Compositions and methods for treating disease using chimeric antigen receptors against B-cell maturation antigen (BCMA)

Abstract

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

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 486,391, filed February 22, 2023, the disclosure of which is incorporated by reference in its entirety.

[0002] (Reference to sequence listing) This application has been filed electronically and includes an electronically submitted Sequence Listing. The Sequence Listing is entitled "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 a part of the present specification and is incorporated herein by reference in its entirety.

[0003] FIELD OF THE INVENTION The present disclosure relates to the treatment of cancer and other diseases using BCMA-targeted chimeric antigen receptor cells. [Background technology]

[0004] B-cell maturation antigen (BCMA) is a tumor necrosis family receptor (TNFR) member 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 plasma cell survival to maintain long-term humoral immunity. BCMA expression is associated with many cancers, autoimmune disorders, and infectious diseases. In particular, BCMA RNA has been ubiquitously detected in multiple myeloma cells, and BCMA protein has been detected on the surface of plasma cells from multiple myeloma patients by several investigators (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)). Thus, BCMA has been investigated as a possible therapeutic target for multiple myeloma and other diseases.

[0005] Chimeric antigen receptor (CAR)-based cell therapy is a specific form of cell-based immunotherapy that uses engineered immune cells to fight disease. Such cell therapies have been transformative for patients with hematologic malignancies in recent years, with the first CAR-based therapy approved by the FDA in 2017 (Larson & Maus, Nat Rev Cancer 21, 145-161 (2021); Yu, et al., Nature Reviews Drug Discovery 19, 583-584 (2020)). Furthermore, the number of clinical trials investigating adoptive cell therapy has rapidly increased over the past few years.

[0006] Although cell therapies have great potential to be curative for patients, many factors limit the widespread development and administration of these drugs. Most cell therapies are currently produced in an autologous manner and are associated with variable cell product quality, cytokine release syndrome and other toxicities, extended manufacturing times, complex supply chain logistics, high costs, and a limited window during which these therapies can be genetically modified to enhance their efficacy (Larson & Maus, Nat Rev Cancer 21, 145-161 (2021)).

[0007] In particular, autologous cell therapies using primary human immune cells (e.g., T cells and NK cells) have a finite potential for expansion. This significantly limits the window for isolating, expanding, manufacturing, and gene-editing these cells while still retaining their functionality upon reinfusion into patients. Predicting the lifespan of these cells through deletion of cell cycle-related genes allows for several manipulations that could widen the window for manufacturing, mitigate cytokine release syndrome and other toxicities, enable cell armoring, and, most importantly, enable the generation of large cell banks of allogeneic edited cells for widespread distribution.

[0008] The currently FDA-approved BCMA CAR-T cell therapy for multiple myeloma (MM) is an autologous product that is transformative for patients refractory to all other available treatments and has reported overall response rates of up to 95%. However, practical challenges in delivering the autologous CAR-T cell product have significantly limited the enrollment of eligible patients. Furthermore, toxicities associated with BCMA CAR-T cell therapy and BCMA T-cell engager-based therapy also occur in over 90% of those treated. Therefore, alternative BCMA CAR cell-based therapies are needed to address these manufacturing constraints while minimizing associated toxicities. Summary of the Invention

[0009] This disclosure describes compositions and methods for using CAR-based cell therapy to treat cancer and other diseases.

[0010] 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: (a) an antigen-binding domain specific for B-cell maturation antigen (BCMA); (b) a transmembrane domain; and (c) comprises one or more intracellular domains.

[0011] 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 fragment variable (scFv), single-chain antibody, VHH, vNAR, nanobody (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 an scFv comprising an amino acid sequence selected from SEQ ID NOs: 9, 36, and 90. In one embodiment of the isolated nucleic acid sequence, the antigen-binding domain is an scFv comprising the amino acid sequence of SEQ ID NO: 9.

[0012] In some embodiments of the isolated nucleic acid sequence, the transmembrane domain comprises a transmembrane domain selected from the transmembrane domains of CD4, CD8α, or CD28. In one embodiment, the transmembrane domain comprises a CD28 transmembrane domain.

[0013] In some implementations of the isolated nucleic acid sequence, one or more intracellular domains comprise a costimulatory domain or a portion thereof. In some embodiments, the costimulatory domain comprises one or more of a CD3z, 4-1BB, CD2, CD27, CD28, OX-40, ICOS, IL-2Rβ, GITR, MyD88 / CD40a costimulatory domain, and / or variants thereof. In one embodiment of the isolated nucleic acid sequence, the intracellular domain comprises a CD3z costimulatory domain and a CD28 costimulatory domain. In another embodiment of the isolated nucleic acid sequence, the intracellular domain comprises a CD3z costimulatory domain and a 4-1BB costimulatory domain. In yet another embodiment of the isolated nucleic acid sequence, the intracellular domain comprises a CD3z costimulatory domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain.

[0014] In some embodiments of the isolated nucleic acid sequence, the CAR further comprises a hinge / spacer domain, optionally located between the antigen-binding domain and the transmembrane domain. In some embodiments, the hinge / spacer domain comprises an IgG1 hinge domain or variant thereof, an IgG2 hinge domain or variant thereof, an IgG3 hinge domain or variant thereof, an IgG4 hinge domain or variant thereof, an IgG4P domain, a CD8 hinge domain or variant thereof, or a CD28 hinge domain or variant thereof. In one embodiment, the hinge / spacer domain is an IgG4 hinge / spacer or variant thereof, optionally an IgG4P hinge / spacer comprising an S241P mutation.

[0015] In one embodiment of the isolated nucleic acid sequence, the nucleic acid sequence encodes a CAR having the amino acid sequence set forth in SEQ ID NO:96.

[0016] In another embodiment of the isolated nucleic acid sequence, the nucleic acid sequence encodes a CAR having the amino acid sequence set forth in SEQ ID NO:97.

[0017] In yet another embodiment of the isolated nucleic acid sequence, the nucleic acid sequence encodes a CAR having the amino acid sequence set forth in SEQ ID NO:98.

[0018] 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); VH comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4, 31, and 85; The VL comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 7, 34, and 88; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 8, 35, and 89.

[0019] In some embodiments of the anti-BCMA CAR, the VH comprises an amino acid sequence selected from SEQ ID NOs: 1, 28, and 82. In some embodiments of the anti-BCMA CAR, the VL comprises an amino acid sequence selected from SEQ ID NOs: 5, 32, and 86.

[0020] 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); VH comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 2; CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and CDR3 comprising the amino acid sequence of SEQ ID NO: 4, 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.

[0021] In some embodiments of the anti-BCMA CARs 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.

[0022] In some embodiments of the anti-BCMA CARs disclosed herein, the CAR comprises a transmembrane domain and one or more intracellular domains. In some embodiments, the transmembrane domain comprises a transmembrane domain selected from the transmembrane domains of CD4, CD8α, or CD28. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain.

[0023] In some embodiments of the anti-BCMA CARs disclosed herein, the one or more intracellular domains comprise a costimulatory domain or a portion thereof. In some embodiments, the costimulatory domain comprises one or more of the following costimulatory domains: CD3z, 4-1BB, CD2, CD27, CD28, OX-40, ICOS, IL-2Rβ, GITR, MyD88 / CD40a costimulatory domains and / or variants thereof.

[0024] In one embodiment of the anti-BCMA CAR disclosed herein, the intracellular domain comprises a CD3z costimulatory domain and a CD28 costimulatory domain.

[0025] In another embodiment of the anti-BCMA CAR disclosed herein, the intracellular domain comprises a CD3z costimulatory domain and a 4-1BB costimulatory domain.

[0026] In yet another embodiment of the anti-BCMA CAR disclosed herein, the intracellular domain comprises a CD3z costimulatory domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain.

[0027] In some embodiments of the anti-BCMA CARs disclosed herein, the CAR further comprises a hinge / spacer domain, optionally located between the antigen-binding domain and the transmembrane domain. In some embodiments, the hinge / spacer domain comprises an IgG1 hinge domain or variant thereof, an IgG2 hinge domain or variant thereof, an IgG3 hinge domain or variant thereof, an IgG4 hinge domain or variant thereof, an IgG4P domain, a CD8a hinge domain or variant thereof, or a CD28 hinge domain or variant thereof. In one embodiment, the hinge / spacer domain is an IgG4 hinge / spacer or variant thereof, optionally an IgG4P hinge / spacer comprising an S241P mutation.

[0028] In one embodiment of the anti-BCMA CAR disclosed herein, the CAR has the amino acid sequence set forth in SEQ ID NO:96.

[0029] In another embodiment of the anti-BCMA CAR disclosed herein, the CAR has the amino acid sequence set forth in SEQ ID NO:97.

[0030] In yet another embodiment of the anti-BCMA CAR disclosed herein, the CAR has the amino acid sequence set forth in SEQ ID NO:98.

[0031] In another aspect, the present disclosure provides a vector comprising an isolated nucleic acid sequence disclosed herein or encoding a chimeric antigen receptor disclosed herein, optionally wherein the vector is a virus, lentivirus, adenovirus, retrovirus, adeno-associated virus (AAV), transposon, DNA vector, mRNA, lipid nanoparticle (LNP), or CRISPR-Cas system.

[0032] In yet another aspect, the present disclosure provides a cell comprising the vector disclosed herein.

[0033] In another aspect, the present disclosure provides a cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) disclosed herein, further comprising a reduction in or knockout of expression of one or more endogenous regulatory factors.

[0034] In some embodiments of the cells disclosed herein, the one or more endogenous regulators are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP).

[0035] In some embodiments of the cells disclosed herein, the cells have reduced expression or knockout of CDKN2A, CDKN2B, and MTAP.

[0036] In some embodiments of the cells disclosed herein, the cells do not express phosphatase and tensin homolog (PTEN).

[0037] In some embodiments of the cells disclosed herein, the cells further comprise a transgene encoding either B-cell lymphoma-ultra-large cell (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0038] In some embodiments of the cells disclosed herein, the cells do not express one or more endogenous immune-related genes, hi some embodiments, the endogenous immune-related genes are beta 2 microglobulin (B2M) and / or T cell receptor alpha constant (TRAC).

[0039] In some embodiments of the cells disclosed herein, the cells do not express cluster of differentiation 38 (CD38).

[0040] 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); VH comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4, 31, and 85; The VL comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 7, 34, and 88; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 8, 35, and 89.

[0041] In some embodiments of the cells disclosed herein, the VH comprises an amino acid sequence selected from SEQ ID NOs: 1, 28, and 82. In some embodiments of the cells disclosed herein, the VL comprises an amino acid sequence selected from SEQ ID NOs: 5, 32, and 86.

[0042] In another aspect, the 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); VH comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 2; CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and CDR3 comprising the amino acid sequence of SEQ ID NO: 4, 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.

[0043] In some embodiments of the cells 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.

[0044] In some embodiments of the cells 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 or knockout expression of CDKN2A, CDKN2B, and MTAP.

[0045] In some embodiments of the cells disclosed herein, the cells do not express phosphatase and tensin homolog (PTEN).

[0046] In some embodiments of the cells disclosed herein, the cells further comprise a transgene encoding either B-cell lymphoma-ultra-large cell (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0047] In some embodiments of the cells disclosed herein, the cells do not express one or more endogenous immune-related genes, hi some embodiments, the endogenous immune-related genes are beta 2 microglobulin (B2M) and / or T cell receptor alpha constant (TRAC).

[0048] In some embodiments of the cells disclosed herein, the cells do not express cluster of differentiation 38 (CD38).

[0049] In another aspect, the 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); VH comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 2; CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and CDR3 comprising the amino acid sequence of SEQ ID NO: 4, 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; The cells comprise reduced or knocked-out expression of CDKN2A, CDKN2B, MTAP, B2M, TRAC, and CD38.

[0050] In one embodiment of the cells 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.

[0051] In one embodiment of the cells disclosed herein, the BCMA-specific antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:96.

[0052] In some embodiments of the cells 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, gamma delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells, and / or combinations thereof.

[0053] In one aspect, the present disclosure provides a method of 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); VH comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4, 31, and 85; The VL comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 7, 34, and 88; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 8, 35, and 89.

[0054] In some embodiments of the methods disclosed herein, the VH comprises an amino acid sequence selected from SEQ ID NOs: 1, 28, and 82. In some embodiments of the methods disclosed herein, the VL comprises an amino acid sequence selected from SEQ ID NOs: 5, 32, and 86.

[0055] In another aspect, the present disclosure provides a method of 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); VH comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 2; CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and CDR3 comprising the amino acid sequence of SEQ ID NO: 4, 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.

[0056] In some embodiments of the methods 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.

[0057] In some embodiments of the methods disclosed herein, the methods further comprise inhibiting cancer growth, inducing cancer regression, and / or prolonging survival in the subject.

[0058] 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 or knockout expression of CDKN2A, CDKN2B, and MTAP.

[0059] In some embodiments of the methods disclosed herein, the cells do not express phosphatase and tensin homolog (PTEN).

[0060] In some embodiments of the methods disclosed herein, the cells further comprise a transgene encoding either B-cell lymphoma-ultra-large cell (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0061] In some embodiments of the methods disclosed herein, the cells do not express one or more endogenous immune-related genes, hi some embodiments, the endogenous immune-related genes are beta 2 microglobulin (B2M) and / or T cell receptor alpha constant (TRAC).

[0062] In some embodiments of the methods disclosed herein, the cells do not express cluster of differentiation 38 (CD38).

[0063] In some embodiments of the methods disclosed herein, the cells are autologous cells.

[0064] In some embodiments of the methods disclosed herein, the cells are allogeneic cells.

[0065] In some embodiments of the methods 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, gamma delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells, and / or combinations thereof.

[0066] In some embodiments of the methods disclosed herein, the disease is a cancer selected from multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphocytic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL). In one embodiment, the cancer is multiple myeloma.

