv).

[0321] Embodiment 4. The isolated nucleic acid sequence of embodiment 3, wherein the antigen binding domain is a scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 36, and 90.

[0322] Embodiment 5. The isolated nucleic acid sequence of embodiment 3, wherein the antigen binding domain is a scFv comprising the amino acid sequence of SEQ ID NO: 9.

[0323] Embodiment 6. The isolated nucleic acid sequence of any one of embodiments 1 to 5, wherein the transmembrane domain comprises a transmembrane domain selected from the transmembrane domains of CD4, CD8α, or CD28.

[0324] Embodiment 7. The isolated nucleic acid sequence of embodiment 6, wherein the transmembrane domain comprises a CD28 transmembrane domain.

[0325] Embodiment 8. The isolated nucleic acid sequence of any one of Embodiments 1 to 7, wherein the one or more intracellular domains comprises a costimulatory domain or a portion thereof.

[0326] Embodiment 9. An isolated nucleic acid sequence according to embodiment 8, wherein the costimulatory domain comprises one or more of the following: CD3z, 4-1BB, CD2, CD27, CD28, OX-40, ICOS, IL-2Rβ, GITR, MyD88 / CD40a costimulatory domains and / or variants thereof.

[0327] Embodiment 10. The isolated nucleic acid sequence of any one of embodiments 1 to 9, wherein the intracellular domain comprises a CD3z co-stimulatory domain and a CD28 co-stimulatory domain.

[0328] Embodiment 11. An isolated nucleic acid sequence according to any one of embodiments 1 to 9, wherein the intracellular domain comprises a CD3z co-stimulatory domain and a 4-1BB co-stimulatory domain.

[0329] Embodiment 12. An isolated nucleic acid sequence according to any one of embodiments 1 to 9, wherein the intracellular domain comprises a CD3z co-stimulatory domain, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain.

[0330] Embodiment 13. An isolated nucleic acid sequence according to any one of embodiments 1 to 12, wherein the CAR further comprises a hinge / spacer domain, optionally wherein the hinge / spacer domain is located between the antigen binding domain and the transmembrane domain.

[0331] Embodiment 14. An isolated nucleic acid sequence according to embodiment 13, wherein the hinge / spacer domain comprises an IgG1 hinge domain or a variant thereof, an IgG2 hinge domain or a variant thereof, an IgG3 hinge domain or a variant thereof, an IgG4 hinge domain or a variant thereof, an IgG4P domain, a CD8 hinge domain or a variant thereof, or a CD28 hinge domain or a variant thereof.

[0332] Embodiment 15. The isolated nucleic acid sequence of embodiment 14, wherein the hinge / spacer domain is an IgG4 hinge / spacer or a variant thereof, optionally an IgG4P hinge / spacer comprising an S241P mutation.

[0333] Embodiment 16. An isolated nucleic acid sequence according to any one of embodiments 1 to 15, wherein the nucleic acid sequence encodes a CAR having an amino acid sequence as shown in SEQ ID NO: 96.

[0334] Embodiment 17. An isolated nucleic acid sequence according to any one of embodiments 1 to 15, wherein the nucleic acid sequence encodes a CAR having an amino acid sequence as shown in SEQ ID NO: 97.

[0335] Embodiment 18. An isolated nucleic acid sequence according to any one of embodiments 1 to 15, wherein the nucleic acid sequence encodes a CAR having an amino acid sequence as shown in SEQ ID NO: 98.

[0336] Embodiment 19. An anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL);

[0337] wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 30, and 84;

[0338] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 31, and 85; and

[0339] wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 34, and 88;

[0340] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 35, and 89.

[0341] Embodiment 20. The anti-BCMACAR of embodiment 19, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 28, and 82.

[0342] Embodiment 21. The anti-BCMACAR of embodiment 19 or 28, wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 32, and 86.

[0343] Embodiment 22. An anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL);

[0344] wherein the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2;

[0345] a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and

[0346] wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6;

[0347] a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0348] Embodiment 23. The anti-BCMACAR of any one of Embodiments 19 to 22, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO: 5.

[0349] Embodiment 24. An anti-BCMACAR according to any one of Embodiments 19 to 23, wherein the CAR comprises a transmembrane domain and one or more intracellular domains.

[0350] Embodiment 25. The anti-BCMACAR of any one of Embodiments 19 to 24, wherein the transmembrane domain comprises a transmembrane domain selected from the transmembrane domains of CD4, CD8α, or CD28.

[0351] Embodiment 26. The anti-BCMACAR of embodiment 25, wherein the transmembrane domain comprises a CD28 transmembrane domain.

[0352] Embodiment 27. An anti-BCMACAR according to any one of embodiments 19 to 26, wherein the one or more intracellular domains comprise a costimulatory domain or a portion thereof.

[0353] Embodiment 28. An anti-BCMACAR according to embodiment 27, wherein the costimulatory domain comprises one or more of the following: CD3z, 4-1BB, CD2, CD27, CD28, OX-40, ICOS, IL-2Rβ, GITR, MyD88 / CD40a costimulatory domains and / or variants thereof.

[0354] Embodiment 29. The anti-BCMACAR of any one of Embodiments 24 to 28, wherein the intracellular domain comprises a CD3z co-stimulatory domain and a CD28 co-stimulatory domain.

[0355] Embodiment 30. An anti-BCMACAR according to any one of embodiments 24 to 28, wherein the intracellular domain comprises a CD3z co-stimulatory domain and a 4-1BB co-stimulatory domain.

[0356] Embodiment 31. An anti-BCMACAR according to any one of Embodiments 24 to 28, wherein the intracellular domain comprises a CD3z co-stimulatory domain, a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain.

[0357] Embodiment 32. An anti-BCMACAR according to any one of Embodiments 19 to 31, wherein the CAR further comprises a hinge / spacer domain, optionally wherein the hinge / spacer domain is located between the antigen binding domain and the transmembrane domain.

[0358] Embodiment 33. An anti-BCMACAR according to embodiment 32, wherein the hinge / spacer domain comprises an IgG1 hinge domain or a variant thereof, an IgG2 hinge domain or a variant thereof, an IgG3 hinge domain or a variant thereof, an IgG4 hinge domain or a variant thereof, an IgG4P domain, a CD8a hinge domain or a variant thereof, or a CD28 hinge domain or a variant thereof.

[0359] Embodiment 34. The anti-BCMACAR of embodiment 33, wherein the hinge / spacer domain is an IgG4 hinge / spacer or a variant thereof, optionally an IgG4P hinge / spacer comprising an S241P mutation.

[0360] Embodiment 35. The anti-BCMACAR of any one of Embodiments 19 to 34, wherein the CAR has the amino acid sequence as shown in SEQ ID NO: 96.

[0361] Embodiment 36. An anti-BCMACAR according to any one of embodiments 19 to 34, wherein the CAR has the amino acid sequence as shown in SEQ ID NO: 97.

[0362] Embodiment 37. An anti-BCMACAR according to any one of embodiments 19 to 34, wherein the CAR has the amino acid sequence as shown in SEQ ID NO: 98.

[0363] Embodiment 38. A vector comprising the isolated nucleic acid sequence of any one of embodiments 1 to 18 or encoding the chimeric antigen receptor of any one of embodiments 19 to 37, wherein the vector is a virus, a lentivirus, an adenovirus, a retrovirus, an adeno-associated virus (AAV), a transposon, a DNA vector, an mRNA, a lipid nanoparticle (LNP), or a CRISPR-Cas system.

[0364] Embodiment 39. A cell comprising the vector according to embodiment 38.

[0365] Embodiment 40. A cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) according to any one of embodiments 19 to 37, further comprising reduced expression or knockout of one or more endogenous regulatory factors.

[0366] Embodiment 41. A cell according to embodiment 40, wherein the one or more endogenous regulatory factors are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B) and S-methyl-5'-thioadenosine phosphorylase (MTAP).

[0367] Embodiment 42. The cell of any one of embodiments 39 to 41, wherein the cell has reduced expression or knockout of CDKN2A, CDKN2B, and MTAP.

[0368] Embodiment 43. The cell of any one of embodiments 39 to 42, wherein the cell does not express phosphatase and tensin homolog (PTEN).

[0369] Embodiment 44. The cell of any one of Embodiments 39 to 43, further comprising a transgene encoding B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0370] Embodiment 45. The cell of any one of embodiments 39 to 44, wherein the cell does not express one or more endogenous immune-related genes.