[0067] In some embodiments of the methods disclosed herein, the disease is an autoimmune disease, hi one embodiment, the autoimmune disease is lupus.

[0068] In one aspect, the present disclosure provides a pharmaceutical composition comprising an isolated nucleic acid disclosed herein, an anti-BCMA CAR disclosed herein, a vector disclosed herein, or a cell disclosed herein, and a pharmaceutically acceptable excipient.

[0069] In some embodiments of the methods disclosed herein, the method comprises administering to a subject an isolated nucleic acid disclosed herein, an anti-BCMA CAR described in any one of the claims disclosed herein, a vector disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed herein. In some embodiments of the methods 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-lymphocytic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL). In one embodiment, the cancer is multiple myeloma. In one embodiment, the autoimmune disease is lupus.

[0070] In one aspect, the present disclosure provides for the use of an isolated nucleic acid disclosed herein, an anti-BCMA CAR disclosed herein, a vector disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed herein in the treatment of a disease in a subject in need thereof.

[0071] 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 lymphocytic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL). In one embodiment, the cancer is multiple myeloma. In one embodiment, the autoimmune disease is lupus.

[0072] In one aspect, the disclosure provides a 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); VH comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4, 31, and 85; The VL comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 7, 34, and 88; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 8, 35, and 89.

[0073] In some embodiments of the uses 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 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.

[0074] In some embodiments of the uses disclosed herein, the VH comprises an amino acid sequence selected from SEQ ID NOs: 1, 28, and 82. In some embodiments of the uses disclosed herein, the VL comprises an amino acid sequence selected from SEQ ID NOs: 5, 32, and 86.

[0075] In some embodiments of the uses 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.

[0076] In one embodiment of the uses disclosed herein, the CAR has the amino acid sequence shown in SEQ ID NO:96.

[0077] In one embodiment of the uses disclosed herein, the CAR has the amino acid sequence shown in SEQ ID NO:97.

[0078] In one embodiment of the uses disclosed herein, the CAR has the amino acid sequence shown in SEQ ID NO:98.

[0079] In some embodiments of the uses disclosed herein, the use further comprises inhibiting cancer growth, inducing cancer regression, and / or prolonging survival in a subject.

[0080] In some embodiments of the uses disclosed herein, the engineered cells further comprise reduced expression or knockout of one or more endogenous regulatory factors. In some embodiments of the uses 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 or knockout expression of CDKN2A, CDKN2B, and MTAP.

[0081] In some embodiments of the uses disclosed herein, the engineered cells do not express phosphatase and tensin homolog (PTEN).

[0082] In some embodiments of the uses disclosed herein, the engineered cells further comprise a transgene encoding either B-cell lymphoma-ultra-large cell (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0083] In some embodiments of the uses disclosed herein, the engineered cells do not express one or more endogenous immune-related genes. In some embodiments of the uses disclosed herein, the endogenous immune-related genes are beta 2 microglobulin (B2M) and / or T cell receptor alpha constant (TRAC).

[0084] In some embodiments of the uses disclosed herein, the engineered cells do not express cluster of differentiation 38 (CD38).

[0085] In some embodiments of the uses disclosed herein, the engineered cells are autologous cells.

[0086] In some embodiments of the uses disclosed herein, the engineered cells are allogeneic cells.

[0087] In some embodiments of the uses 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, gamma delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells, and / or combinations thereof.

[0088] In some embodiments of the uses disclosed herein, the disease is a cancer selected from multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphocytic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL). In some embodiments of the uses disclosed herein, the cancer is multiple myeloma. In some embodiments of the uses disclosed herein, the disease is an autoimmune disease. In some embodiments of the uses disclosed herein, the autoimmune disease is lupus.

[0089] In one aspect, the present disclosure provides 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); VH comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4, 31, and 85; The VL comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 7, 34, and 88; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 8, 35, and 89.

[0090] In some embodiments of the engineered cells 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 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.

[0091] In some embodiments of the engineered cells disclosed herein, the VH comprises an amino acid sequence selected from SEQ ID NOs: 1, 28, and 82. In some embodiments of the engineered cells disclosed herein, the VL comprises an amino acid sequence selected from SEQ ID NOs: 5, 32, and 86.

[0092] In one embodiment of the engineered cells 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.

[0093] In one embodiment of the engineered cells disclosed herein, the CAR has the amino acid sequence set forth in SEQ ID NO:96.

[0094] In one embodiment of the engineered cells disclosed herein, the CAR has the amino acid sequence set forth in SEQ ID NO:97.

[0095] In one embodiment of the engineered cells disclosed herein, the CAR has the amino acid sequence set forth in SEQ ID NO:98.

[0096] In some embodiments of the engineered cells disclosed herein, the cells further comprise inhibiting cancer growth, inducing cancer regression, and / or prolonging survival in a subject.

[0097] In some embodiments of the engineered cells 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 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 or knockout expression of CDKN2A, CDKN2B, and MTAP.

[0098] In some embodiments of the engineered cells disclosed herein, the engineered cells do not express phosphatase and tensin homolog (PTEN).

[0099] In some embodiments of the engineered cells disclosed herein, the engineered cells further comprise a transgene encoding either B-cell lymphoma-ultra-large cell (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0100] In some embodiments of the engineered cells disclosed herein, the engineered cells do not express one or more endogenous immune-related genes. In some embodiments of the engineered cells disclosed herein, the endogenous immune-related genes are beta 2 microglobulin (B2M) and / or T cell receptor alpha constant (TRAC).

[0101] In some embodiments of the engineered cells disclosed herein, the engineered cells do not express cluster of differentiation 38 (CD38).

[0102] In some embodiments of the engineered cells disclosed herein, the engineered cells are autologous cells.

[0103] In some embodiments of the engineered cells disclosed herein, the engineered cells are allogeneic cells.

[0104] In some embodiments of the engineered cells 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, gamma delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells, and / or combinations thereof.

[0105] In some embodiments of the engineered cells disclosed herein, the disease is a cancer selected from multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphocytic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML), and acute lymphocytic 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.

[0106] 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, which should be noted that the claims are defined by the recitation therein, rather than the specific description of the features and advantages set forth herein. [Brief explanation of the drawings]

[0107] 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 operation of the present disclosure. [Figure 1] Figure 1 shows surface plasmon resonance binding of anti-BCMA scFv-Fc to soluble BCMA protein. Mean values ​​of ka, kd and KD are reported. [Figure 2] Figure 2 shows a membrane proteome array for anti-BCMA scFv-Fc specificity for BCMA. Anti-BCMA scFv-Fc was used in a cell-based membrane proteome array of over 6,000 membrane proteins to investigate off-target binding. Figure 2 shows that the scFv used in anti-BCMA CAR-T is specific for BCMA. [Figure 3-01] Figure 3 shows anti-BCMA CAR staining of primary T cells. Figure 3 shows that CAR is successfully expressed on the surface of T cells. Staining is representative of five different donors. [Figure 3-02] Figure 3 shows anti-BCMA CAR staining of primary T cells. Figure 3 shows that CAR is successfully expressed on the surface of T cells. Staining is representative of five different donors. [Figure 3-03] Figure 3 shows anti-BCMA CAR staining of primary T cells. Figure 3 shows that CAR is successfully expressed on the surface of T cells. Staining is representative of five different donors. [Figure 4] Figure 4 shows the BCMA CAR-T binding curves. Figure 4 shows the binding characteristics of each anti-BCMA CAR-T clone to soluble BCMA antigen. [Figure 5] Anti-BCMA CAR-T expansion. Figure 5 shows the different growth kinetics and length of expansion of anti-BCMA CAR-T clones in primary T cells using IL-2-containing medium. [Figure 6A] Figures 6A and 6B show in vitro cytotoxicity of multiple myeloma cell lines. Figures 6A and 6B show BCMA CAR-T clone cytotoxicity across a spectrum of multiple myeloma (MM) cell lines and compare it to two clinical benchmarks. Figure 6A is a heat map of the mean % cytotoxicity of anti-BCMA CAR-T clones from four donors across different MM cell lines at an E:T ratio of 1:2. Figure 6B shows a bar graph of the mean % cytotoxicity and standard deviation from the four donors. [Figure 6B]Figures 6A and 6B show in vitro cytotoxicity of multiple myeloma cell lines. Figures 6A and 6B show BCMA CAR-T clone cytotoxicity across a spectrum of multiple myeloma (MM) cell lines and compare it to two clinical benchmarks. Figure 6A is a heat map of the mean % cytotoxicity of anti-BCMA CAR-T clones from four donors across different MM cell lines at an E:T ratio of 1:2. Figure 6B shows a bar graph of the mean % cytotoxicity and standard deviation from the four donors. [Figure 7] Figure 7 shows effector cytokine production from anti-BCMA CAR-T clones. Figure 7 shows representative cytokine production from anti-BCMA CAR-T cells when challenged with BCMA expressing huh7 engineered cell lines. [Figure 8] Showing resistance to soluble BCMA, Figure 8 shows the difference in % cytolysis observed after 40 hours of co-culture with BCMA-expressing huh7 cells in the presence or absence of soluble BCMA protein (sBCMA). [Figure 9] Figure 9 shows effector cytokine production in the presence of soluble BCMA. Figure 9 shows the effect of soluble BCMA on CAR-T production of IFNy and IL-2 effector cytokines. [Figure 10A] Figure 10 shows CAR-T cell persistence and expansion after repeated antigen stimulation in the presence or absence of soluble BCMA. Figure 10A shows anti-BCMA CAR-T cell proliferation after repeated antigen stimulation (JJN3 co-culture) over 12 days. During this period, the % control of tumor cells was used as a measure of T cell function and persistence. Figure 10B similarly illustrates antigen-dependent CAR-T cell proliferation in the presence of soluble antigen (JJN3 co-culture). The % cytolysis of target cells highlights the impact of soluble BCMA protein on the function and persistence of anti-BCMA CAR-T clones. [Figure 10B]Figure 10 shows CAR-T cell persistence and expansion after repeated antigen stimulation in the presence or absence of soluble BCMA. Figure 10A shows anti-BCMA CAR-T cell proliferation after repeated antigen stimulation (JJN3 co-culture) over 12 days. During this period, the % control of tumor cells was used as a measure of T cell function and persistence. Figure 10B similarly illustrates antigen-dependent CAR-T cell proliferation in the presence of soluble antigen (JJN3 co-culture). The % cytolysis of target cells highlights the impact of soluble BCMA protein on the function and persistence of anti-BCMA CAR-T clones. [Figure 11] Figure 11 shows in vivo tumor control and clinical benchmarks of anti-BCMA CAR-T clones. Figure 11 shows the functional efficacy of CAR-T in vivo compared to clinical benchmarks at both high and low doses of CAR-T cells in a disseminated MM model (MM1.S). [Figure 12]