[0371] Embodiment 46. The cell of embodiment 45, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

[0372] Embodiment 47. The cell of any one of Embodiments 39 to 46, wherein the cell does not express cluster of differentiation 38 (CD38).

[0373] Embodiment 48. A cell comprising a BCMA-specific antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL);

[0374] wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 30, and 84;

[0375] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 31, and 85; and

[0376] wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 34, and 88;

[0377] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 35, and 89.

[0378] Embodiment 49. A cell according to embodiment 48, wherein the VH comprises an amino acid sequence selected from SEQ ID NO: 1, 28 and 82.

[0379] Embodiment 50. The cell of embodiment 48 or 49, wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 32, and 86.

[0380] Embodiment 51. A cell comprising a BCMA-specific antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL);

[0381] wherein the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2;

[0382] a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and

[0383] wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6;

[0384] a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0385] Embodiment 52. The cell of any one of embodiments 48 to 51, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO: 5.

[0386] Embodiment 53. The cell of any one of embodiments 48 to 52, further comprising reduced expression or knockout of one or more endogenous regulatory factors.

[0387] Embodiment 54. A cell according to embodiment 53, wherein the one or more endogenous regulatory factors are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B) and S-methyl-5'-thioadenosine phosphorylase (MTAP).

[0388] Embodiment 55. The cell of any one of embodiments 48 to 54, wherein the cell has reduced expression or knockout of CDKN2A, CDKN2B, and MTAP.

[0389] Embodiment 56. The cell of any one of embodiments 48 to 55, wherein the cell does not express phosphatase and tensin homolog (PTEN).

[0390] Embodiment 57. The cell of any one of embodiments 48 to 56, wherein the cell further comprises a transgene encoding B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0391] Embodiment 58. The cell of any one of embodiments 48 to 57, wherein the cell does not express one or more endogenous immune-related genes.

[0392] Embodiment 59. The cell of embodiment 58, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

[0393] Embodiment 60. The cell of any one of embodiments 48 to 59, wherein the cell does not express cluster of differentiation 38 (CD38).

[0394] Embodiment 61. A cell comprising a BCMA-specific antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL);

[0395] wherein the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2;

[0396] a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and

[0397] wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6;

[0398] a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and

[0399] wherein the cells comprise reduced expression or knockout of CDKN2A, CDKN2B, MTAP, B2M, TRAC, and CD38.

[0400] Embodiment 62. A cell according to embodiment 61, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1 and the VL comprises the amino acid sequence of SEQ ID NO: 5.

[0401] Embodiment 63. The cell of Embodiment 61 or Embodiment 62, wherein the BCMA-specific antigen binding domain comprises the amino acid sequence shown in SEQ ID NO: 96.

[0402] Embodiment 64. The cell of any one of embodiments 48 to 63, wherein the cell is selected from T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes, regulatory T cells, CD8+ T cells, CD4+ T cells, γδ T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells, and / or combinations thereof.

[0403] Embodiment 65. A method of treating a disease, the method comprising:

[0404] administering to a subject in need thereof an effective amount of cells comprising an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL),

[0405] wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 30, and 84;

[0406] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 31, and 85; and

[0407] wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 34, and 88;

[0408] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 35, and 89.

[0409] Embodiment 66. A method according to embodiment 65, wherein the VH comprises an amino acid sequence selected from SEQ ID NO: 1, 28 and 82.

[0410] Embodiment 67. A method according to embodiment 65 or embodiment 66, wherein the VL comprises an amino acid sequence selected from SEQ ID NO: 5, 32 and 86.

[0411] Embodiment 68. A method of treating a disease, the method comprising:

[0412] administering to a subject in need thereof an effective amount of cells comprising an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL),

[0413] wherein the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2;

[0414] a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and

[0415] wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6;

[0416] a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0417] Embodiment 69. The method of any one of embodiments 65 to 68, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO: 5.

[0418] Embodiment 70. The method of any one of Embodiments 65 to 69, further comprising inhibiting cancer growth, inducing cancer regression, and / or prolonging the survival of the subject.

[0419] Embodiment 71. The method of any one of Embodiments 65 to 70, wherein the cell further comprises reduced expression or knockout of one or more endogenous regulatory factors.

[0420] Embodiment 72. A method according to embodiment 71, wherein the one or more endogenous regulatory factors are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B) and S-methyl-5'-thioadenosine phosphorylase (MTAP).

[0421] Embodiment 73. The method of any one of Embodiments 65 to 72, wherein the cell has reduced expression or knockout of CDKN2A, CDKN2B, and MTAP.

[0422] Embodiment 74. The method of any one of Embodiments 65 to 73, wherein the cell does not express phosphatase and tensin homolog (PTEN).

[0423] Embodiment 75. The method of any one of Embodiments 65 to 74, wherein the cell further comprises a transgene encoding B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0424] Embodiment 76. The method of any one of Embodiments 65 to 75, wherein the cell does not express one or more endogenous immune-related genes.

[0425] Embodiment 77. The method of embodiment 76, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

[0426] Embodiment 78. The cell of any one of Embodiments 65 to 77, wherein the cell does not express cluster of differentiation 38 (CD38).

[0427] Embodiment 79. The method of any one of Embodiments 65 to 78, wherein the cells are autologous cells.

[0428] Embodiment 80. The method of any one of Embodiments 65 to 78, wherein the cells are allogeneic cells.

[0429] Embodiment 81. The method of any one of Embodiments 65 to 80, wherein the cells are selected from T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes, regulatory T cells, CD8+ T cells, CD4+ T cells, γδ T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells, and / or combinations thereof.

[0430] Embodiment 82. The method of any one of Embodiments 65 to 81, wherein the disease is a cancer selected from multiple myeloma (MM), chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).

[0431] Embodiment 83. The method of embodiment 82, wherein the cancer is multiple myeloma.

[0432] Embodiment 84. The method of any one of Embodiments 65 to 81, wherein the disease is an autoimmune disease.

[0433] Embodiment 85. The method of embodiment 84, wherein the autoimmune disease is lupus.

[0434] Embodiment 86. A pharmaceutical composition comprising the isolated nucleic acid of any one of embodiments 1 to 18, the anti-BCMACAR of any one of embodiments 19 to 37, the vector of embodiment 38, or the cell of any one of embodiments 39 to 64, and a pharmaceutically acceptable excipient.

[0435] Embodiment 87. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject the isolated nucleic acid of any one of embodiments 1 to 18, the anti-BCMACAR of any one of embodiments 19 to 37, the vector of embodiment 38, the cell of any one of embodiments 39 to 64, or the pharmaceutical composition of embodiment 78.

[0436] Embodiment 88. The method of embodiment 87, wherein the disease is cancer or an autoimmune disease.

[0437] Embodiment 89. The method of embodiment 88, wherein the cancer is selected from multiple myeloma (MM), chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).

[0438] Embodiment 90. The method of embodiment 89, wherein the cancer is multiple myeloma.

[0439] Embodiment 91. The method of embodiment 88, wherein the autoimmune disease is lupus.

[0440] Embodiment 92. Use of the isolated nucleic acid of any one of embodiments 1 to 18, the anti-BCMACAR of any one of embodiments 19 to 37, the vector of embodiment 38, the cell of any one of embodiments 39 to 64, or the pharmaceutical composition of embodiment 78 for treating a disease in a subject in need thereof.

[0441] Embodiment 93. The use according to embodiment 92, wherein the disease is cancer or an autoimmune disease.

[0442] Embodiment 94. The use according to embodiment 93, wherein the cancer is selected from multiple myeloma (MM), chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).

[0443] Embodiment 95. The use according to embodiment 94, wherein the cancer is multiple myeloma.

[0444] Embodiment 96. The use according to embodiment 93, wherein the autoimmune disease is lupus.

[0445] Embodiment 97. Use of an engineered cell for the manufacture of a medicament for treating a disease in a patient, wherein the engineered cell comprises an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL),

[0446] wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 30, and 84;

[0447] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 31, and 85; and

[0448] wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 34, and 88;

[0449] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 35, and 89.

[0450] Embodiment 98. The use according to embodiment 99, wherein the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0451] Embodiment 99. The use according to embodiment 97, wherein the VH comprises an amino acid sequence selected from SEQ ID NO: 1, 28 and 82.

[0452] Embodiment 100. The use according to embodiment 97, wherein the VL comprises an amino acid sequence selected from SEQ ID NO: 5, 32 and 86.

[0453] Embodiment 101. The use of any one of Embodiments 97 to 100, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO: 5.

[0454] Embodiment 102. The use according to any one of Embodiments 97 to 101, wherein the CAR has the amino acid sequence as shown in SEQ ID NO:96.

[0455] Embodiment 103. The use according to any one of Embodiments 97 to 100, wherein the CAR has the amino acid sequence as shown in SEQ ID NO:97.

[0456] Embodiment 104. The use according to any one of Embodiments 97 to 100, wherein the CAR has the amino acid sequence as shown in SEQ ID NO:98.

[0457] Embodiment 105. The use according to any one of Embodiments 97 to 104, further comprising inhibiting cancer growth, inducing cancer regression and / or prolonging the survival of the subject.

[0458] Embodiment 106. The use according to any one of embodiments 97 to 105, wherein the engineered cell further comprises reduced expression or knockout of one or more endogenous regulatory factors.

[0459] Embodiment 107. The use according to embodiment 106, wherein the one or more endogenous regulatory factors are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B) and S-methyl-5'-thioadenosine phosphorylase (MTAP).

[0460] Embodiment 108. The use according to any one of embodiments 97 to 107, wherein the engineered cell has reduced expression or knockout of CDKN2A, CDKN2B and MTAP.

[0461] Embodiment 109. The use according to any one of embodiments 97 to 108, wherein the engineered cell does not express phosphatase and tensin homolog (PTEN).

[0462] Embodiment 110. The use of any one of Embodiments 97 to 109, wherein the engineered cell further comprises a transgene encoding B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0463] Embodiment 111. The use of any one of Embodiments 97 to 110, wherein the engineered cell does not express one or more endogenous immune-related genes.

[0464] Embodiment 112. The use according to embodiment 111, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

[0465] Embodiment 113. The use according to any one of Embodiments 97 to 112, wherein the engineered cells do not express cluster of differentiation 38 (CD38).

[0466] Embodiment 114. The use according to any one of embodiments 97 to 113, wherein the engineered cells are autologous cells.

[0467] Embodiment 115. The use of any one of Embodiments 97 to 113, wherein the engineered cells are allogeneic cells.

[0468] Embodiment 116. The use according to any one of Embodiments 97 to 115, wherein the engineered cells are selected from T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes, regulatory T cells, CD8+ T cells, CD4+ T cells, γδ T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells and / or combinations thereof.

[0469] Embodiment 117. The use according to any one of Embodiments 97 to 116, wherein the disease is a cancer selected from multiple myeloma (MM), chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).

[0470] Embodiment 118. The use according to embodiment 117, wherein the cancer is multiple myeloma.

[0471] Embodiment 119. The use according to any one of Embodiments 97 to 116, wherein the disease is an autoimmune disease.

[0472] Embodiment 120. The use according to embodiment 119, wherein the autoimmune disease is lupus.

[0473] Embodiment 121. An engineered cell for use in the manufacture of a medicament for treating a disease in a patient, wherein the engineered cell comprises an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL),

[0474] wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 30, and 84;

[0475] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 31, and 85; and

[0476] wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 34, and 88;

[0477] and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 35, and 89.

[0478] Embodiment 122. An engineered cell according to Embodiment 121, wherein the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0479] Embodiment 123. An engineered cell according to embodiment 121, wherein the VH comprises an amino acid sequence selected from SEQ ID NO: 1, 28 and 82.

[0480] Embodiment 124. An engineered cell according to embodiment 121, wherein the VL comprises an amino acid sequence selected from SEQ ID NO: 5, 32 and 86.

[0481] Embodiment 125. The engineered cell of any one of Embodiments 121 to 124, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO: 5.

[0482] Embodiment 126. An engineered cell according to any one of Embodiments 121 to 125, wherein the CAR has an amino acid sequence as shown in SEQ ID NO:96.

[0483] Embodiment 127. An engineered cell according to any one of Embodiments 121 to 124, wherein the CAR has an amino acid sequence as shown in SEQ ID NO:97.

[0484] Embodiment 128. An engineered cell according to any one of Embodiments 121 to 124, wherein the CAR has an amino acid sequence as shown in SEQ ID NO:98.

[0485] Embodiment 129. The engineered cell of any one of Embodiments 121 to 128, wherein the method further comprises inhibiting cancer growth, inducing cancer regression, and / or prolonging the survival of the subject.

[0486] Embodiment 130. The engineered cell of any one of Embodiments 121 to 129, wherein the engineered cell further comprises reduced expression or knockout of one or more endogenous regulatory factors.

[0487] Embodiment 131. An engineered cell according to embodiment 130, wherein the one or more endogenous regulatory factors are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B) and S-methyl-5'-thioadenosine phosphorylase (MTAP).

[0488] Embodiment 132. The engineered cell of any one of Embodiments 121 to 131, wherein the engineered cell has reduced expression or knockout of CDKN2A, CDKN2B, and MTAP.

[0489] Embodiment 133. The engineered cell of any one of Embodiments 121 to 132, wherein the engineered cell does not express phosphatase and tensin homolog (PTEN).

[0490] Embodiment 134. The engineered cell of any one of Embodiments 121 to 133, wherein the engineered cell further comprises a transgene encoding B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0491] Embodiment 135. The engineered cell of any one of Embodiments 121 to 134, wherein the engineered cell does not express one or more endogenous immune-related genes.

[0492] Embodiment 136. The engineered cell of embodiment 135, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

[0493] Embodiment 137. The engineered cell of any one of Embodiments 121 to 136, wherein the engineered cell does not express cluster of differentiation 38 (CD38).

[0494] Embodiment 138. The engineered cell of any one of Embodiments 121 to 137, wherein the engineered cell is an autologous cell.

[0495] Embodiment 139. The engineered cell of any one of Embodiments 121 to 138, wherein the engineered cell is an allogeneic cell.

[0496] Embodiment 140. The engineered cell of any one of Embodiments 121 to 139, wherein the engineered cell is selected from T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes, regulatory T cells, CD8+ T cells, CD4+ T cells, γδ T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells, and / or combinations thereof.

[0497] Embodiment 141. The engineered cell of any one of Embodiments 121 to 140, wherein the disease is a cancer selected from multiple myeloma (MM), chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL).

[0498] Embodiment 142. The engineered cell of Embodiment 141, wherein the cancer is multiple myeloma.

[0499] Embodiment 143. The engineered cell of any one of Embodiments 121 to 142, wherein the disease is an autoimmune disease.

[0500] Embodiment 144. The engineered cell of Embodiment 143, wherein the autoimmune disease is lupus.

[0501] Example

[0502] The following examples are illustrations of specific embodiments of the present disclosure and their various uses. They are set forth for illustrative purposes only and should not be construed as limiting the scope of the present disclosure in any way.

[0503] Materials and methods :

[0504] cell lines :

[0505] All cells were cultured in culture medium according to the supplier's recommendations and maintained in tissue culture flasks at 37°C in a humidified atmosphere of 5% CO. JJN3, U226B1, MM1S, KMS12BM, KMS34, KMS11, RPMI 8226, NCIH929, and Huh7 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) (DSMZ, Braunschweig, Germany).

[0506] Lentivirus preparation

[0507] Lentiviral vectors were prepared by co-transfecting suspension-adapted HEK293 cells with a proprietary lentiviral transfer vector and a commercially available packaging plasmid mixture (pPACKH1, System Biosciences, Palo Alto, CA, USA). The transfected cells were cultured for 24 hours and then transferred to fresh medium. 48 hours after transfection, the cells were cleared by centrifugation and the supernatant containing the lentivirus was recovered.

[0508] The scheme that uses PEG-it precipitation reagent (System Biosciences) to provide according to manufacturer is purified and concentrated slow virus particle by precipitation.After precipitation from culture supernatant, by centrifugal collection virus particle and it is resuspended in the culture medium of 1 / 100 original volume.Determine functional titer by the serial dilution transduction HEK-293 cell with the concentrated virus of purification.After transduction, the rBCMA labeling cell of fluorophore conjugation is used to determine the percentage of the cell transduced with anti-BCMACAR construct, and then determine its number.The linear fit of the cell number transduced and the virus volume added is calculated titer value (in tfu / mL).

[0509] Example 1. Development and Characterization of Anti-BCMA Antigen Binding Fragments

[0510] BCMA-reactive human antibodies were generated by immunizing transgenic mice (human variable domain repertoire) and generating hybridomas. Antibodies produced by individual hybridoma clones were assayed for binding to human BCMA in an ELISA assay, and antibody V region sequences were recovered for binding to competent clones.