[0033] Figure 12 shows serum cytokine levels from an in vivo tumor challenge model. Figure 12 demonstrates CAR-T function at two doses via effector cytokine production after in vivo MM1.S tumor challenge. [Figure 13] Figure 13 shows the BCMA binding curve of BCMA CAR-TREX. Figure 13 shows the BCMA binding properties of the 7A8.11 CAR when expressed in a CAR-TREX chassis and benchmarked against primary CAR-T and clinical products. [Figure 14] Figure 14 illustrates in vitro cytotoxicity of multiple myeloma cell lines. Figure 14 illustrates BCMA CAR-TREX cytotoxicity across a panel of multiple myeloma cell lines and compares it to primary T cells and two clinical benchmarks. [Figure 15]Figure 15 shows the effector cytokine profile from BCMA CAR-TREX cells compared to primary T cells. Figure 15 shows representative effector cytokine production from BCMA CAR-TREX cells when encountering the multiple myeloma target cell line JJN3 at a 1:1 E:T ratio. BCMA CAR-TREX cells showed 2-15% effector cytokine production compared to primary T cells of the same CAR and less than 5% of clinical comparator cells, suggesting a potentially safer cytokine profile. [Figure 16] Figure 16 shows in vivo tumor control of MM1.S cells by BCMA CAR-TREX compared to clinical benchmarks. Figure 16 shows comparable or improved tumor clearance kinetics of BCMA CAR-TREX cells compared to primary T cells of the same CAR and clinical benchmark. [Figure 17A] Daratumumab (Dara) treatment protects anti-BCMA-TREX cell numbers, demonstrating that the remaining TREX cells are functional. After 5 hours, cocultures were assessed by flow cytometry to quantify NK cell and anti-BCMA-TREX cell numbers, demonstrating Dara-mediated protection of anti-BCMA-TREX cell numbers (Figure 17A). Cells further proceeded to either two consecutive killings of JJN3 target cells at the indicated E:T ratios (Figure 17B), or a single killing of the BCMA-ectopically expressing SNU-182 adherent cell line (Figure 17C), as assessed by Xcelligence®, with % tumor cell lysis measured by luciferase assay. [Figure 17B] Daratumumab (Dara) treatment protects anti-BCMA-TREX cell numbers, demonstrating that the remaining TREX cells are functional. After 5 hours, cocultures were assessed by flow cytometry to quantify NK cell and anti-BCMA-TREX cell numbers, demonstrating Dara-mediated protection of anti-BCMA-TREX cell numbers (Figure 17A). Cells further proceeded to either two consecutive killings of JJN3 target cells at the indicated E:T ratios (Figure 17B), or a single killing of the BCMA-ectopically expressing SNU-182 adherent cell line (Figure 17C), as assessed by Xcelligence®, with % tumor cell lysis measured by luciferase assay. [Figure 17C] Daratumumab (Dara) treatment protects anti-BCMA-TREX cell numbers, demonstrating that the remaining TREX cells are functional. After 5 hours, cocultures were assessed by flow cytometry to quantify NK cell and anti-BCMA-TREX cell numbers, demonstrating Dara-mediated protection of anti-BCMA-TREX cell numbers (Figure 17A). Cells further proceeded to either two consecutive killings of JJN3 target cells at the indicated E:T ratios (Figure 17B), or a single killing of the BCMA-ectopically expressing SNU-182 adherent cell line (Figure 17C), as assessed by Xcelligence®, with % tumor cell lysis measured by luciferase assay. [Figure 18] These results demonstrate that BCMA CAR-TREX cells effectively clear tumors from the bone marrow, even when administered immediately after cryo-recovery. BCMA CAR-TREX cells administered immediately after cryo-recovery demonstrated comparable tumor clearance and bone marrow recovery in mice compared to other groups. [Figure 19] We show that BCMA CAR-TREX cells demonstrate deep tumor clearance in vivo when administered immediately after cryo-recovery. NSG mice were inoculated with MM1S-luciferase tumor cells, and 3 days later, primary BCMA CAR-T cells from two donors or BCMA CAR-TREX cells (7A8.11) were administered immediately after cryo-recovery at the indicated doses. [Figure 20] Characterization of BCMA expression and B cell subsets in blood from healthy and SLE donors is shown. PBMCs from healthy and SLE donors were isolated from fresh whole blood and stained with antibodies. Cells shown are lymphocytes, viable cells, dump gate negative, CD20+ and CD19+ / -, as defined by FSC / SSC. DN2 cells (IgD-, CD27-), memory B cells (CD27+IgD-), naive B cells (IgD+CD27-), NCSM (non-class-switched memory, IgD+CD27+), plasma cells (CD19low / negative, CD138+CD38low), and plasmablasts (CD19low / negative, CD138-CD38+CD27+). [Figure 21]We demonstrate that BCMA CAR-T cells deplete healthy human plasma cells to the same extent as MM1S (BCMA+) tumor cells. Primary plasmablasts, plasma cells, and multiple myeloma MM1S cells were co-cultured with BCMA CAR-T cells or untransduced T cells at effector:target ratios of 1:1, 1:2, or 1:4 for 24 hours, then stained and evaluated by flow cytometry. T cells were gated as live, dump-positive cells. Plasma cells were pre-gated as live, dump-negative cells, CD19+, CD20low, CD38+, CD27+ (plasmocytes+plasmablasts). [Figure 22] We demonstrate that in vitro differentiated plasmablasts from SLE and healthy donors exhibit dose-dependent depletion (E:T) by BCMA CAR-T cells. Frozen naive B cells from healthy donors and SLE patients were isolated and plated using a proprietary mixture of cytokines to drive B cell differentiation. After 5 days of differentiation, differentiated B cells were incubated with BCMA CAR-T cells or untransduced T cells at effector:target ratios of 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32 for 24 hours, then stained and assayed by flow cytometry. Percent depletion was calculated by 1 - (% plasmablasts in experimental wells / average % plasmablasts in stem cell-only wells) * 100. [Figure 23] Figure 1 shows that BCMA-targeting CAR-T cells reduced BCMA+ cells in a xenogeneic model of graft-versus-host disease. Whole blood was collected for FACS analysis at the end of the study, 12 days after engraftment. The top panel shows a representative FACS histogram of splenic CD27+ memory B cells expressing BCMA. The graph in the bottom panel shows the percent BCMA+ CD27+ memory B cells in the spleen, blood, and bone marrow. The mean + / - standard error is shown for each treatment group. One-way ANOVA with Tukey's multiple comparison test was performed to determine statistical significance. *P<0.05, ****P<0.0001. [Figure 24]Serum cytokines / cytolytic granzymes were increased with BCMA CAR-T treatment. Serum collected from PBS-, UTT-, and BCMA CAR-T-treated mice was collected 12 days post-engraftment, and serum levels of IFN-γ, GM-CSF, TNF-α, IL-2, granzyme A, and granzyme B were assessed by ELISA. Mean + / - standard error for each cytokine from each treatment group is shown. One-way ANOVA with Sidak's multiple comparison test was performed to determine statistical significance. *P<0.05, **P<0.01.

[0108] Those skilled in the art will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements to help to improve understanding of embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0109] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this disclosure: 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, the following terms have the meanings set forth below, unless otherwise specified.

[0110] As used herein, the terms "comprise" and "include" and variations thereof (e.g., "comprises," "comprising," "includes," and "including") are understood to indicate the inclusion of a stated component, feature, element, or step, or group of components, features, elements, or steps, but not the exclusion of any other component, feature, element, or step, or group of components, features, elements, or steps. The terms "comprising," "consisting essentially of," and "consisting of" may be substituted for either of the other two terms while retaining their ordinary meaning.

[0111] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0112] The percentages disclosed herein can vary from the disclosed values ​​by ±10, 20, or 30% amounts and still be within the contemplated range of the disclosure.

[0113] Unless otherwise indicated or apparent from the context and the understanding of one of ordinary skill in the art, values ​​herein expressed as ranges can contemplate in different embodiments of the present disclosure any specific value or subrange within the stated range, down to one-tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0114] As used herein, ranges and amounts can be expressed as "about" a particular value or range. The term "about" also includes the exact amount. For example, "about 5%" means "about 5%" and also "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% to 5.5%. Furthermore, "about" or "essentially comprising" can refer to a range of up to ±10%. Furthermore, particularly with respect to biological systems or processes, these terms can mean up to an order of magnitude or up to five times the value. When a specific value or composition is provided in the present application and claims, unless otherwise specified, the meaning of "about" or "essentially comprising" should be assumed to be within an acceptable error range for that particular value or composition. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0115] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof (such as integer tenths and hundredths), where appropriate, unless otherwise indicated.

[0116] Units, prefixes, and symbols are denoted in the format accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limiting of the various aspects of this disclosure, which may be obtained by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0117] As used herein, the terms "or" and "and / or" can describe multiple components in combination with or exclusive of each other. For example, "x, y, and / or z" can refer to "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."

[0118] 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 chain or chains of two or more amino acids. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with any of these terms.

[0119] As used herein, "protein" can refer to a single polypeptide, i.e., a single amino acid chain as defined above, but can also refer to two or more polypeptides associated, for example, by disulfide bonds, hydrogen bonds, or hydrophobic interactions, to produce a multimeric protein.

[0120] An "isolated" material, e.g., an isolated nucleic acid, is material that is not in its natural environment, although 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. An isolated material can be separated, fractionated, or at least partially purified by any suitable technique.

[0121] As used herein, the terms "antibody" and "antigen-binding fragment thereof" refer to at least the minimum portion of an antibody capable of binding to a specific antigen targeted by the antibody, e.g., at least some of the complementarity-determining regions (CDRs) of the heavy chain variable domain (VH) and light chain variable domain (VL) in the context of a typical antibody produced by a B cell. In some antibodies, e.g., naturally occurring IgG antibodies, the heavy chain constant region is composed of a hinge and three domains, CH1, CH2, and CH3. In some antibodies, e.g., 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 is composed of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are embedded in more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin 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 specified herein, amino acids in the variable regions are numbered using the Kabat numbering system, and amino acids in the constant regions are numbered using the EU system.

[0122] The antibody or antigen-binding fragment thereof may be a polyclonal antibody, a monoclonal antibody, a human antibody, a humanized antibody or a chimeric antibody, a single chain antibody, an epitope-binding fragment such as Fab, Fab' and F(ab'), Fd, Fv, a single chain fragment variable (scFv), a single chain antibody, a V HAntibody molecules encompassed by the present disclosure may be or may be derived from H, vNAR, nanobodies, (single-domain antibodies), disulfide-linked Fvs (sdFvs), fragments comprising either the VL or VH domain alone or in combination with a portion of the opposing domain (e.g., an entire VL domain and a partial VH domain with one, two, or three CDRs), and fragments produced by a Fab expression library. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019. Antibody molecules encompassed by the present disclosure may be of or may be derived from any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule.

[0123] In a particular 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), wherein the VH comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 2, 11, 20, 29, 38, 47, 56, 65, 74, and 83; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 3, 12, 21, 30, 39, 48, 57, 66, 75, and 84; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4, 12, 21, 30, 39, 48, 57, 66, 75, and 84. and the VL comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 6, 15, 24, 33, 42, 51, 60, 69, 78, and 87; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 7, 16, 25, 34, 43, 52, 61, 70, 79, and 88; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 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 NOs: 1, 10, 19, 28, 37, 46, 55, 64, 73, and 82, and the VL comprises an amino acid sequence selected from SEQ ID NOs: 5, 14, 23, 32, 41, 50, 59, 68, 77, and 86.

[0124] In one aspect, the disclosure provides an anti-BCMA CAR comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 96. In another aspect, the disclosure provides an anti-BCMA CAR comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 97. In yet another aspect, the disclosure provides an anti-BCMA CAR comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 98.

[0125] As used herein, antibodies or antigen-binding fragments thereof also include "single-domain antibodies," which are antibodies whose complementarity-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 traditional 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 known as heavy-chain antibodies lacking light chains. In particular, camelid species, such as camels, dromedaries, llamas, alpacas, and guanacos, produce heavy-chain antibodies naturally lacking light chains. The variable heavy chains of single-domain antibodies lacking light chains are known as "VHHs" or "nanobodies." Similar to traditional VH domains, VHHs contain four FRs and three CDRs. Nanobodies have advantages over conventional antibodies: they are smaller than IgG molecules, and as a result, properly folded, functional nanobodies can be produced by in vitro expression with high yields. For example, VHH domains, nanobodies, and proteins / polypeptides containing them can be produced using microbial fermentation, eliminating the need for mammalian expression systems. VHH domains and nanobodies are relatively small (approximately 15 kDa, or 10-fold smaller than conventional IgG), and therefore exhibit higher tissue penetration (including, but not limited to, solid tumors and other dense tissues) than conventional four-chain antibodies and their antigen-binding fragments. VHH domains and nanobodies can exhibit so-called cavity-binding properties (due, inter alia, to their elongated CDR3 loops compared to conventional VH domains), and therefore can access targets and epitopes inaccessible to conventional four-chain antibodies and their antigen-binding fragments. Furthermore, nanobodies are highly stable and resistant to the action of proteases.

[0126] As used herein, "VHH domain" refers to the variable domain present in a naturally occurring heavy chain antibody, to distinguish between the heavy chain variable domain (herein referred to as "VH domain") present in a conventional four-chain antibody and the light chain variable domain (herein referred to as "VL domain") present in a conventional four-chain antibody. 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" by substituting one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence with one or more amino acid residues present at the corresponding position in a VH domain from a conventional four-chain antibody of human origin. This can be done by methods known in the art.

[0127] In one embodiment, the 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), a) CDR1 is the 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 amino acid sequences having at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity with the amino acid sequences of SEQ ID NO: 2, 11, 20, 29, 38, 47, 56, 65, 74 and 83, or is selected from the group consisting of amino acid sequences having two or only one amino acid difference compared to the amino acid sequences of SEQ ID NO: 2, 11, 20, 29, 38, 47, 56, 65, 74 and 83, b) CDR2 is the 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 amino acid sequences having at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity with the amino acid sequences of SEQ ID NO: 3, 12, 21, 30, 39, 48, 57, 66, 75 and 84, or is selected from the group consisting of amino acid sequences having two or only one amino acid difference compared to the amino acid sequences of SEQ ID NO: 3, 12, 21, 30, 39, 48, 57, 66, 75 and 84, c) CDR3 is the 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 amino acid sequences having at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity with the amino acid sequences of SEQ ID NO: 4, 13, 22, 31, 40, 49, 58, 67, 76 and 85, or is selected from the group consisting of amino acid sequences having two or only one amino acid difference compared to the amino acid sequences of SEQ ID NO: 4, 13, 22, 31, 40, 49, 58, 67, 76 and 85; The framework sequences may be any suitable framework sequences, such as those of single domain antibodies, in particular VHH antibodies.

[0128] B-cell maturation antigen (BCMA; also known as BCM; CD269; and TNFRSF13A) is a member of the TNF receptor superfamily. The receptor is expressed on mature B lymphocytes and may be important for B-cell development and autoimmune responses. BCMA, also known as TNF receptor superfamily member 17, has been shown to bind to tumor necrosis factor superfamily member 13b, resulting in NF-κB and MAPK8 / JNK activation. This receptor also binds to various TRAF family members, thus transmitting signals for cell survival and proliferation.

[0129] The term "antigen-binding portion" of an antibody, as used herein, 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 fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody, such as the anti-BCMA antibodies described herein, include (i) a Fab fragment (a fragment derived from papain cleavage) or a V L , V H (ii) a F(ab')2 fragment (a pepsin cleavage fragment) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H (iv) a Fd fragment consisting of a V domain and a CH1 domain; L and V H Fv fragment consisting of domains; (v) V H (vi) isolated complementarity-determining regions (CDRs); and (vii) combinations of two or more isolated CDRs, optionally joined by a synthetic linker. Additionally, the two domains of the Fv fragment, V, L and V H are encoded by separate genes, but they can be synthesized using recombinant methods. L and V HThese regions may be joined by a synthetic linker that allows them to be produced as a single protein chain, pairing to form a monovalent molecule (known as a 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 intended to be encompassed by terms such as "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments 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.