[0511] The candidate antibodies from the above hybridoma screening were converted into scFv-Fc formats, recombinantly expressed, and their target binding affinity was characterized by surface plasmon resonance (SPR). Briefly, anti-human IgG Fc specific antibodies were immobilized on S series CM5 sensor chips (Cytiva, Marlborough, MA, USA) to allow subsequent capture of candidate scFv-Fc molecules. In order to measure the kinetics of BCMA binding, recombinant human BCMA (Acro Biosystems, Newark, DE USA) was passed through the fixed scFv-Fc prepared as described above at a flow rate of 30 μL / min and a concentration ranging from 0.03 nM to 30 nM. After 150 seconds, the injection was stopped and the dissociation was monitored for up to 400 seconds. A global fit across multiple concentrations was used to determine the kinetic parameters of binding as well as K D value( Figure 1 ). Clone 7A8.11 and clone L15 have higher affinity for human BCMA than comparator I, K D The values ​​were 0.44 nM and 0.37 nM, respectively.

[0512] To evaluate the specificity of scFv binding to BCMA, the binding of scFv-Fc molecules to the 6,000-element membrane proteome array (Integral Molecular, Philidelphia, PA USA) was evaluated. Briefly, cDNAs encoding 6,000 unique human membrane proteins were individually transfected into HEK-293T cells and the binding of scFv-Fc molecules binding to BCMA to these transfected cells was evaluated using flow cytometry ( Figure 2 Off-target binding was verified by transfecting 293 cells with plasmids encoding the identified target, protein A, or vector alone. After 36 hours, four-fold serial dilutions of each ligand, starting at 20 μg / ml, were added to the transfected cells to detect ligand binding using flow cytometry. Based on these results, no off-target binding was identified for any of the internally developed clones or the comparator, Benchmark 1 (see US 2017 / 0226216).

[0513] Example 2. CAR conversion of anti-BCMA antigen-binding fragments and functional demonstration in primary T cells .

[0514] Of the prospective BCMA-binding antibodies characterized, 10 were preferentially converted into scFv format and incorporated into CAR constructs for in vitro and in vivo evaluation. To generate a lentiviral expression vector encoding a BCMA-reactive CAR, the BCMA scFv was fused to a human IgG4P hinge, followed by the human CD28 transmembrane domain, the human 4-1BB intracellular domain, and the human CD3z intracellular domain from the N-terminus to the C-terminus. A signal peptide from human CD33 was used to direct the secretion and membrane insertion of the CAR. As described above, sequence-verified constructs were used to generate lentivirus.

[0515] Alternative CAR formats have also been explored, including alternative hinges (CD8, CD28), transmembrane domains (CD8), costimulatory domains (CD28), and CD3ζ domains.

[0516] CAR-T manufacturing

[0517] Primary T cells were activated for 24 hours with Dynabeads (human T-activator CD3 / CD28), and then transduced with a lentivirus encoding anti-BCMACAR. Cells expressing CAR were identified and purified using recombinant BCMA protein labeled with AF647. CAR purity was confirmed via flow cytometry. Purified CAR-T cells were used for the following research. In total, 10 unique scFv CAR constructs were tested for 3 benchmark controls.

[0518] CAR-T cells were labeled with 1 ug / ml of rBCMA conjugated with AF647 (BCMA-AF647), and the fluorescence intensity was measured using flow cytometry. Figure 3 Surface expression of each CAR clone is shown. Clones 33G12.1-1, 33G12,1-2, 13F4.3-1 failed to express CAR on the cell surface, while clone 7a8.11 had the brightest BCMA staining intensity and expression comparable to comparator C (see WO 2018 / 028647).

[0519] CAR-T cells were labeled with BCMA-AF647 at protein concentrations of 0.3 nM–1000 nM, and the fluorescence intensity was measured by flow cytometry. Figure 4 The BCMA binding properties of each anti-BCMACAR-T clone to soluble BCMA antigen are shown. These binding curves demonstrate that the 7a8.11 clone has comparable BCMA binding to the Comparator C benchmark.

[0520] Using representative T cell donors, the expansion kinetics of different BCMA CAR-T clones were followed when cultured in AIM-V medium containing human serum and IL2. Figure 5 Viable cell counts taken every 2-3 days over 80 days to track expansion are shown. Figure 5 Differences in growth kinetics and expansion length of anti-BC MA CAR-T clones in primary T cells are illustrated, and the superior expansion profile of the 7a8.11 CAR-T clone relative to Comparator C and Comparator I is highlighted.

[0521] The evaluation of in vitro CAR-T activity was performed using multiple myeloma cell lines (JJN3, RPMI8226, U226B1, KMS11, KMS12, KMS34, NCIH929, MM1S) engineered to stably express luciferase. In brief, cells were transduced with a lentivirus expressing mCherry / luciferase. mCherry positive cells were selected via FACS sorting, and pure mCherry positive cell groups expressing luciferase were derived. The cells were then co-cultured with BCMACAR-T cells from several donors with different E:T ratios. 24 hours after co-cultivation, the cytotoxicity of multiple myeloma target cells was evaluated by adding luciferin substrate and measuring luminescence by microplate reader assay. Figure 6A and Figure 6B Representative data showing the cytotoxicity of target cell lines when co-cultured with each CAR-T clone and compared with two BCMA CAR-T benchmarks, Comparator I and Comparator C. Figure 6A Figure 2 is a heatmap of the average percentage cytotoxicity of anti-BCMA CAR-T clones across four donors in eight multiple myeloma cell lines after 24 hours at a 1:2 E:T ratio. Figure 6B The bar graph of these identical data of the average cytotoxicity percentage and standard deviation from these 4 donors is shown.All internally derived BCMACAR-T clones demonstrate the killing of multiple myeloma cells, and 7a8.11BCMACAR-T has the cytotoxic effect suitable with comparator 1 and comparator C in all multiple myeloma lines.More specifically, in three cell lines, clone 7a8.11 has the cytotoxicity stronger than comparator 1, and compares the cytotoxicity that only slightly reduces with comparator C.

[0522] In order to measure effector cytokine output, Huh7 target cells are engineered to express BCMA antigen, and parental cells are used as negative control. In brief, Huh7 cells are transfected with a slow virus containing TNFRSF17 and puromycin resistance box, and the cells successfully integrated into the gene are selected based on puromycin resistance. The Huh7BCMA target cells are then co-cultured with anti-BCMACAR-T clones with different E:T, and supernatants are collected at 24 hours to quantitatively assay IFN γ and IL2 output via Meso Scale Discovery assay (MSD). Figure 7Illustrated is a representative effector cytokine production from anti-BCMACAR-T cells when co-cultured with a 1:2 E:T ratio of a huh7 engineered cell line expressing BCMA. All anti-BCMACAR-T cells showed effector cytokine production in response to CAR-T cell activation and killing. The 7a8.11 clone showed the highest level of cytokine production in response to antigen, comparable to the IFNγ and IL2 levels produced by comparator 1 under the same conditions.

[0523] To determine the effect of soluble BCMA on CAR-T killing, Huh7 cells expressing BCMA were seeded on RTCA E plates (Agilent) for impedance-based cytotoxicity measurements by xCELLigence. Soluble recombinant BCMA protein (10 μg / ml) was added to the cells and subsequently co-cultured with BCMA CART cells. Figure 8 The difference in percentage of cell lysis observed 40 hours after co-culture with huh7 cells expressing BCMA in the presence or absence of soluble BCMA protein is illustrated. These data demonstrate that 7A8.11 BCMA CAR-T cells have the highest level of cell lysis across all BCMA CAR-T clones and also show comparable levels of cell lysis to Comparator C in the presence and absence of soluble protein. Supernatants were collected from the co-culture 24 hours after the addition of CAR-T cells, and IFNγ and IL-2 levels were measured by MSD to compare the differences in effector cytokine production in the presence and absence of soluble BCMA antigen in the culture. Figure 9 The results show that soluble BCMA protein has different effects on effector cytokine production. In the presence of soluble antigen, all BCMA CAR-T cells evaluated showed low levels of IFNγ and IL2 production; however, relative to other internally developed CAR clones, 7A8.11CAR-T clones were least negatively affected by the presence of soluble proteins. In order to evaluate the difference in antigen-driven CAR-T amplification and cell persistence, an in vitro continuous killing experiment was carried out, in which CAR-T cells were co-cultured with a 1: 1 E: T ratio with multiple myeloma target cell line JJN3. Every 2-3 days, the co-culture was sampled and the count and viability of T cells and tumor cells were evaluated by flow cytometry. After this determination, the CAR-T / tumor cell co-culture was then "fed" with live JJN3 cells to restore the co-culture to an E: T ratio of 1: 1. Figure 10A Shown are the CAR-T expansion of each clone in a 12-day serial killing experiment (upper panel) and the percentage of cell lysis of target JJN3 cells after each round of co-culture as a measure of CAR-T cell function and persistence (lower panel). Figure 10AThe results showed that 7A8.11 CAR-T cells have antigen-driven expansion and persistence that are superior to other BCMACAR-T clones, with a sustained expansion and prolonged cell lysis based on the target. In addition, 7A8.11 CAR-T cells showed expansion and functional persistence that were equivalent to Comparator C and superior to Comparator I. Figure 10B Shown are the persistence and expansion of CAR-T cells (upper panel) and cytolysis of target cells (lower panel) after repeated antigen stimulation in the presence of soluble BCMA. Figure 10B Similarly, antigen-dependent CAR-T cell expansion and functional persistence were evaluated by co-culture with JJN3 in the presence of soluble recombinant BCMA protein. Compared with the expansion in the absence of soluble BCMA ( Figure 9 A), all CAR-T cells had reduced expansion and shortened persistence. However, 7A8.11 CAR-T cells were still superior in expansion compared to other internal clones and comparator 1. Figure 10A and Figure 10B Based on the two sets of data, 7A8.11 CAR-T cells demonstrated favorable functional persistence and expansion in response to antigens, and exhibited functionality comparable to the comparator benchmarks, Comparator C and Comparator I.

[0524] In vivo animal studies :

[0525] All animal experiments were performed in an institution approved by the Association for the Assessment of Laboratory Animal Care (AALAC) under the guidelines of the Institutional Animal Care and Use Committee (IACUC) and appropriate animal research approval. In order to evaluate BCMACAR-T function in vivo in the in vivo dissemination model of multiple myeloma, we intravenously infused 10e6 multiple myeloma 1S-luc cells into NSG mice. Four days after tumor cell infusion, mice were imaged and then intravenously infused with 0.3e6 or 3e6 CAR-T cells. Every 3-4 days, animals were weighed and imaged (via intraperitoneal injection of fluorescein) to find signs of morbidity and track tumor cell growth. Figure 11 Shown in disseminated multiple myeloma model (MM1.S) under high dose and low dose of CAR-T cells, anti-BCMACAR-T clone and the in vivo tumor control of clinical benchmark.Under both high dose and low dose, 7A8.11 CAR-T cells show better tumor control than any comparator benchmark (comparator C and comparator I), and are functionally equivalent to the clone derived from L15 internally.In addition, under these dosage levels, the administration of CAR-T cells does not show any treatment-related toxicity, such as cytokine release syndrome (CRS).Three days after CAR-T infusion, animals were bled and serum cytokine levels were quantitatively measured using MSD. Figure 12Demonstrated dose-responsive effector cytokine production by BCMACAR-T clones in the in vivo MM1.S tumor model.

[0526] Example 3. BCMACAR-T REX cell

[0527] In order to characterize the REX BCMA binding on the surface of 7A8.11 CAR expressed in the framework was detected by flow cytometry. CAR-T cells were labeled with BCMA-AF647 at a protein concentration of 0.3 nM-1000 nM, and the fluorescence intensity was measured by flow cytometry. Figure 13 The results show the 7A8.11 CAR-T cell proliferation and differentiation compared with primary 7A8.11 CAR-T cells and compared with Comparator I and Comparator C. REX BCMA binding curves of 7A8.11 CAR expressed in primary T cells and Comparators I and C. REX 7A8.11 CAR expressed in T cells showed the highest ability to bind to BCMA on the cell surface. REX The EC50 in [alpha] was higher than that when expressed in primary T cells, higher than Comparator 1, and similar to Comparator C.

[0528] Example 4. BCMACAR-T REX In vitro cytotoxicity of cells in multiple myeloma cell lines .

[0529] CAR-T cells were inoculated with each of the following luciferase-expressing target cell lines at four different E:T ratios: KMS12, U226B1, JJN3, and RPMI8226. Cytotoxicity was measured 24 hours after addition of CAR-T cells, and viability was measured after addition of luciferin substrate. Luminescence of live cells was measured by a microplate reader. 7A8.11 CAR-T REX The cells exhibited cytotoxic function comparable to primary T cells expressing the 7A8.11 CAR and to Comparators I and C across all E:T ratios and for all target cell lines (see Figure 14 ).

[0530] Example 5. BCMACAR-T REX Cytokine profile of cells .

[0531] Figure 15 The results show that 7A8.11 CAR-T cells expressed 7A8.11, compared with primary T cells expressing 7A8.11 and Comparator Benchmark I and Comparator Benchmark C when co-cultured with the target cell line JJN3. REX Example of effector cytokine profile of cells. CAR-T cells were seeded with JJN3 at a 1:1 E:T ratio, and supernatants were sampled 24 hours after co-culture to measure IFNγ and IL2 cytokine production by MSD. 7A8.11 CAR-T REXThe cells displayed 2%-15% of the effector cytokine production compared to 7A8.11 primary T cells and less than 5% of the effector cytokine production of the clinical comparator, indicating that 7A8.11 CAR-T REX Cells have a potentially safer cytokine profile.

[0532] Example 6. BCMACAR-T REX In vivo tumor control of multiple myeloma cells by cell-mediated .

[0533] Figure 16 Compared with the clinical benchmarks, Comparator I and Comparator C, 7A8.11 CAR-T REX In vivo tumor control of MM1.S cells. A non-targeted HER2 CAR-T was used as a control. In this in vivo disseminated model of multiple myeloma, 10e6 MM1S-luc cells were intravenously infused into NSG mice. Four days after tumor cell infusion, mice were imaged and then intravenously infused with 7A8.11 CAR-T. REX Cells or 7A8.11 primary T cells or infusions of Comparators I and C. Animals were weighed and imaged (via intraperitoneal injection of luciferin) every 3-4 days to look for signs of pathogenesis and to track tumor cell growth. Figure 16 7A8.11 CAR-T REX The cells showed the same tumor clearance kinetics as 7A8.11 primary T cells, and the same or better tumor clearance kinetics relative to Comparator I and Comparator C.

[0534] Example 7. Daratumumab treatment protects against BCMA-T cells REX Cells are protected from NK cells .

[0535] FIG. 17 shows that daratumumab treatment protects against BCMA-T cells. REX cells are protected from NK cells, and the remaining T REX The cells were functional. Briefly, purified NK cells were cultured overnight (NK-Xpander medium, 500 IU / mL IL-2) in the presence or absence of Dara (10 ug / mL). The next day, NK cells were washed and incubated with anti-BCMA-T REX Cells were mixed at a 1:1 or 0.5:1 NK:T ratio. REX After 5 h, the co-cultures were evaluated by flow cytometry to quantify NK cells and anti-BCMA-T REX Cell number, demonstrating Dara-mediated anti-BCMA-T REX Protection of cell number ( Figure 17A Cells were further progressed by two rounds of serial killing of JJN3 target cells at the specified E:T ratios, where the percentage of tumor cell lysis was measured by luciferase assay ( Figure 17B); or further progression of cells by a single round of cell killing with the SNU-182 adherent cell line ectopically expressing BCMA, where tumor cell killing was assessed by Xcelligence ( Figure 17C ). Data are representative of studies using NK cells from three different donors. As shown in Figure 17, Dara treatment was able to protect anti-BCMA-T cells. REX cell numbers while maintaining the cytotoxic capacity of these cells.

[0536] Example 8. Low-temperature recovery of BCMACAR-T REX Cells demonstrate tumor clearance in vivo .

[0537] NSG mice were inoculated with 10E6 MM1S-luciferase tumor cells. After 3 days, the cells were recovered at low temperature and cultured to obtain primary BCMACAR-T cells (7A8.11 CAR-T cells), BCMACAR-T REX cells (7A8.11) or BCMA CAR-T administered immediately after cryopreservation REX (7A8.11) (cryo) was administered at 10E6 cells per mouse. 25 days after MM1S cell administration, bone marrow was harvested from the mice and analyzed for the presence of MM1S tumor cells, healthy bone marrow cells, or other populations. BCMACAR-T cells were cryogenically recovered and immediately administered. REX The cells showed comparable tumor clearance and mouse bone marrow recovery to other groups (see Figure 18 ).