[0130] 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, a CAR can optionally comprise 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, a CAR can optionally comprise an armor domain comprising a nucleic acid sequence encoding an armor molecule. Expression of a CAR on the surface of a cell, e.g., an immune cell, enables the cell to target and bind to a specific antigen. In some embodiments, a CAR is expressed by an immune cell, e.g., a T cell. In some embodiments, the antigen-binding domain comprises a Fab, Fab', F(ab')2, Fd, Fv, single-chain fragment variable (scFv), single-chain antibody, VHH, vNAR, nanobody (single-domain antibody), or any combination thereof. In some embodiments, the transmembrane domain comprises a transmembrane domain selected from the transmembrane domains of CD4, CD8α, or CD28. In some embodiments, one or more intracellular domains comprise a costimulatory domain or a portion thereof. In some embodiments, the intracellular domain comprises a costimulatory domain or a portion thereof. In some embodiments, the intracellular domain comprises a costimulatory domain of CD3z or a variant thereof. For example, a CD3z costimulatory domain variant may contain only one or two functional immunoreceptor tyrosine-based activation motifs (ITAMs) of the three ITAMs present in wild-type CD3z. In some embodiments, the intracellular domain comprises a costimulatory domain selected from the group consisting of a CD3 zeta costimulatory domain, a CD28 costimulatory domain, a CD27 costimulatory domain, a 4-1BB costimulatory domain, an ICOS costimulatory domain, an OX-40 costimulatory domain, a GITR costimulatory domain, a CD2 costimulatory domain, an IL-2Rβ costimulatory domain, a MyD88 / CD40 costimulatory domain, and any combination thereof. The CAR can further comprise a "hinge" or "spacer" domain.Non-limiting examples of hinge / spacer domains include immunoglobulin hinge / spacer domains such as an IgG1 hinge domain, an IgG2 hinge domain, an IgG3 hinge domain, an IgG4 hinge domain, an IgG4P hinge domain (an IgG4 hinge domain containing an S241P mutation), a CD8a hinge domain, or a CD28 hinge domain. In one embodiment, the CAR comprises a hinge comprising the sequence of SEQ ID NO: 93.

[0131] As used herein, the term "polynucleotide" includes single nucleic acids as well as multiple nucleic acids and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). The term "nucleic acid" includes any type of nucleic acid, such as DNA or RNA. A "conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, a predicted non-essential amino acid residue in a BCMA binding moiety (e.g., an anti-BCMA CAR or antibody) is replaced with another amino acid residue from the same side chain family.

[0132] The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0133] 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 gap weights of 40, 50, 60, 70, or 80 and length weights 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)) as 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. Furthermore, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm, which is incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0134] The nucleic acid and protein sequences described herein can further be used as "query sequences" to perform searches against public databases, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (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 (eg, XBLAST and NBLAST) can be used.

[0135] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (e.g., lentiviral vectors, replication-defective retroviruses, adenoviruses and adeno-associated viruses) or transposons (e.g., DNA transposons or retrotransposons), which perform equivalent functions, are also included. In certain embodiments, the CAR and / or antibody or antigen-binding fragment thereof is contained in and / or delivered to cells and / or patients using viruses, lentiviruses, adenoviruses, retroviruses, adeno-associated viruses (AAV), transposons, DNA vectors, mRNA, lipid nanoparticles (LNPs), or CRISPR-Cas systems. In one embodiment, a lentiviral vector is used.

[0136] As used herein, the term "vector" can refer to a nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A vector can include a nucleic acid sequence that enables it to replicate in the host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Certain types of vectors contemplated herein can be associated with or incorporated into viruses to facilitate cell transformation.

[0137] A "transformed" cell or "host" cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. All techniques that can introduce a nucleic acid molecule into such a cell are contemplated herein, including transfection with a viral vector, transformation with a plasmid vector, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. In certain embodiments, the cell is transformed by one or more techniques using a virus, lentivirus, adenovirus, retrovirus, adeno-associated virus (AAV), transposon, DNA vector, mRNA, lipid nanoparticle (LNP), and CRISPR-Cas system.

[0138] As used herein, the term "affinity" refers to a measure of the strength of binding of an antigen or target (e.g., an epitope) to its cognate binding domain (e.g., a paratope). As used herein, the term "avidity" refers to the overall stability of the complex between a collection of epitopes and a paratope (i.e., an antigen and an antigen-binding domain).

[0139] The term "epitope" refers to a site on an antigen (e.g., BCMA) to which a chimeric antigen receptor, immunoglobulin, or antibody specifically binds, as defined, for example, by the particular method used to identify it. Epitopes can be formed both from contiguous amino acids (usually linear epitopes) or from noncontiguous amino acids juxtaposed by tertiary folding of a protein (usually conformational epitopes). Epitopes formed from adjacent amino acids are typically, but not always, retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation.

[0140] "Immunotherapy" refers to the treatment of a subject suffering from, or at risk of suffering from, a disease or a recurrence of a disease by methods involving inducing, enhancing, suppressing or otherwise modifying the immune system or immune response.

[0141] "Immune response," as understood in the art, generally refers to a biological response in a vertebrate to foreign or abnormal, e.g., cancerous, cells, which protects the organism from these agents and the diseases they cause. An immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by either these cells or the liver, resulting in the selective targeting, binding, damaging, destroying, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. An immune response can include, for example, the activation or inhibition of T cells, e.g., effector T cells, Th cells, CD4+ cells, CD8+ T cells, or Treg cells, or the activation or inhibition of any other cells of the immune system, e.g., NK cells.

[0142] As used herein, the terms "treat," "treatment," or "treatment of," when used in the context of treating cancer, refer to alleviating the pathology of the disease, reducing or eliminating the symptoms of the disease, promoting increased survival, and / or alleviating discomfort. For example, treating can refer to the ability of a treatment, when administered to a subject, to alleviate the symptoms, signs, or causes of a disease. Treating can also refer to the alleviation or reduction of 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 disorder.

[0143] As used herein, the terms "subject," "individual," or "patient" refer to any subject for whom diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, etc.

[0144] As used herein, the term "effective amount" or "therapeutically effective amount" of an administered therapeutic agent, such as CAR T cells, is an amount sufficient to carry out a specifically stated or intended purpose, such as treating or managing cancer. An "effective amount" can be empirically determined in relation to the stated purpose. In certain embodiments, a therapeutically effective amount can refer to the number of cells administered to a subject in need of treatment. The number of cells per dose, number of doses, and frequency of administration will depend on various parameters, such as the patient's age, weight, clinical evaluation, type of disease, type of cancer, type of tumor, tumor burden, and / or other factors, including the judgment of the attending physician.

[0145] The term "T cell" or "T lymphocyte" is art-recognized and is intended to include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, e.g., T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be T helper cells (HT1; 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 - The T cells may be T cells, or any other subset of T cells. Other exemplary populations of T cells suitable for use in certain embodiments include naive T cells and memory T cells.

[0146] As used herein, the term "proliferation" refers to an increase in cell division, either symmetric or asymmetric division of cells. In certain embodiments, "proliferation" refers to symmetric or asymmetric division of T cells. "Increased proliferation" occurs when there is an increase in the number of cells in a treated sample compared to cells in an untreated sample.

[0147] The term "growing" in the methods of the present disclosure refers to the process of increasing the number of cells in a cell culture. During the growth step, in one embodiment, the cells are fed and the culture medium is replaced at regular intervals according to a feeding regimen. The specific timing and amount of medium added in a particular feeding regimen depends on the number of cells and the level of metabolites in the culture.

[0148] As used herein, the term "differentiation" refers to a method of reducing the potency or proliferation of a cell or transitioning the cell to a more developmentally restricted state. In certain embodiments, differentiated T cells acquire immune effector cell function.

[0149] 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). Exemplary immune effector cells contemplated herein are NK cells or T lymphocytes, particularly cytotoxic T cells (CTL; ​​CD8 + T cells), TIL, and helper T cells (HTL; CD4 + T cells).

[0150] "Modified T cells" refers to T cells that have been modified by the introduction of a polynucleotide encoding an engineered CAR as contemplated herein. Modified T cells include both genetically modified and non-genetically modified (e.g., episomal or extrachromosomal).

[0151] As used herein, the terms "genetically engineered" or "genetically modified" refer to the addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell.

[0152] The terms "genetically modified cells," "modified cells," and "redirected cells" are used interchangeably.

[0153] The acronym "SMART" (Shorty-Manipulated Auto-Replicating T-Cells) refers to an abbreviated T-cell production and expansion process in which cells are cultured in the presence of IL-21 (and optionally IL-2).

[0154] The acronym "TNT" (conventional expanded T cells) refers to a conventional T cell expansion process that does not use IL-21, typically involves cell culture for more than 7 days, and / or typically involves the use of IL-2.

[0155] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating signal transduction events, including, but not limited to, signal transduction through the TCR / CD3 complex.

[0156] "Stimulatory molecule" refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand.

[0157] As used herein, "stimulatory ligand" means a ligand that, when present on an antigen-presenting cell (e.g., APC, dendritic cell, B cell, etc.), is capable of specifically binding to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response by 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), and peptides (e.g., CMV, HPV, EBV peptides).

[0158] The term "activated" refers to a state of T cells that have been stimulated sufficiently to induce detectable cell proliferation. In certain embodiments, activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to proliferating T cells. Signals generated through the TCR alone are insufficient for full activation of T cells; one or more secondary or costimulatory signals are also required. Thus, T cell activation includes a primary stimulatory signal via the TCR / CD3 complex and one or more secondary costimulatory signals. Costimulation can be evidenced by proliferation and / or cytokine production by T cells that have received a primary activation signal, such as stimulation via the CD3 / TCR complex or CD2.

[0159] A "costimulatory 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 upregulation or downregulation of specific molecules (e.g., CD28).

[0160] A "costimulatory ligand" refers to a molecule that binds to a costimulatory molecule. The costimulatory ligand may be soluble or may be provided on a surface. A "costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand (e.g., an anti-CD28 antibody).

[0161] "Autologous," as used herein, refers to cells derived from the same subject. In some embodiments, the cells of the present disclosure are autologous.

[0162] "Allogeneic," as used herein, refers to cells of the same species that are genetically distinct from the cell in comparison. In some embodiments, the cells of the present disclosure are allogeneic.

[0163] "Syngeneic," as used herein, refers to cells of a different subject that are genetically identical to the cell in comparison. In some embodiments, the cells of the present disclosure are syngeneic.

[0164] "Xenogeneic," as used herein, refers to a cell of a different species than the cell in comparison. In some embodiments, the cells of the present disclosure are xenogeneic.

[0165] 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 therapeutics, and methods disclosed elsewhere herein. Suitable subjects (e.g., patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), livestock, and domestic animals or pets (such as cats or dogs). Included are non-human primates, preferably human patients. Typical subjects include human patients who have cancer, have been diagnosed with cancer, or are at risk for or have cancer.

[0166] "Enhance" or "promote" or "increase" or "expand" generally refers to the ability of a composition contemplated herein to produce, induce, or cause a greater physiological response (i.e., downstream effect) compared to the response caused by either a vehicle or a control molecule / composition. A measurable physiological response can include increased T cell proliferation, activation, persistence, and / or increased ability to kill cancer cells, among other things that are apparent from understanding in the art and the description herein. An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include an increase in the response caused by a vehicle or control composition of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) (all integers and decimal points between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.).

[0167] "Decrease" or "lower" or "lessen" or "reduce" or "attenuate" generally refers to the ability of a composition contemplated herein to produce, induce, or cause a smaller physiological response (i.e., downstream effect) compared to the response caused by either a vehicle or a control molecule / composition. A "decreased" or "reduced" amount is typically a "statistically significant" amount and can include a reduction that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) (all integers and decimal points between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response caused by the vehicle, the control composition (the reference response), or the response in a particular cell lineage.

[0168] "Maintain" or "preserve" or "maintain" or "no change" or "no substantial change" or "no substantial decrease" generally refers to the ability of a composition contemplated herein to produce, induce, or cause a lesser physiological response (i.e., downstream effect) in a cell compared to the response caused by either a vehicle, a control molecule / composition, or the response in a particular cell lineage. An equivalent response is one that is not significantly or measurably different from the reference response.

[0169] overview In some aspects, the present disclosure relates to compositions and methods for treating disease using chimeric antigen receptor (CAR) cell therapy. More specifically, the present disclosure relates to CAR cell therapy in which transformed cells, such as T cells or NK cells, express a CAR that targets BCMA. Furthermore, CAR constructs, transformed cells expressing the constructs, and treatments utilizing the transformed cells disclosed herein can provide robust treatments for cancer, autoimmune diseases, or other diseases that express BCMA.

[0170] Without wishing to be bound by theory, BCMA is believed to be a viable disease target across multiple modalities, and BCMA is believed to be a promising target for CAR cell therapy.

[0171] CAR construct design The CAR constructs of the present disclosure can have several components, many of which can be selected based on the desired or improved function of the resulting CAR construct. In addition to the antigen-binding domain, the CAR construct can 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, the CAR can optionally include an armor domain comprising a nucleic acid sequence encoding an armor molecule. The selection of one component over another (i.e., the selection of a particular costimulatory domain from one receptor versus a costimulatory domain from a different receptor) can affect clinical efficacy and safety profile.

[0172] antigen-binding domain The antigen-binding domain contemplated herein may comprise an antibody or one or more antigen-binding fragments thereof. One contemplated CAR construct targeting BCMA comprises a single-chain variable fragment (scFv) containing light and heavy chain variable regions from one or more antibodies specific for BCMA, linked together either directly or via a flexible linker (e.g., G4S repeats having 1, 2, 3 or more repeats). In one embodiment, the linker comprises the sequence of SEQ ID NO: 92.

[0173] The antigen-binding domains of the CARs targeting BCMA disclosed herein may differ in their binding affinity to the BCMA protein. The relationship between binding affinity and efficacy may be more nuanced in the context of CARs compared to antibodies, where higher affinity is typically desirable. For example, preclinical studies on receptor tyrosine kinase-like orphan receptor 1 (ROR1)-CARs derived from a high-affinity scFv (dissociation constant 0.56 nM) resulted in an increased therapeutic index compared to low-affinity variants. Conversely, other examples have reported that engineering scFvs for lower affinity improved discrimination between cells with varying antigen densities. This may be useful for improving therapeutic specificity for antigens differentially expressed on tumors versus normal tissues.

[0174] Various methods can be used to confirm the binding affinity of an antigen-binding domain. In some embodiments, a method can be used to exclude avidity effects. Avidity effects often involve multiple antigen-binding sites simultaneously interacting with multiple target epitopes in a multimerized structure. Thus, avidity functionally represents the cumulative strength of multiple interactions. One example of a methodology to exclude avidity effects is any approach in which one or both interacting proteins are monomeric / monovalent, since multiple simultaneous interactions are impossible if one or both partners contain only a single interaction site.