[0538] NSG mice were inoculated with 10E6 MM1S-luciferase tumor cells. Three days later, primary BCMA CAR-T cells (benchmark C CAR-T cells) or BCMA CAR-T cells from two donors were administered at the indicated doses immediately after cryo-recovery (cryo-recovery). REX cells (7A8.11). Tumor burden was monitored twice weekly using IVIS imaging. When administered immediately after cryopreservation, BCMACAR-T REX Cells demonstrated profound tumor clearance in vivo (see Figure 19 ).

[0539] Example 9. BCMA antigen density and cell composition between SLE and healthy donors .

[0540] The ability of cells targeting BCMA to deplete BCMA+ cells is thought to depend on the antigen density on the cell surface. Therefore, the BCMA antigen density and cell composition of patients with systemic lupus erythematosus (SLE) were compared with healthy donors. PBMCs were isolated from fresh whole blood from patients with SLE and healthy donors, and the BCMA receptor density and B cell subset percentages of PBMCs were assessed. Figure 20As shown, the cellularity of the B cell compartment is largely similar between healthy donors and SLE patients. In addition, target expression (BCMA receptor density) is also similar between healthy donors and SLE patients (or slightly higher in SLE patients; especially on plasmablasts).

[0541] Example 10. BCMA CAR-T cells deplete healthy human plasma cells expressing the target to bind to MM1S (BCMA+) tumor cells. The degree of cell similarity

[0542] BCMA CART-T cell research batches are manufactured from fresh peripheral blood from individual healthy donors. Briefly, PBMCs from healthy donors were harvested from the blood by Ficoll gradient centrifugation, and CD4- and CD8-positive T cells were enriched from the leukocyte fraction. The isolated T cells were then activated, transduced with the 7A8.11 BCMA lentiviral vector, and expanded in culture flasks. The cells were then washed, harvested, and frozen in cryopreservation medium.

[0543] Primary human plasma cells (CD138+ selection) were isolated from fresh whole blood of healthy donors. Plasma cells were then co-cultured with 7A8.11 BCMA CAR-T cells or untransduced T cells (untargeted) at different effector: target ratios. As a control, MM1S cells (a multiple myeloma cell line expressing BCMA) were co-cultured with the product or untransduced T cells at different effector: target ratios. Figure 21 As shown, BCMACAR-T cells were able to deplete primary human plasma cells or MM1S tumor cells to a similar extent (89% and 95% at an effector:target ratio of 1:1 and 93% and 86% at an effector:target ratio of 1:2, respectively), whereas untransduced T cells mediated little depletion of primary human plasma cells or the MM1S tumor cell line.

[0544] Example 11. In vitro differentiated plasmablasts from SLE and healthy donors express BCMA CAR-T cells Dose-dependent depletion (E:T)

[0545] Primary human naive B cells (IgD+CD27-selection) were isolated from fresh or frozen PBMCs from healthy donors or SLE donors. A proprietary mixture of cytokines was then used to differentiate naive B cells to drive plasmablast differentiation (BCMA+B cells). After 5 days of differentiation, the cells were co-cultured with 7A8.11-transduced CAR-T cells or untransduced T cells (untargeted) at different effector: target ratios. 7A8.11-transduced CAR-T cells were able to deplete healthy or SLE primary human differentiation plasmablasts to a similar degree (see Figure 22 ).

[0546] Example 12. CAR-T cells targeting BCMA reduce BCMA+ cells in an allogeneic model of graft-versus-host disease. cell

[0547] To evaluate the ability of CAR-T cells targeting BCMA to deplete primary B cells expressing BCMA in vivo, a pharmacodynamic (PD) study was performed in an allogeneic model of graft-versus-host disease (XenoGvHD). To prepare for this XenoGvHD PD study, healthy human donor PBMCs were isolated from fresh leukopak (StemExpress) and cryopreserved until transplantation. Autologous untransformed T cells (UTT) or autologous BCMA-targeted CAR-T cells (BCMACAR-T) were made from PBMCs from the same donor. In brief, isolated T cells were activated, transduced with the 16C6 BCMA lentiviral vector, and expanded in culture flasks, and the cells were then washed, harvested, and frozen in cryopreservation medium.

[0548] On day -1, 8-10 week old female NOD scidγ mice (NSG; Jackson Laboratories) were preconditioned with sublethal whole body irradiation (1 Gy). On day 0, 15 million total PBMCs of healthy human donors were injected intravenously. After 4 hours, mice were intravenously injected with phosphate buffered saline (PBS), 3 million autologous UTT cells or 3 million autologous BCMACAR-T cells (n=5 mice / group). On day 12, mice were euthanized and tissues were collected for FACS analysis to determine whether these BCMACAR-T cells consumed the BCMA+ cells observed to be present in the in vivo model.

[0549] Mice treated with the BCMACAR-T product showed a significant decrease in the percentage of BCMA-expressing CD27+ memory B cells in the spleen and whole blood compared to PBS- and UTT-treated mice ( Figure 23 Serum cytokines and cytolytic granzymes commonly associated with CAR-T therapy were assessed using a multiplex ELISA from Meso-scale Discovery and found to be significantly elevated in mice treated with BCMA-targeting CAR-T cells compared to mice treated with PBS and UTT ( Figure 24 ).

[0550] Table sequence

[0551]

[0552]

[0553]

[0554]

[0555]

[0556] The embodiments described herein can be implemented in the absence of any one or more elements, one or more restrictions not specifically disclosed herein. The terms and phrases that have been adopted have been used as descriptive terms rather than restrictive terms, and the use of such terms and phrases is not intended to exclude any equivalents of the features shown and described or parts thereof, but it should be recognized that various modifications are possible within the scope of the claimed embodiments. Therefore, it should be understood that although this specification has been specifically disclosed by the embodiments, those skilled in the art can resort to the optional features, modifications and changes of the concepts disclosed herein, and it is believed that such modifications and changes are within the scope of these embodiments defined by the specification and the appended claims. Although some aspects of the present disclosure can be identified as particularly advantageous herein, it is contemplated that the present disclosure is not limited to these particular aspects of the disclosure.

[0557] Unless indicated to the contrary or otherwise obvious from the context, a claim or description that includes "or" between one or more members of the group is considered satisfied if one, more than one, or all of the members of the group are present in, used in, or otherwise related to a given product or method. The disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise related to a given product or method. The disclosure includes embodiments in which more than one or all of the members of the group are present in, used in, or otherwise related to a given product or method.

[0558] Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more listed claims are introduced into another claim. For example, any claim that is dependent on another claim may be amended to include one or more limitations found in any other claim that is dependent on the same basic claim. Where elements are presented as a list, such as in Markush group format, each subgroup of these elements is also disclosed, and any one or more elements may be removed from the group.

[0559] It will be understood that, in general, where the present disclosure or aspects of the present disclosure are referred to as including particular elements and / or features, certain embodiments of the present disclosure or aspects of the present disclosure consist of or consist essentially of such elements and / or features. For the sake of simplicity, those embodiments are not specifically set forth herein.

[0560] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference. Citation or identification of any reference in any part of this application shall not be construed as an admission that such reference is available as prior art to the present disclosure.

Claims

1. An isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) an antigen-binding domain specific for B-cell maturation antigen (BCMA); (b) a transmembrane domain; and (c) one or more intracellular domains.

2. The isolated nucleic acid sequence of claim 1, wherein the antigen binding domain comprises an antibody or antigen binding fragment thereof, Fab, Fab', F(ab')2, Fd, Fv, single chain variable fragment (scFv), single chain antibody, V H H, vNAR, Nanobody (single domain antibody) or any combination thereof.

3. The isolated nucleic acid sequence of claim 2, wherein the antigen binding domain is a single-chain variable fragment (scFv).

4. The isolated nucleic acid sequence of claim 3, wherein the antigen binding domain is a scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 36 and 90.

5. The isolated nucleic acid sequence of claim 3, wherein the antigen binding domain is a scFv comprising the amino acid sequence of SEQ ID NO:

9.

6. The isolated nucleic acid sequence according to any one of claims 1 to 5, wherein the transmembrane domain comprises a transmembrane domain selected from the transmembrane domains of CD4, CD8α or CD28.

7. The isolated nucleic acid sequence of claim 6, wherein the transmembrane domain comprises a CD28 transmembrane domain.

8. The isolated nucleic acid sequence of any one of claims 1 to 7, wherein the one or more intracellular domains comprises a costimulatory domain or a portion thereof.

9. The isolated nucleic acid sequence of claim 8, wherein the costimulatory domain comprises one or more of the following: CD3z, 4-1BB, CD2, CD27, CD28, OX-40, ICOS, IL-2Rβ, GITR, MyD88 / CD40a costimulatory domains and / or variants thereof.

10. The isolated nucleic acid sequence of any one of claims 1 to 9, wherein the intracellular domain comprises a CD3z co-stimulatory domain and a CD28 co-stimulatory domain.

11. The isolated nucleic acid sequence of any one of claims 1 to 9, wherein the intracellular domain comprises a CD3z co-stimulatory domain and a 4-1BB co-stimulatory domain.

12. The isolated nucleic acid sequence of any one of claims 1 to 9, wherein the intracellular domain comprises a CD3z co-stimulatory domain, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain.

13. The isolated nucleic acid sequence of any one of claims 1 to 12, wherein the CAR further comprises a hinge / spacer domain, optionally wherein the hinge / spacer domain is located between the antigen binding domain and the transmembrane domain.

14. The isolated nucleic acid sequence of claim 13, wherein the hinge / spacer domain comprises an IgG1 hinge domain or a variant thereof, an IgG2 hinge domain or a variant thereof, an IgG3 hinge domain or a variant thereof, an IgG4 hinge domain or a variant thereof, an IgG4P domain, a CD8 hinge domain or a variant thereof, or a CD28 hinge domain or a variant thereof.

15. The isolated nucleic acid sequence of claim 14, wherein the hinge / spacer domain is an IgG4 hinge / spacer or a variant thereof, optionally an IgG4P hinge / spacer comprising an S241P mutation.

16. The isolated nucleic acid sequence according to any one of claims 1 to 15, wherein the nucleic acid sequence encodes a CAR having the amino acid sequence as shown in SEQ ID NO:

96.

17. The isolated nucleic acid sequence according to any one of claims 1 to 15, wherein the nucleic acid sequence encodes a CAR having the amino acid sequence as shown in SEQ ID NO:

97.

18. The isolated nucleic acid sequence according to any one of claims 1 to 15, wherein the nucleic acid sequence encodes a CAR having the amino acid sequence as shown in SEQ ID NO:

98.

19. An anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 31, and 85; and wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 34, and 88; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 35, and 89.

20. The anti-BCMACAR of claim 19, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 28, and 82.

21. The anti-BCMACAR of claim 19 or 28, wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 32, and 86.

22. An anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO:

8.

23. The anti-BCMACAR according to any one of claims 19 to 22, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO:

5.

24. The anti-BCMACAR of any one of claims 19 to 23, wherein the CAR comprises a transmembrane domain and one or more intracellular domains.

25. The anti-BCMACAR according to any one of claims 19 to 24, wherein the transmembrane domain comprises a transmembrane domain selected from the transmembrane domains of CD4, CD8α or CD28.

26. The anti-BCMACAR of claim 25, wherein the transmembrane domain comprises a CD28 transmembrane domain.

27. The anti-BCMACAR of any one of claims 19 to 26, wherein the one or more intracellular domains comprise a costimulatory domain or a portion thereof.

28. The anti-BCMACAR of claim 27, wherein the costimulatory domain comprises one or more of the following: CD3z, 4-1BB, CD2, CD27, CD28, OX-40, ICOS, IL-2Rβ, GITR, MyD88 / CD40a costimulatory domains and / or variants thereof.

29. The anti-BCMACAR according to any one of claims 24 to 28, wherein the intracellular domain comprises a CD3z co-stimulatory domain and a CD28 co-stimulatory domain.

30. The anti-BCMACAR of any one of claims 24 to 28, wherein the intracellular domain comprises a CD3z co-stimulatory domain and a 4-1BB co-stimulatory domain.

31. The anti-BCMACAR of any one of claims 24 to 28, wherein the intracellular domain comprises a CD3z co-stimulatory domain, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain.

32. The anti-BCMACAR of any one of claims 19 to 31, wherein the CAR further comprises a hinge / spacer domain, optionally wherein the hinge / spacer domain is located between the antigen binding domain and the transmembrane domain.

33. The anti-BCMACAR of claim 32, wherein the hinge / spacer domain comprises an IgG1 hinge domain or a variant thereof, an IgG2 hinge domain or a variant thereof, an IgG3 hinge domain or a variant thereof, an IgG4 hinge domain or a variant thereof, an IgG4P domain, a CD8a hinge domain or a variant thereof, or a CD28 hinge domain or a variant thereof.

34. The anti-BCMACAR of claim 33, wherein the hinge / spacer domain is an IgG4 hinge / spacer or a variant thereof, optionally an IgG4P hinge / spacer comprising an S241P mutation.

35. The anti-BCMACAR of any one of claims 19 to 34, wherein the CAR has the amino acid sequence shown in SEQ ID NO:

96.

36. The anti-BCMACAR of any one of claims 19 to 34, wherein the CAR has the amino acid sequence shown in SEQ ID NO:

97.

37. The anti-BCMACAR of any one of claims 19 to 34, wherein the CAR has the amino acid sequence shown in SEQ ID NO:

98.

38. A vector comprising an isolated nucleic acid sequence according to any one of claims 1 to 18 or encoding a chimeric antigen receptor according to any one of claims 19 to 37, wherein the vector is a virus, a lentivirus, an adenovirus, a retrovirus, an adeno-associated virus (AAV), a transposon, a DNA vector, mRNA, a lipid nanoparticle (LNP), or a CRISPR-Cas system.

39. A cell comprising the vector of claim 38.

40. A cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) according to any one of claims 19 to 37, further comprising reduced expression or knockout of one or more endogenous regulatory factors.

41. The cell of claim 40, wherein the one or more endogenous regulatory factors are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP).

42. The cell of any one of claims 39 to 41, wherein the cell has reduced expression or knockout of CDKN2A, CDKN2B and MTAP.

43. The cell of any one of claims 39 to 42, wherein the cell does not express phosphatase and tensin homolog (PTEN).

44. The cell of any one of claims 39 to 43, further comprising a transgene encoding B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

45. The cell of any one of claims 39 to 44, wherein the cell does not express one or more endogenous immune-related genes.

46. ​​The cell of claim 45, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

47. The cell of any one of claims 39 to 46, wherein the cell does not express cluster of differentiation 38 (CD38).

48. A cell comprising a BCMA-specific antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 31, and 85; and wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 34, and 88; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 35, and 89.

49. The cell of claim 48, wherein the VH comprises a polypeptide selected from the group consisting of SEQ ID NO: 1, The amino acid sequences of 28 and 82.

50. The cell of claim 48 or 49, wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 32, and 86.

51. A cell comprising a BCMA-specific antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO:

8.

52. The cell of any one of claims 48 to 51, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO:

5.

53. The cell of any one of claims 48 to 52, further comprising reduced expression or knockout of one or more endogenous regulatory factors.

54. The cell of claim 53, wherein the one or more endogenous regulatory factors are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP).

55. The cell of any one of claims 48 to 54, wherein the cell has reduced expression or knockout of CDKN2A, CDKN2B and MTAP.

56. The cell of any one of claims 48 to 55, wherein the cell does not express phosphatase and tensin homolog (PTEN).

57. The cell of any one of claims 48 to 56, wherein the cell further comprises a transgene encoding B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

58. The cell of any one of claims 48 to 57, wherein the cell does not express one or more endogenous immune-related genes.

59. The cell of claim 58, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

60. The cell of any one of claims 48 to 59, wherein the cell does not express cluster of differentiation 38 (CD38).

61. A cell comprising a BCMA-specific antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and wherein the cells comprise reduced expression or knockout of CDKN2A, CDKN2B, MTAP, B2M, TRAC, and CD38.

62. The cell of claim 61, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO:

5.

63. The cell of claim 61 or claim 62, wherein the BCMA-specific antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:

96.

64. The cell according to any one of claims 48 to 63, wherein the cell is selected from T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes, regulatory T cells, CD8 + T cells, CD4 + T cells, γδ T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells and / or combinations thereof.

65. A method for treating a disease, the method comprising: administering to a subject in need thereof an effective amount of cells comprising an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 31, and 85; and wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 34, and 88; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 35, and 89.

66. The method of claim 65, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 28, and 82.

67. The method of claim 65 or claim 66, wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 32, and 86.

68. A method for treating a disease, the method comprising: administering to a subject in need thereof an effective amount of cells comprising an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO:

8.