[0175] spacer domain The CAR constructs of the present disclosure can have a spacer domain to provide conformational freedom to facilitate binding to the target antigen on the target cell. The optimal length of the spacer domain can 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 facilitating binding of the CAR to the target antigen, achieving an optimal distance between the CAR cell and the cancer cell can also help sterically block large inhibitory molecules from the immune synapse formed between the CAR cell and the target cancer cell. BCMA-targeting CARs can have long, intermediate, or short spacers. While long spacers can include the CH2CH3 domains (approximately 220 amino acids) of immunoglobulin G1 (IgG1) or IgG4 (either native or with a modification common in therapeutic antibodies, such as the S228P mutation), the CH3 region itself can be used to construct an intermediate spacer (approximately 120 amino acids). Shorter spacers can be derived from segments (<60 amino acids) of CD28, CD8α, CD3, or CD4. Short spacers 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 common to therapeutic antibodies, such as the S228P mutation described above. For example, the hinge domain can comprise an IgG1 hinge domain or mutant thereof, an IgG2 hinge domain or mutant thereof, an IgG3 hinge domain or mutant thereof, an IgG4 hinge domain or mutant thereof, a CD8 hinge domain or mutant thereof, or a CD28 hinge domain or mutant thereof.

[0176] Hinge domain BCMA-targeting CARs may 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 often used interchangeably; for example, an IgG4 sequence can be considered both a "hinge" and a "spacer" sequence (i.e., a hinge / spacer sequence). In some embodiments, 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 (particularly 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 comprises the sequence of SEQ ID NO: 93.

[0177] signal peptide A BCMA-targeting CAR can further comprise a sequence comprising a signal peptide. The signal peptide functions to prompt the cell to translocate the CAR to the cell membrane. Examples include an IgG1 heavy chain signal polypeptide, an Ig kappa or lambda light chain signal peptide, a granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2 or CSFR2) signal peptide, a CD8a signal polypeptide, or a CD33 signal peptide. In one embodiment, the signal peptide comprises the sequence of SEQ ID NO:91.

[0178] Transmembrane domain The CAR-targeting BCMA can further comprise a sequence comprising a transmembrane domain. The transmembrane domain can comprise a hydrophobic alpha helix spanning the cell membrane. Although the properties of the transmembrane domain have not been as thoroughly studied as other aspects of the CAR construct, they can potentially affect CAR expression and association with endogenous membrane proteins. The transmembrane domain can be derived from, for example, CD3, CD4, CD8α, or CD28. Any transmembrane domain can be used in the compositions disclosed herein. In some embodiments, the transmembrane domain comprises a transmembrane domain selected from the transmembrane domains of CD3, CD4, CD8α, or CD28. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO: 94.

[0179] Intracellular domain / costimulatory domain BCMA-targeting CARs can further comprise one or more sequences forming an intracellular domain and / or a costimulatory domain (sometimes referred to as a signaling domain). A costimulatory domain is a domain that can enhance or modulate (i.e., initiate) an immune effector cell response. In some embodiments, the costimulatory domain and / or signaling domain comprises a primary activation signal derived from the cytoplasmic domain of CD3ζ, which contains a sequence motif called an immunoreceptor tyrosine-based activation motif (ITAM). In certain embodiments, the intracellular domain refers to a combination of a costimulatory domain (e.g., a costimulatory domain derived from 4-1BB or CD28) plus a primary activation signal of CD3 zeta (CD3z or CD3 zeta). The costimulatory domain can comprise sequences, e.g., a costimulatory domain derived from one or more of CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ, and MyD88 / CD40. In certain embodiments, a costimulatory domain selected from CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ, and MyD88 / CD40 is combined with a primary activation signal of CD3 zeta (CD3z or CD3 zeta). In some embodiments, the costimulatory domain may comprise a variant of one or more costimulatory domains of CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ, and MyD88 / CD40. In certain embodiments, the costimulatory domain variant is selected from a costimulatory domain variant of CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ, and MyD88 / CD40, and is combined with a primary activation signal of CD3 zeta (CD3z or CD3 zeta). In one embodiment, the CAR costimulatory domain may further comprise a modification to the CD3z domain. For example, a CD3z signaling domain variant can contain one or two functional immunoreceptor tyrosine-based activation motifs (ITAMs) of the three ITAMs present in wild-type CD3z. The choice of costimulatory domain influences the phenotype and metabolic signature of the CAR cell.For example, CD28 costimulation results in a potent but short-lived effector-like phenotype with high levels of cytolytic capacity, interleukin-2 (IL-2) secretion, and glycolysis. In contrast, T cells engineered with CARs containing a 4-1BB costimulatory domain tend to proliferate and persist longer in vivo, have increased oxidative metabolism, are less prone to exhaustion, and have an increased ability to generate central memory T cells. In some embodiments, the intracellular signaling domain comprises a costimulatory domain or a portion thereof. In one embodiment, the intracellular domain comprises the sequence of SEQ ID NO:95.

[0180] In some embodiments, the intracellular domain comprises a costimulatory domain selected from the group consisting of an intracellular domain of a CD28 costimulatory domain, a CD27 costimulatory domain, a 4-1BB costimulatory domain, an ICOS costimulatory domain, an OX-40 costimulatory domain, a GITR costimulatory domain, a CD2 costimulatory domain, an IL-2Rβ costimulatory domain, a 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 CD3 zeta. In some embodiments, the intracellular domain comprises a 4-1BB costimulatory domain in combination with CD3 zeta. In one embodiment, the intracellular domain comprises the sequence of SEQ ID NO: 95.

[0181] In certain embodiments, the intracellular domain comprises a costimulatory domain, CD3 zeta (or CD3z; the CD3z signaling domain is also referred to herein as a "CD3z costimulatory domain"), that comprises a portion of the intracellular T cell receptor (TCR) signaling domain. In some embodiments, the CD3 zeta comprises one or more modifications to the CD3z format. For example, the CD3z signaling domain variant can contain one or two functional immunoreceptor tyrosine-based activation motifs (ITAMs) of the three ITAMs (e.g., 1XX, X1X, or X2X) present in wild-type CD3z.

[0182] Exemplary CAR According to all aspects of the invention, the CAR may comprise or consist of the amino acid sequence set forth as SEQ ID NO: 96. According to all aspects of the invention, the CAR may comprise or consist of the amino acid sequence set forth as SEQ ID NO: 97. According to all aspects of the invention, the CAR may comprise or consist of the amino acid sequence set forth as SEQ ID NO: 98.

[0183] In a particular 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), wherein the VH comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 2, 11, 20, 29, 38, 47, 56, 65, 74, and 83; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 3, 12, 21, 30, 39, 48, 57, 66, 75, and 84; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4, 12, 21, 30, 39, 48, 57, 66, 75, and 84. and the VL comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 6, 15, 24, 33, 42, 51, 60, 69, 78, and 87; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 7, 16, 25, 34, 43, 52, 61, 70, 79, and 88; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 8, 17, 26, 35, 44, 53, 62, 71, 80, and 89.

[0184] 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 NOs: 1, 10, 19, 28, 37, 46, 55, 64, 73, and 82, and the VL comprises an amino acid sequence selected from SEQ ID NOs: 5, 14, 23, 32, 41, 50, 59, 68, 77, and 86.

[0185] The CAR constructs of the present disclosure can comprise several combinations of the modular components described herein. For example, in some embodiments of the present disclosure, the CAR construct comprises a BCMA scFv antigen-binding domain. In some embodiments of the present disclosure, the CAR construct comprises a CD33 signal peptide. In one embodiment, the CAR comprises a signal peptide comprising the sequence of SEQ ID NO: 91. In some embodiments, the CAR construct comprises an IgG4 hinge / spacer domain with a S241P mutation (IgG4P). In one embodiment, the CAR comprises a hinge domain comprising the sequence of SEQ ID NO: 93. In some embodiments, the CAR construct comprises a CD28 transmembrane domain. In one embodiment, the CAR comprises a transmembrane domain comprising the sequence of SEQ ID NO: 94.

[0186] Different costimulatory domains can be utilized in the CAR constructs of the present disclosure. In some embodiments, the CAR construct comprises a costimulatory domain comprising a signaling domain from the intracellular domain of CD3z (e.g., a portion of the intracellular T cell receptor (TCR) signaling domain, CD3 zeta (or CD3z) or a variant thereof). In some embodiments, the CAR construct comprises a CD28 costimulatory domain. In some embodiments, the CAR construct comprises a 4-1BB costimulatory domain. In some embodiments, the CAR construct comprises costimulatory domains from CD3z and CD28 as described herein. In some embodiments, the CAR construct comprises costimulatory domains from CD3z and 4-1BB as described herein. In some embodiments, the CAR construct comprises costimulatory domains from all of CD3z, CD28, and 4-1BB as described herein. In some embodiments, the CAR construct comprises costimulatory domains from ICOS, OX-40, and / or GITR. In one embodiment, the CAR comprises an intracellular domain comprising the sequence of SEQ ID NO: 95.

[0187] cell CAR-based cell therapy can be used with a variety of cell types, such as lymphocytes. Specific types of cells that can be used include T cells, natural killer (NK) cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, alpha beta T cells, gamma delta T cells, virus-specific T (VST) cells, cytotoxic T lymphocytes (CTLs), tumor-infiltrating lymphocytes, and regulatory T cells (Tregs). In some embodiments, the cells are autologous. In certain embodiments, the cells are allogeneic. In other embodiments, the cells can be derived from a genetically similar, but not identical, donor (allogeneic).

[0188] In some embodiments, the population of cells may also include expanded populations and / or engineered T cells. In some embodiments, the population of cells may include total T cells, CD4+ T cells, CD8+ T cells, regulatory T cells, gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells, or natural killer T (NKT) cells. T cells are broadly classified into cells that express CD4 on their surface (also called CD4+ cells) and cells that express CD8 on their surface (also called CD8+ cells).

[0189] In some embodiments, T cells suitable for use with the methods provided herein are mononuclear lymphocytes derived from the bone marrow (BM), peripheral blood (PB), or umbilical cord blood (CB) of a human donor. These cells can be collected directly from the BM, PB, or CB, or can be collected after mobilization or stimulation by administering growth factors and / or cytokines, such as granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), to an allogeneic or autologous donor. Those skilled in the art will appreciate that there are many established protocols for isolating peripheral blood mononuclear cells (PBMCs) from peripheral blood. PBMC isolation can be assisted by density gradient separation protocols, typically employing density gradient centrifugation techniques using Ficoll®-Hypaque or Histopaque® to separate lymphocytes from other components in the blood. Preferably, PBMC isolation is performed under sterile conditions. PBMC isolation can also utilize a negative selection kit. Alternatively, cell lysis methods can be used to isolate mononuclear cell populations. In some embodiments, the population of cells are human cells. In certain embodiments, the population of cells are human primary immune cells.

[0190] In some embodiments, the cell compositions and methods of the present disclosure may include cells genetically engineered to be resistant to replicative senescence (RRS). In some embodiments, cells resistant to replicative senescence may include a transgene encoding B-cell lymphoma-ultra-large cell (Bcl-xL). In certain embodiments, cells resistant to replicative senescence may include a transgene encoding B-cell lymphoma-ultra-large cell (Bcl-xL) and / or B-cell lymphoma 2 (Bcl-2). In some embodiments, cells resistant to replicative senescence may include a knockout of, or inhibition of expression of, one or more endogenous regulators selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP). In some embodiments, cells resistant to replicative senescence may include a knockout of, or inhibition of expression of, one or more endogenous immune-related genes in primary immune cells. In certain embodiments, the endogenous immune-related gene is beta 2 microglobulin (B2M) or T cell receptor alpha constant (TRAC). In some embodiments, cells resistant to replicative senescence can comprise knockout or inhibited expression of CD38.

[0191] The term "genetically engineered" refers to changes to the genetic material of a cell. Gene editing involves adding, removing, or modifying genetic material in a genetically engineered cell. In certain embodiments, gene editing involves introducing a transgene into the cell and / or inhibiting expression of a gene in the cell. In certain embodiments, introducing one or more gene edits involves introducing one or more transgenes into the cell that encode anti-apoptotic factors or virus-derived factors.

[0192] The term "transgene" refers to any nucleic acid sequence introduced into a cell by experimental manipulation. A transgene may be an "endogenous DNA sequence" or a "heterologous DNA sequence." The term "endogenous" refers to originating within a cell, tissue, or organism or part of a cell, tissue, or organism. A transgene can be isolated or obtained in suitable quantities using one or more methods known in the art. These and other methods useful for isolating transgenes are described, for example, in Sambrook et al. (supra) and Berger and Kimmel (Methods in Enzymology: Guide to Molecular Cloning Techniques, vol. 152, Academic Press, Inc., San Diego, CA (1987)). A transgene can be incorporated into a "transgene construct," which contains the gene of interest along with other regulatory DNA sequences required for either transient, cell-specific, or enhanced expression of the transgene of interest. A transgene can be introduced into a cell by any suitable method or technique known in the art. In some embodiments, the transgene is introduced using a plasmid-based DNA transposon, a lentiviral platform, or CRISPR-mediated site-specific integration. Transgene expression in cells can be constitutive or inducible.

[0193] In certain embodiments, the transgene encodes a virus-derived factor. "Virus-derived factor" refers to both naturally occurring viral peptides, polypeptides, or proteins, as well as peptides, polypeptides, or proteins that exhibit a degree of sequence identity and / or similarity with a viral protein and / or maintain one or more structural, mechanistic, or antigenic properties of the viral protein. In certain embodiments, the virus-derived factor is derived from Saimiri gammaherpesvirus 2 StpA A11, herpesvirus Saimiri StpC, herpesvirus Saimiri Tip, or modified herpesvirus Ateles-Epstein-Barr virus Tio-LMP1.

[0194] In some embodiments, the cells described herein further comprise inhibition of expression of one or more endogenous regulatory factors in the cells such that the activity of the endogenous regulatory factors is eliminated or reduced. As used herein, "regulator" 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 or reducing the activity of a factor include, but are not limited to, CRISPR / Cas (including cytosine and adenine base editors), microRNA, shRNA, RNAi, TALEN, zinc finger nucleases, meganucleases, neutralizing antibodies, small molecule inhibitors, chemical inhibitors that block downstream signaling pathways, and the like. Inhibition of endogenous regulatory factors can be complete inhibition, partial inhibition, downregulation of gene expression, or reduction of the activity of the factor. In some embodiments, endogenous regulator activity or gene expression is reduced by 1% to 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%). Regulators include genes encoding proteins involved in regulating cell cycle arrest, cell death, or signal suppression. In certain aspects, the one or more endogenous regulators 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 regulators are RB transcriptional corepressor 1 (RB1), TP53, autophagy and beclin 1 regulator 1 (AMBRA1), neurofibromatosis type 1 (NF1), tyrosine-protein phosphatase non-receptor type 2 (PTPN2), or suppressor of cytokine signaling 1 (SOCS1).

[0195] In some embodiments, the cells disclosed herein comprise inhibition of expression of one or more endogenous immune-related genes in the cells such that the activity of the immune-related genes is eliminated or reduced. As used herein, "immune-related gene" refers to a gene encoding a protein involved in bringing about an immune response. In certain aspects, the immune-related gene encodes a protein involved in host-versus-graft (HvG) and graft-versus-host (GvH) allogeneic immune responses. Immune-related genes can be downregulated or blocked by any suitable method or technique known in the art. Known methods for downregulating gene expression 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, meganucleases, neutralizing antibodies, small molecule inhibitors, chemical inhibitors that block downstream signaling pathways, and the like. Inhibition of endogenous immune-related genes can be complete inhibition, partial inhibition, downregulation of gene expression, or reduction of the activity of a factor. In some embodiments, the activity or gene expression of endogenous immune-related genes is reduced by 1% to 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 encoding 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 certain embodiments, the one or more endogenous immune-related genes are beta 2 microglobulin (B2M) or T-cell receptor alpha constant (TRAC).In certain embodiments, the one or more endogenous immune-related genes are 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 receptor), 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-1 (PD-1), TIM3, or TIGIT genes.

[0196] In further embodiments, the cells disclosed herein comprise inhibition of expression of cluster of differentiation 38 (CD38) in the cells such that CD38 activity is eliminated or reduced. CD38 can be downregulated or blocked by any suitable method or technique known in the art. Known methods for downregulating gene expression or reducing CD38 activity include, but are not limited to, CRISPR / Cas (including cytosine and adenine base editors), microRNA, shRNA, RNAi, TALEN, zinc finger nucleases, meganucleases, neutralizing antibodies, small molecule inhibitors, chemical inhibitors that block downstream signaling pathways, and the like. In some embodiments, CD38 activity or gene expression is reduced by between 1% and 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%).

[0197] In further embodiments, the cells disclosed herein comprise inhibition of phosphatase and tensin homolog (PTEN) expression in primary immune cells such that PTEN activity is eliminated or reduced. PTEN can be downregulated or blocked by any suitable method or technique known in the art. Known methods for downregulating gene expression or reducing PTEN activity include, but are not limited to, CRISPR / Cas (including cytosine and adenine base editors), microRNA, shRNA, RNAi, TALEN, zinc finger nucleases, meganucleases, neutralizing antibodies, small molecule inhibitors, chemical inhibitors that block downstream signaling pathways, and the like. In some embodiments, PTEN activity or gene expression is reduced by between 1% and 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%).

[0198] "T REX The term "newly expandable T cells" refers to "newly expandable T cells" using, for example, the techniques and genetic modifications provided herein. More specifically, T REX The cells refer to cells in which 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 absent.

[0199] In some embodiments, the inhibition of expression of one or more endogenous regulatory factors occurs after introducing one or more transgenes into the cells. In some embodiments, 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 before the inhibition of one or more endogenous regulatory factors occurs. In further embodiments, the inhibition of expression of PTEN occurs after introducing one or more transgenes into the cells. In some embodiments, the method comprises the sequential steps of: 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, or at least 20 days; ii) inhibiting one or more endogenous regulators of PTEN expression while 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, or at least 20 days; and iii) inhibiting PTEN expression.

[0200] SMART / T cells / T REX Cell activation and proliferation The present disclosure also relates to a method for culturing chimeric antigen receptor (CAR)-transduced T cells that generates a sustained population of T cells that exhibits increased antigen-independent activation. The acronym "SMART" (Shorty-Manipulated Auto-Replicating T-Cells) refers to an abbreviated T cell manufacturing and expansion process in which cells are cultured in the presence of IL-21 (and optionally IL-2).

[0201] Some aspects of the present disclosure relate to cells comprising a polynucleotide or polypeptide disclosed herein. Some aspects of the present disclosure relate to cells 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 autologous to the recipient. In some embodiments, the cell is selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, γδ T 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.

[0202] Prior to the expansion and genetic modification of T cells of the present disclosure, a source of T cells is obtained from a 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 an infection site, ascites, pleural effusion, spleen tissue, and tumors. Certain embodiments of the present disclosure can use any number of T cell lines available in the art. In certain embodiments of the present disclosure, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation. In one embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In another embodiment, the wash solution lacks calcium, and may lack magnesium, or may lack many, but not all, divalent cations. Again, an initial activation step in the absence of calcium results in expanded activation. As those skilled in the art will readily appreciate, the wash 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., a Cobe 2991 cell processor, a Baxter CytoMate, or a 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 buffers. Alternatively, undesirable components of the apheresis sample can be removed and the cells resuspended directly in culture medium.

[0203] In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or counterflow centrifugal elution. Specific subpopulations of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. In some embodiments, T cells are isolated by positive selection for CD4 and CD8 expression. For example, in one embodiment, T cells are isolated by incubation with anti-CD4 / anti-CD8 conjugated beads for a time sufficient to positively select the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or more, and all integer values ​​therebetween. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the time period is 10 to 24 hours. In any situation where T cells are scarce relative to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals, longer incubation times can be used to isolate T cells. Furthermore, using longer incubation times can increase the efficiency of CD8+ T cell capture. Thus, simply by shortening or lengthening the time T cells are allowed to bind to CD4 / CD8 beads and / or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for at the beginning of culture or at other times during the process. Furthermore, by increasing or decreasing the ratio of anti-CD4 and / or anti-CD8 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for at the beginning of culture or at other desired times. 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 expansion process. "Unselected" cells can also be subjected to additional rounds of selection.

[0204] Enrichment of T cell populations by negative selection can be achieved using a combination of antibodies against surface markers unique to the negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, and HLA-DR. In certain embodiments, it may be desirable to enrich or positively select regulatory T cells, which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain embodiments, T regulatory cells are depleted using anti-C25 conjugated beads or other similar selection methods.

[0205] For isolation of desired cell populations by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the cells and the beads. 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 a further embodiment, greater than 100 million cells / mL is used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / mL is used. In further embodiments, concentrations of 125 or 150 million cells / mL can be used. The use of higher concentrations can result in increased cell yield, cell activation, and cell proliferation.

[0206] In related embodiments, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells are minimized. This selects for cells that express large amounts of the desired antigen that binds to the particles. For example, CD4+ T cells express higher levels of CD28 than CD8+ T cells at dilute concentrations and are captured more efficiently. In one embodiment, the concentration of cells used is 5x10 6 / mL. In other embodiments, the concentration used is about 1x10 5 / mL ~ 1x10 6 / mL, and any integer value therebetween.

[0207] In other embodiments, cells may be incubated on a rotator at various speeds for various lengths of time at either 2-10°C or room temperature.

[0208] T cells for stimulation may also be frozen after a washing step. In some embodiments, the freezing and subsequent thawing step can provide a more uniform product by removing granulocytes and some monocytes from the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and are 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. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen, can be used.

[0209] In certain embodiments, cryopreserved cells are thawed, washed, and allowed to stand at room temperature for 1 hour before activation using the methods of the present disclosure.

[0210] In the context of the present invention, collection of a blood sample or apheresis product from a subject at a time before the expanded cells described in this disclosure may be needed is also contemplated. Thus, a source of expanded cells can be collected at any time needed, and desired cells, such as T cells, can be isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy (e.g., diseases or conditions described herein). In one embodiment, a blood sample or apheresis is taken from a generally healthy subject. In a specific embodiment, a blood sample or apheresis is taken from a generally healthy subject who is at risk for developing a disease but has not yet developed the disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, T cells can be expanded, frozen, and used later. In certain embodiments, a sample is collected from a patient shortly after diagnosis of a particular disease described herein, but prior to any treatment. In further embodiments, the cells are isolated from a blood sample or apheresis from the subject prior to any number of relevant therapeutic modalities, including, but not limited to, treatment with drugs such as natalizumab, efalizumab, antivirals, chemotherapy, radiation, drugs such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation. These drugs either inhibit calcineurin, a calcium-dependent phosphatase (cyclosporine and FK506), or inhibit p70S6 kinase (rapamycin), which is important in growth factor-induced signal transduction (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 a further embodiment, the cells are isolated for a patient and frozen for later use in conjunction with (e.g., before, simultaneously with, or after) T cell depleting therapy using either bone marrow or stem cell transplant, chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cells may be isolated prior to B cell depleting therapy, such as an agent reactive with CD20, e.g., Rituxan, and frozen for use in subsequent treatment.

[0211] In further embodiments of the present disclosure, T cells are obtained from a patient immediately after treatment. In this regard, it has been observed that following certain cancer treatments, particularly treatment with drugs that damage the immune system, the quality of T cells obtained immediately after treatment, during the period when patients are typically recovering from treatment, can be optimal or improved in terms of their ability to expand ex vivo. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present disclosure to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic system, during this recovery period. Furthermore, in certain embodiments, mobilization (e.g., mobilization with GM-CSF or G-CSF) and pretreatment regimens can be used to create conditions in a subject that favor the repopulation, recirculation, regeneration, and / or expansion of specific cell types, particularly during a defined time frame after treatment. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0212] Either before or after genetic modification of the T cells to express a desired CAR, the T cells can be engineered to express a desired CAR, as described, for example, 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; 7,066; Activation and propagation can generally be achieved using methods such as those described in U.S. Patent Application Publication Nos. 7,318; 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.

[0213] Generally, T cells of the present disclosure are expanded by contacting them with a surface bearing an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells. In particular, T cell populations can be stimulated, for example, by contact with a surface-immobilized anti-CD3 antibody or antigen-binding fragment thereof or anti-CD2 antibody, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore, as described herein. For costimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besangon, France), and can be used in a similar manner to other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0214] In certain embodiments, the primary stimulatory signal and the costimulatory signal for T cells can be provided by different protocols. For example, the agents providing each signal can be in solution or bound to a surface. If bound to a surface, the agents can be bound to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one agent can be bound to a surface and the other agent can be in solution. In one embodiment, the agent providing the costimulatory signal is bound to a cell surface, and the agent providing the primary activation signal is in solution or bound to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents can be in soluble form and then crosslinked to a surface, such as a cell expressing an Fc receptor or an antibody or other binding agent that binds the agent. In this regard, see, e.g., U.S. Patent Application Publication Nos. 20040101519 and 20060034810, for artificial antigen-presenting cells (aAPCs) contemplated for use in the activation and expansion of T cells in the present disclosure.

[0215] In one embodiment, the two agents are immobilized on beads, either on the same bead (i.e., "cis") or on separate beads (i.e., "trans"). By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof, with both agents co-immobilized on the same bead in equimolar amounts. In one embodiment, a 1:1 ratio of each antibody bound to beads is used for CD4+ T cell expansion and T cell proliferation. In certain embodiments of the present disclosure, bead-bound anti-CD3:CD28 antibodies are used such that increased T cell proliferation is observed compared to the proliferation observed using a 1:1 ratio.

[0216] In further embodiments of the present disclosure, cells, such as T cells, are combined with drug-coated beads, the beads and cells are subsequently separated, and the cells are then cultured. In another embodiment, the drug-coated beads and cells are cultured together without separation prior to culture. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0217] Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)), which may contain factors necessary for growth 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 additive for the growth of cells known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The culture medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, and is supplemented with amino acids, sodium pyruvate, and vitamins, and may be serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in amounts sufficient for T cell growth and proliferation. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells infused into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2). In one embodiment, the culture medium is X-Vivo 15 serum-free medium containing 1% (v / v) recombinant serum substitute (ITSE-A).

[0218] In one embodiment, T cells are cultured in medium containing 10 to 300 IU / mL of recombinant human IL-2. In one embodiment, T cells are cultured in medium containing 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, or 300 IU / mL of recombinant human IL-2. In another embodiment, T cells are cultured in medium also containing 0.1 to 0.3 U / mL of recombinant IL-21. In another embodiment, T cells are cultured in medium containing IL-2 and 0.1, 0.2, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 75, or 100 U / mL of recombinant human IL-21. In another embodiment, T cells are cultured in medium containing IL-2 and 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.30 U / mL of recombinant human IL-21. In one embodiment, T cells are cultured in medium containing 40 IU / mL of recombinant human IL-2 and 0.24 U / mL of recombinant human IL-21.

[0219] In one embodiment of the present disclosure, the cells were cultured for up to 14 days. In another embodiment, the mixture may be cultured for 4 days. The T cells can be agitated during any stage of culture. In one embodiment, the cells are agitated during cell culture in a medium containing IL-2 and IL-21. In a specific embodiment, T cells harvested on day 4 exhibit higher target-independent killing activity compared to CAR-T cells harvested on day 6.