69. The method of any one of claims 65 to 68, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO:

5.

70. The method of any one of claims 65 to 69, further comprising inhibiting cancer growth, inducing cancer regression, and / or prolonging the subject's survival.

71. The method of any one of claims 65 to 70, wherein the cell further comprises reduced expression or knockout of one or more endogenous regulatory factors.

72. The method of claim 71, wherein the one or more endogenous regulatory factors are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP).

73. The method of any one of claims 65 to 72, wherein the cells have reduced expression or knockout of CDKN2A, CDKN2B, and MTAP.

74. The method of any one of claims 65 to 73, wherein the cell does not express phosphatase and tensin homolog (PTEN).

75. The method of any one of claims 65 to 74, wherein the cells further comprise a transgene encoding B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

76. The method of any one of claims 65 to 75, wherein the cell does not express one or more endogenous immune-related genes.

77. The method of claim 76, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

78. The cell of any one of claims 65 to 77, wherein the cell does not express cluster of differentiation 38 (CD38).

79. The method of any one of claims 65 to 78, wherein the cells are autologous cells.

80. The method of any one of claims 65 to 78, wherein the cells are allogeneic cells.

81. The method of any one of claims 65 to 80, wherein the cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes, regulatory T cells, CD8 + T cells, CD4 + T cells, γδ T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells and / or combinations thereof.

82. The method of any one of claims 65 to 81, wherein the disease is a cancer selected from the group consisting of multiple myeloma (MM), chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL).

83. The method of claim 82, wherein the cancer is multiple myeloma.

84. The method of any one of claims 65 to 81, wherein the disease is an autoimmune disease.

85. The method of claim 84, wherein the autoimmune disease is lupus.

86. A pharmaceutical composition comprising the isolated nucleic acid of any one of claims 1 to 18, the anti-BCMACAR of any one of claims 19 to 37, the vector of claim 38 or the cell of any one of claims 39 to 64, and a pharmaceutically acceptable excipient.

87. A method for treating a disease in a subject in need thereof, comprising administering to the subject an isolated nucleic acid according to any one of claims 1 to 18, an anti-BCMACAR according to any one of claims 19 to 37, a vector according to claim 38, a cell according to any one of claims 39 to 64, or a pharmaceutical composition according to claim 78.

88. The method of claim 87, wherein the disease is cancer or an autoimmune disease.

89. The method of claim 88, wherein the cancer is selected from the group consisting of multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL).

90. The method of claim 89, wherein the cancer is multiple myeloma.

91. The method of claim 88, wherein the autoimmune disease is lupus.

92. according to claim 1 to any one of the isolated nucleic acid, the anti-BCMACAR of any one of claims 19 to 37, the vector according to claim 38, the cell according to any one of claims 39 to 64 or the pharmaceutical composition according to claim 78 for the treatment of a disease in a subject in need thereof.

93. The use according to claim 92, wherein the disease is cancer or an autoimmune disease.

94. The use of claim 93, wherein the cancer is selected from the group consisting of multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).

95. The use of claim 94, wherein the cancer is multiple myeloma.

96. The use according to claim 93, wherein the autoimmune disease is lupus.

97. Use of an engineered cell for the manufacture of a medicament for treating a disease in a patient, wherein the engineered cell comprises an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 31, and 85; and wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 34, and 88; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 35, and 89.

98. The use of claim 99, wherein the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO:

8.

99. The use according to claim 97, wherein the VH comprises an amino acid sequence selected from SEQ ID NO: 1, 28 and 82.

100. The use according to claim 97, wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 32 and 86.

101. The use according to any one of claims 97 to 100, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO:

5.

102. The use according to any one of claims 97 to 101, wherein the CAR has the amino acid sequence as shown in SEQ ID NO:

96.

103. The use according to any one of claims 97 to 100, wherein the CAR has the amino acid sequence as shown in SEQ ID NO:

97.

104. The use according to any one of claims 97 to 100, wherein the CAR has the amino acid sequence as shown in SEQ ID NO:

98.

105. The use of any one of claims 97 to 104, further comprising inhibiting cancer growth, inducing cancer regression and / or prolonging the survival of the subject.

106. The use of any one of claims 97 to 105, wherein the engineered cell further comprises reduced expression or knockout of one or more endogenous regulatory factors.

107. The use according to claim 106, wherein the one or more endogenous regulatory factors are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B) and S-methyl-5'-thioadenosine phosphorylase (MTAP).

108. The use of any one of claims 97 to 107, wherein the engineered cell has reduced expression or knockout of CDKN2A, CDKN2B and MTAP.

109. The use of any one of claims 97 to 108, wherein the engineered cell does not express phosphatase and tensin homolog (PTEN).

110. The use of any one of claims 97 to 109, wherein the engineered cell further comprises a transgene encoding B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

111. The use according to any one of claims 97 to 110, wherein the engineered cells do not express one or more endogenous immune-related genes.

112. The use according to claim 111, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

113. The use of any one of claims 97 to 112, wherein the engineered cells do not express cluster of differentiation 38 (CD38).

114. The use according to any one of claims 97 to 113, wherein the engineered cells are autologous cells.

115. The use according to any one of claims 97 to 113, wherein the engineered cells are allogeneic cells.

116. The use according to any one of claims 97 to 115, wherein the engineered cells are selected from T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes, regulatory T cells, CD8 + T cells, CD4 + T cells, γδ T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells and / or combinations thereof.

117. The method of any one of claims 97 to 116, wherein the disease is a cancer selected from the group consisting of multiple myeloma (MM), chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).

118. The use according to claim 117, wherein the cancer is multiple myeloma.

119. The use according to any one of claims 97 to 116, wherein the disease is an autoimmune disease.

120. The use according to claim 119, wherein the autoimmune disease is lupus.

121. An engineered cell for use in the manufacture of a medicament for treating a disease in a patient, wherein the engineered cell comprises an anti-BCMA chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 31, and 85; and wherein the VL comprises: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 34, and 88; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 35, and 89.

122. The engineered cell of claim 121, wherein the VH comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and wherein the VL comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO:

8.

123. The engineered cell of claim 121, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 28, and 82.

124. The engineered cell of claim 121, wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 32, and 86.

125. The engineered cell of any one of claims 121 to 124, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO:

5.

126. The engineered cell of any one of claims 121 to 125, wherein the CAR has an amino acid sequence as shown in SEQ ID NO:

96.

127. The engineered cell of any one of claims 121 to 124, wherein the CAR has an amino acid sequence as shown in SEQ ID NO:

97.

128. The engineered cell of any one of claims 121 to 124, wherein the CAR has an amino acid sequence as shown in SEQ ID NO:

98.

129. The engineered cell of any one of claims 121 to 128, further comprising inhibiting cancer growth, inducing cancer regression, and / or prolonging survival of the subject.

130. The engineered cell of any one of claims 121 to 129, wherein the engineered cell further comprises reduced expression or knockout of one or more endogenous regulatory factors.

131. The engineered cell of claim 130, wherein the one or more endogenous regulatory factors are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP).

132. The engineered cell of any one of claims 121 to 131, wherein the engineered cell has reduced expression or knockout of CDKN2A, CDKN2B, and MTAP.

133. The engineered cell of any one of claims 121 to 132, wherein the engineered cell does not express phosphatase and tensin homolog (PTEN).

134. The engineered cell of any one of claims 121 to 133, wherein the engineered cell further comprises a transgene encoding B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

135. The engineered cell of any one of claims 121 to 134, wherein the engineered cell does not express one or more endogenous immune-related genes.

136. The engineered cell of claim 135, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

137. The engineered cell of any one of claims 121 to 136, wherein the engineered cell does not express cluster of differentiation 38 (CD38).

138. The engineered cell of any one of claims 121 to 137, wherein the engineered cell is an autologous cell.

139. The engineered cell of any one of claims 121 to 138, wherein the engineered cell is an allogeneic cell.

140. The engineered cell of any one of claims 121 to 139, wherein the engineered cell is selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes, regulatory T cells, CD8 + T cells, CD4 + T cells, γδ T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells and / or combinations thereof.

141. The engineered cell of any one of claims 121 to 140, wherein the disease is a cancer selected from multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).

142. The engineered cell of claim 141, wherein the cancer is multiple myeloma.

143. The engineered cell of any one of claims 121 to 142, wherein the disease is an autoimmune disease.

144. The engineered cell of claim 143, wherein the autoimmune disease is lupus.

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