[0220] In one embodiment of the present disclosure, CD8 +T cells are isolated from total PBMCs. Cells are either cryopreserved or activated immediately after isolation using CD3 / CD28 stimulation. After 3 days of activation, CRISPR knockout of CDKN2A, CDKN2B, and MTAP (called REX editing) is performed to confer resistance to replicative senescence. Cells are further engineered with site-specific CRISPR knockin of BCMA-CAR. In some embodiments, knockout of B2M enhances the patient's CD8 + This is done to limit T cell recognition of these donor cells. In some embodiments, CD38 is additionally knocked out to allow resistance to daratumumab. In some embodiments, cells are edited at the TRAC locus to eliminate TCR αβ expression and eliminate the risk of graft-versus-host disease.

[0221] Vectors, host cells and pharmaceutical compositions of the present disclosure In some embodiments, the polynucleotides of the present disclosure are present in a vector. Thus, provided herein are vectors comprising the polynucleotides of the present disclosure. In some embodiments, the present disclosure relates to a vector or set of vectors comprising a polynucleotide encoding a CAR as described herein.

[0222] Any vector known in the art may be suitable for the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an 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, the CAR and / or antibody or antigen-binding fragment thereof is contained in and / or delivered to cells and / or patients using a virus, lentivirus, adenovirus, retrovirus, adeno-associated virus (AAV), a transposon, a DNA vector, mRNA, a lipid nanoparticle (LNP), or a CRISPR-Cas system.

[0223] In other embodiments, provided herein are host cells comprising a polynucleotide or vector of the present disclosure. In some embodiments, the present disclosure relates to host cells, e.g., in vitro cells, comprising a polynucleotide encoding a CAR or TCR described herein. In other embodiments, the present disclosure relates to in vitro cells comprising a polypeptide encoded by a polynucleotide encoding a CAR that specifically binds to BCMA. In other embodiments, the present disclosure relates to cells, e.g., in vitro cells, comprising a polypeptide encoded by a polynucleotide encoding an antibody or antigen-binding molecule thereof that specifically binds to BCMA, as disclosed herein.

[0224] Any cell can be used as a host cell for the polynucleotides, vectors, or polypeptides of the present disclosure, hi 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, e.g., gram-negative or gram-positive organisms, such as Enterobacteriaceae, e.g., Escherichia, e.g., E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g., Salmonella typhimurium; Serratia, e.g., Serratia marcescens and Shigella; Bacillus, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell.

[0225] Other embodiments of the present disclosure relate to compositions comprising a polynucleotide described herein, a vector described herein, a polypeptide described herein, or a cell described herein. In some embodiments, the composition comprises a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and / or adjuvant. In some embodiments, the composition comprises an excipient. In one embodiment, the composition comprises a polynucleotide encoding a CAR, wherein the CAR comprises an antigen binding molecule that specifically binds to BCMA. In another embodiment, the composition comprises a CAR encoded by a polynucleotide of the present disclosure, wherein the CAR comprises an antigen binding molecule that specifically binds to BCMA. In another embodiment, the composition comprises a T cell comprising a polynucleotide encoding a CAR, wherein the CAR comprises an antigen binding molecule that specifically binds to BCMA. In another embodiment, the composition comprises a cell (e.g., a T cell, e.g., a CAR-T cell) comprising a polynucleotide encoding a CAR comprising an antigen binding domain that specifically binds to BCMA, as disclosed herein.

[0226] In other embodiments, the compositions are formulated for parenteral delivery, inhalation, or delivery via the digestive tract, such as orally. Preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art. In certain embodiments, a buffer is used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8. In certain embodiments, when parenteral administration is intended, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution in a pharmaceutically acceptable vehicle, with or without additional therapeutic agents. In certain embodiments, the parenteral injection vehicle is sterile distilled water, with or without at least one additional therapeutic agent, formulated as a properly preserved, sterile, isotonic solution. In certain embodiments, preparation involves formulating the desired molecule using beads or liposomes, which are polymeric compounds (such as polylactic acid or polyglycolic acid) that allow for controlled or sustained release of the product, which are then delivered by depot injection. In certain embodiments, an implantable drug delivery device is used to introduce the desired molecule.

[0227] Treating Disease with CARs In some embodiments, the present disclosure provides CAR cells for treating disease. 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 BCMA-expressing cancer and / or hematological malignancies. The compositions (e.g., CAR constructs and CAR cells) and methods of use described herein are particularly useful for inhibiting the growth or spread of neoplastic cells, particularly neoplastic cell proliferation in which BCMA plays a role.

[0228] In one embodiment, cancers contemplated for treatment herein include any that express BCMA on the cell surface of cancer cells. Cancers contemplated for treatment herein may include, but are not limited to, multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphocytic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL). In one embodiment, the present disclosure provides CAR cells for treating multiple myeloma.

[0229] In some embodiments, the present disclosure provides CAR cells for the treatment of autoimmune diseases. In certain embodiments, the present disclosure provides CAR cells for the treatment of autoimmune diseases in which BCMA is involved. The compositions (e.g., CAR constructs and CAR cells) and methods of use thereof described herein are particularly useful for the treatment of autoimmune diseases in which BCMA plays a role. In certain embodiments, the present disclosure provides CAR cells for the treatment of lupus.

[0230] Treatment method The CAR-modified cells of the present disclosure, such as CAR T cells, can be administered alone or as a pharmaceutical composition together with a diluent and / or cytokines or other components associated with the cell population. Briefly, the pharmaceutical composition of the present disclosure can include, for example, the CAR cells described herein together with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include a buffer, such as neutral buffered saline, buffered saline, etc.; sulfate salts; 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 can be adapted for treatment (or prevention).

[0231] In some embodiments, the present disclosure provides methods of treating disease by 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, which may 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 SEQ ID NOs: 2, 11, 20, 29, 38, 47, 56, 65, 74, and 83; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 3, 12, 21, 30, 39, 48, 57, 66, 75, and 84; and a CDR3 comprising an amino acid sequence selected from 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 SEQ ID NOs: 6, 15, 24, 33, 42, 51, 60, 69, 78, and 87; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 7, 16, 25, 34, 43, 52, 61, 70, 79, and 88; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 8, 17, 26, 35, 44, 53, 62, 71, 80, and 89. In certain embodiments, the VH comprises an amino acid sequence selected from SEQ ID NOs: 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 NOs: 5, 14, 23, 32, 41, 50, 59, 68, 77, and 86. In one aspect, the disclosure provides an anti-BCMA CAR comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 96. In another aspect, the disclosure provides an anti-BCMA CAR comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 97. In yet another aspect, the disclosure provides an anti-BCMA CAR comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 98.

[0232] As used herein, the term "effective amount" or "therapeutically effective amount" of an administered therapeutic agent, such as CAR T cells, is an amount sufficient to carry out a specifically stated or intended purpose, such as treating a disease or curing a disease. An "effective amount" can be empirically determined in relation to the stated purpose. In certain embodiments, a therapeutically effective amount can refer to the number of cells administered to a subject in need of treatment. The number of cells per dose, number of doses, and frequency of administration will depend on various parameters, such as the patient's age, weight, clinical evaluation, type of disease, type of cancer, type of tumor, tumor burden, and / or other factors, including the judgment of the attending physician.

[0233] In some embodiments, the cancer treated by the methods is multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphocytic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML), or acute lymphocytic leukemia (ALL). In one embodiment, the present disclosure provides CAR cells for treating multiple myeloma.

[0234] In some embodiments, the present disclosure provides CAR cells for the treatment of autoimmune diseases. In certain embodiments, the methods of the present disclosure provide CAR cells for the treatment of autoimmune diseases in which BCMA is involved. The compositions (e.g., CAR constructs and CAR cells) and methods of use thereof described herein are particularly useful for the treatment of autoimmune diseases in which BCMA plays a role. In certain embodiments, the present disclosure provides CAR cells for the treatment of lupus.

[0235] It is to be understood that certain aspects of the present specification are not limited to the specific embodiments presented and may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting unless specifically defined herein. Furthermore, certain embodiments disclosed herein can be combined with other embodiments disclosed herein without limitation, as recognized by one of ordinary skill in the art.

[0236] Embodiments: Embodiment 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 An isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), comprising:

[0237] 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, single-chain fragment variable (scFv), single-chain antibody, VHH, vNAR, nanobody (single-domain antibody), or any combination thereof.

[0238] Embodiment 3. The isolated nucleic acid sequence of embodiment 2, wherein the antigen-binding domain is a single-chain variable fragment (scFv).

[0239] Embodiment 4. The isolated nucleic acid sequence of embodiment 3, wherein the antigen-binding domain is an scFv comprising an amino acid sequence selected from SEQ ID NOs: 9, 36, and 90.

[0240] Embodiment 5. The isolated nucleic acid sequence of embodiment 3, wherein the antigen-binding domain is an scFv comprising the amino acid sequence of SEQ ID NO:9.

[0241] 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.

[0242] Embodiment 7. The isolated nucleic acid sequence of embodiment 6, wherein the transmembrane domain comprises a CD28 transmembrane domain.

[0243] Embodiment 8. The isolated nucleic acid sequence of any one of embodiments 1 to 7, wherein the one or more intracellular domains comprise a costimulatory domain or a portion thereof.

[0244] Embodiment 9. The isolated nucleic acid sequence of embodiment 8, wherein the costimulatory domain comprises one or more of the CD3z, 4-1BB, CD2, CD27, CD28, OX-40, ICOS, IL-2Rβ, GITR, MyD88 / CD40a costimulatory domains and / or variants thereof.

[0245] Embodiment 10. The isolated nucleic acid sequence of any one of embodiments 1 to 9, wherein the intracellular domain comprises a CD3z costimulatory domain and a CD28 costimulatory domain.

[0246] Embodiment 11. The isolated nucleic acid sequence of any one of embodiments 1 to 9, wherein the intracellular domain comprises a CD3z costimulatory domain and a 4-1BB costimulatory domain.

[0247] Embodiment 12. The isolated nucleic acid sequence of any one of embodiments 1 to 9, wherein the intracellular domain comprises a CD3z costimulatory domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain.

[0248] Embodiment 13. The isolated nucleic acid sequence of 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.

[0249] Embodiment 14. The isolated nucleic acid sequence of embodiment 13, wherein the hinge / spacer domain comprises an IgG1 hinge domain or variant thereof, an IgG2 hinge domain or variant thereof, an IgG3 hinge domain or variant thereof, an IgG4 hinge domain or variant thereof, an IgG4P domain, a CD8 hinge domain or variant thereof, or a CD28 hinge domain or variant thereof.

[0250] 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 the S241P mutation.

[0251] Embodiment 16. The isolated nucleic acid sequence of any one of embodiments 1 to 15, wherein the nucleic acid sequence encodes a CAR having the amino acid sequence set forth in SEQ ID NO: 96.

[0252] Embodiment 17. The isolated nucleic acid sequence of any one of embodiments 1 to 15, wherein the nucleic acid sequence encodes a CAR having the amino acid sequence set forth in SEQ ID NO: 97.

[0253] Embodiment 18. The isolated nucleic acid sequence of any one of embodiments 1 to 15, wherein the nucleic acid sequence encodes a CAR having the amino acid sequence set forth in SEQ ID NO: 98.

[0254] 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), VH comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4, 31, and 85; An anti-BCMA chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the VL comprises: CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 6, 33, and 87; CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 7, 34, and 88; and CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 8, 35, and 89.

[0255] Embodiment 20. An anti-BCMA CAR according to embodiment 19, wherein the VH comprises an amino acid sequence selected from SEQ ID NOs: 1, 28 and 82.

[0256] Embodiment 21. An anti-BCMA CAR according to embodiment 19 or 28, wherein the VL comprises an amino acid sequence selected from SEQ ID NOs: 5, 32 and 86.

[0257] 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), VH comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 2; CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and CDR3 comprising the amino acid sequence of SEQ ID NO: 4, An anti-BCMA chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the VL comprises: CDR1 comprising the amino acid sequence of SEQ ID NO: 6; CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0258] Embodiment 23. An anti-BCMA CAR according to 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.

[0259] Embodiment 24. An anti-BCMA CAR according to embodiments 19 to 23, wherein the CAR comprises a transmembrane domain and one or more intracellular domains.

[0260] Embodiment 25. An anti-BCMA CAR according to 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.

[0261] Embodiment 26. The anti-BCMA CAR of embodiment 25, wherein the transmembrane domain comprises a CD28 transmembrane domain.

[0262] Embodiment 27. An anti-BCMA CAR according to any one of embodiments 19 to 26, wherein the one or more intracellular domains comprise a costimulatory domain or a portion thereof.

[0263] Embodiment 28. The anti-BCMA CAR of embodiment 27, wherein the costimulatory domain comprises one or more of the CD3z, 4-1BB, CD2, CD27, CD28, OX-40, ICOS, IL-2Rβ, GITR, MyD88 / CD40a costimulatory domains and / or variants thereof.

[0264] Embodiment 29. An anti-BCMA CAR according to any one of embodiments 24 to 28, wherein the intracellular domain comprises a CD3z costimulatory domain and a CD28 costimulatory domain.

[0265] Embodiment 30. An anti-BCMA CAR according to any one of embodiments 24 to 28, wherein the intracellular domain comprises a CD3z costimulatory domain and a 4-1BB costimulatory domain.

[0266] Embodiment 31. An anti-BCMA CAR according to any one of embodiments 24 to 28, wherein the intracellular domain comprises a CD3z costimulatory domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain.

[0267] Embodiment 32. An anti-BCMA CAR according to any one of embodiments 19 to 31, wherein the CAR further comprises a hinge / spacer domain, optionally located between the antigen-binding domain and the transmembrane domain.

[0268] Embodiment 33. An anti-BCMA CAR according to embodiment 32, wherein the hinge / spacer domain comprises an IgG1 hinge domain or variant thereof, an IgG2 hinge domain or variant thereof, an IgG3 hinge domain or variant thereof, an IgG4 hinge domain or variant thereof, an IgG4P domain, a CD8a hinge domain or variant thereof, or a CD28 hinge domain or variant thereof.

[0269] Embodiment 34. An anti-BCMA CAR according to embodiment 33, wherein the hinge / spacer domain is an IgG4 hinge / spacer or a variant thereof, optionally an IgG4P hinge / spacer comprising the S241P mutation.

[0270] Embodiment 35. An anti-BCMA CAR according to any one of embodiments 19 to 34, wherein the CAR has the amino acid sequence set forth in SEQ ID NO: 96.

[0271] Embodiment 36. An anti-BCMA CAR described in any one of embodiments 19 to 34, wherein the CAR has the amino acid sequence set forth in SEQ ID NO: 97.

[0272] Embodiment 37. An anti-BCMA CAR described in any one of embodiments 19 to 34, wherein the CAR has the amino acid sequence set forth in SEQ ID NO: 98.

[0273] 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, optionally wherein the vector is a virus, lentivirus, adenovirus, retrovirus, adeno-associated virus (AAV), transposon, DNA vector, mRNA, lipid nanoparticle (LNP), or CRISPR-Cas System.

[0274] Embodiment 39. A cell comprising the vector of embodiment 38.

[0275] 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 a reduction or knockout of expression of one or more endogenous regulatory factors.

[0276] Embodiment 41. The cell of embodiment 40, wherein the one or more endogenous regulators are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP).

[0277] Embodiment 42. A cell according to any one of embodiments 39 to 41, having reduced or knocked out expression of CDKN2A, CDKN2B and MTAP.

[0278] Embodiment 43. A cell according to any one of embodiments 39 to 42, wherein the cell does not express phosphatase and tensin homolog (PTEN).

[0279] Embodiment 44. The cell of any one of embodiments 39 to 43, further comprising a transgene encoding either B-cell lymphoma-extra-large cell (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0280] 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.

[0281] 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 (TRAC).

[0282] Embodiment 47. The cells of any one of embodiments 39 to 46, wherein the cells do not express Cluster of Differentiation 38 (CD38).

[0283] 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), VH comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4, 31, and 85; A cell comprising a BCMA-specific antigen-binding domain, wherein the VL comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 7, 34, and 88; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 8, 35, and 89.

[0284] Embodiment 49. The cell of embodiment 48, wherein the VH comprises an amino acid sequence selected from SEQ ID NOs: 1, 28, and 82.

[0285] Embodiment 50. A cell described in embodiment 48 or 49, wherein the VL comprises an amino acid sequence selected from SEQ ID NOs: 5, 32 and 86.

[0286] 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), VH comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 2; CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and CDR3 comprising the amino acid sequence of SEQ ID NO: 4, A cell comprising a BCMA-specific antigen-binding domain, wherein the VL comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 6; CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0287] Embodiment 52. A cell described in any one of embodiments 48 to 51, wherein VH comprises the amino acid sequence of SEQ ID NO: 1 and VL comprises the amino acid sequence of SEQ ID NO: 5.

[0288] Embodiment 53. A cell according to any one of embodiments 48 to 52, further comprising a reduction in or knockout of expression of one or more endogenous regulatory factors.

[0289] Embodiment 54. The cell of 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).

[0290] Embodiment 55. A cell according to any one of embodiments 48 to 54, having reduced or knocked out expression of CDKN2A, CDKN2B and MTAP.

[0291] Embodiment 56. A cell according to any one of embodiments 48 to 55, wherein the cell does not express phosphatase and tensin homolog (PTEN).

[0292] Embodiment 57. The cell of any one of embodiments 48 to 56, wherein the cell further comprises a transgene encoding either B-cell lymphoma-ultra-large cell (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0293] 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.

[0294] 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 (TRAC).

[0295] Embodiment 60. The cells of any one of embodiments 48 to 59, wherein the cells do not express Cluster of Differentiation 38 (CD38).

[0296] 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), VH comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 2; CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and CDR3 comprising the amino acid sequence of SEQ ID NO: 4, 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; The cells comprise reduced or knocked-out expression of CDKN2A, CDKN2B, MTAP, B2M, TRAC, and CD38. Cells containing a BCMA-specific antigen-binding domain.

[0297] Embodiment 62. The cell of 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.

[0298] Embodiment 63. The cell of embodiment 61 or embodiment 62, wherein the BCMA-specific antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 96.

[0299] 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, gamma delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells, and / or combinations thereof.

[0300] Embodiment 65. 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); VH comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4, 31, and 85; A method for treating a disease, wherein the VL comprises CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 6, 33, and 87; CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 7, 34, and 88; and CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 8, 35, and 89.

[0301] Embodiment 66. The method of embodiment 65, wherein the VH comprises an amino acid sequence selected from SEQ ID NOs: 1, 28, and 82.

[0302] Embodiment 67. The method of embodiment 65 or embodiment 66, wherein the VL comprises an amino acid sequence selected from SEQ ID NOs: 5, 32 and 86.

[0303] Embodiment 68. 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); VH comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 2; CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and CDR3 comprising the amino acid sequence of SEQ ID NO: 4, A method for treating a disease, wherein the VL comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 6; CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0304] Embodiment 69. The method of any one of embodiments 65 to 68, wherein VH comprises the amino acid sequence of SEQ ID NO: 1 and VL comprises the amino acid sequence of SEQ ID NO: 5.

[0305] Embodiment 70. The method of any one of embodiments 65-69, further comprising inhibiting cancer growth, inducing cancer regression, and / or prolonging survival in the subject.

[0306] Embodiment 71. The method of any one of embodiments 65 to 70, wherein the cells further comprise a reduction in or knockout of expression of one or more endogenous regulatory factors.

[0307] Embodiment 72. The method of embodiment 71, wherein the one or more endogenous regulators are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP).

[0308] Embodiment 73. The method of any one of embodiments 65 to 72, having reduced or knocked out expression of CDKN2A, CDKN2B and MTAP.

[0309] Embodiment 74. The method of any one of embodiments 65 to 73, wherein the cells do not express phosphatase and tensin homolog (PTEN).

[0310] Embodiment 75. The method of any one of embodiments 65 to 74, wherein the cells further comprise a transgene encoding either B-cell lymphoma-ultra-large cell (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0311] Embodiment 76. The method of any one of embodiments 65 to 75, wherein the cells do not express one or more endogenous immune-related genes.

[0312] 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 (TRAC).

[0313] Embodiment 78. The cells of any one of embodiments 65 to 77, wherein the cells do not express cluster of differentiation 38 (CD38).

[0314] Embodiment 79. The method of any one of embodiments 65 to 78, wherein the cells are autologous cells.

[0315] Embodiment 80. The method of any one of embodiments 65 to 78, wherein the cells are allogeneic cells.

[0316] 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, gamma delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells, and / or combinations thereof.

[0317] 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 lymphocytic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL).

[0318] Embodiment 83. The method of embodiment 82, wherein the cancer is multiple myeloma.

[0319] Embodiment 84. The method of any one of embodiments 65 to 81, wherein the disease is an autoimmune disease.

[0320] Embodiment 85. The method of embodiment 84, wherein the autoimmune disease is lupus.

[0321] Embodiment 86. A pharmaceutical composition comprising an isolated nucleic acid according to any one of embodiments 1 to 18, an anti-BCMA CAR according to any one of embodiments 19 to 37, a vector according to embodiment 38, or a cell according to any one of embodiments 39 to 64, and a pharmaceutically acceptable excipient.

[0322] Embodiment 87. A method for treating a disease in a subject in need thereof, comprising administering to the subject an isolated nucleic acid described in any one of embodiments 1 to 18, an anti-BCMA CAR described in any one of embodiments 19 to 37, a vector described in embodiment 38, a cell described in any one of embodiments 39 to 64, or a pharmaceutical composition described in embodiment 78.

[0323] Embodiment 88. The method of embodiment 87, wherein the disease is cancer or an autoimmune disease.

[0324] Embodiment 89. The method of embodiment 88, wherein the cancer is selected from multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphocytic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL).

[0325] Embodiment 90. The method of embodiment 89, wherein the cancer is multiple myeloma.

[0326] Embodiment 91. The method of embodiment 88, wherein the autoimmune disease is lupus.

[0327] Embodiment 92. Use of an isolated nucleic acid according to any one of embodiments 1 to 18, an anti-BCMA CAR according to any one of embodiments 19 to 37, a vector according to embodiment 38, a cell according to any one of embodiments 39 to 64 or a pharmaceutical composition according to embodiment 78 in the treatment of a disease in a subject in need thereof.

[0328] Embodiment 93. The use according to embodiment 92, wherein the disease is cancer or an autoimmune disease.

[0329] Embodiment 94. The use of embodiment 93, wherein the cancer is selected from multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphocytic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL).

[0330] Embodiment 95. The use of embodiment 94, wherein the cancer is multiple myeloma.

[0331] Embodiment 96. The use of embodiment 93, wherein the autoimmune disease is lupus.

[0332] Embodiment 97. Use of engineered cells for the manufacture of a medicament for treating a disease in a patient, wherein the engineered cells comprise 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); VH comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4, 31, and 85; Use of engineered cells for producing a pharmaceutical for treating a disease in a patient, wherein the VL comprises CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 6, 33, and 87; CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 7, 34, and 88; and CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 8, 35, and 89.

[0333] Embodiment 98. The use described in 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 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.

[0334] Embodiment 99. The use of embodiment 97, wherein the VH comprises an amino acid sequence selected from SEQ ID NOs: 1, 28, and 82.

[0335] Embodiment 100. The use of embodiment 97, wherein the VL comprises an amino acid sequence selected from SEQ ID NOs: 5, 32, and 86.

[0336] Embodiment 101. The use according to any one of embodiments 97 to 100, wherein VH comprises the amino acid sequence of SEQ ID NO: 1 and VL comprises the amino acid sequence of SEQ ID NO: 5.

[0337] Embodiment 102. The use of any one of embodiments 97 to 101, wherein the CAR has the amino acid sequence set forth in SEQ ID NO: 96.

[0338] Embodiment 103. The use of any one of embodiments 97 to 100, wherein the CAR has the amino acid sequence set forth in SEQ ID NO: 97.

[0339] Embodiment 104. The use of any one of embodiments 97 to 100, wherein the CAR has the amino acid sequence set forth in SEQ ID NO: 98.

[0340] Embodiment 105. The use of any one of embodiments 97 to 104, further comprising inhibiting cancer growth, inducing cancer regression, and / or prolonging survival in a subject.

[0341] Embodiment 106. The use according to any one of embodiments 97 to 105, wherein the engineered cells further comprise a reduction in or knockout of expression of one or more endogenous regulatory factors.

[0342] Embodiment 107. The use according to embodiment 106, wherein the one or more endogenous regulators are selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B) and S-methyl-5'-thioadenosine phosphorylase (MTAP).

[0343] Embodiment 108. The use according to any one of embodiments 97 to 107, wherein the engineered cells have reduced or knocked-out expression of CDKN2A, CDKN2B and MTAP.

[0344] Embodiment 109. The use according to any one of embodiments 97 to 108, wherein the engineered cells do not express phosphatase and tensin homolog (PTEN).

[0345] Embodiment 110. The use of any one of embodiments 97 to 109, wherein the engineered cells further comprise a transgene encoding either B-cell lymphoma-ultra-large cell (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0346] Embodiment 111. The use of any one of embodiments 97 to 110, wherein the engineered cells do not express one or more endogenous immune-related genes.

[0347] 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 (TRAC).

[0348] 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).

[0349] Embodiment 114. The use according to any one of embodiments 97 to 113, wherein the engineered cells are autologous cells.

[0350] Embodiment 115. The use according to any one of embodiments 97 to 113, wherein the engineered cells are allogeneic cells.

[0351] 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, gamma delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells and / or combinations thereof.

[0352] 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 lymphocytic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL).

[0353] Embodiment 118. The use according to embodiment 117, wherein the cancer is multiple myeloma.

[0354] Embodiment 119. The use according to any one of embodiments 97 to 116, wherein the disease is an autoimmune disease.

[0355] Embodiment 120. The use according to embodiment 119, wherein the autoimmune disease is lupus.

[0356] Embodiment 121. 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); VH comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4, 31, and 85; An engineered cell for producing a pharmaceutical for treating a disease in a patient, wherein the VL comprises CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 6, 33, and 87; CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 7, 34, and 88; and CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 8, 35, and 89.

[0357] Embodiment 122. The engineered cell of 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 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.

[0358] Embodiment 123. The engineered cell of embodiment 121, wherein the VH comprises an amino acid sequence selected from SEQ ID NOs: 1, 28, and 82.

[0359] Embodiment 124. The engineered cell of embodiment 121, wherein the VL comprises an amino acid sequence selected from SEQ ID NOs: 5, 32, and 86.

[0360] Embodiment 125. An engineered cell according to 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.

[0361] Embodiment 126. An engineered cell described in any one of embodiments 121 to 125, wherein the CAR has the amino acid sequence set forth in SEQ ID NO: 96.

[0362] Embodiment 127. An engineered cell described in any one of embodiments 121 to 124, wherein the CAR has the amino acid sequence set forth in SEQ ID NO: 97.

[0363] Embodiment 128. An engineered cell described in any one of embodiments 121 to 124, wherein the CAR has the amino acid sequence set forth in SEQ ID NO: 98.

[0364] Embodiment 129. The engineered cell of any one of embodiments 121 to 128, further comprising inhibiting cancer growth, inducing cancer regression, and / or prolonging survival in a subject.

[0365] Embodiment 130. The engineered cell of any one of embodiments 121 to 129, wherein the engineered cell further comprises a reduction in or knockout of expression of one or more endogenous regulatory factors.

[0366] Embodiment 131. The engineered cell of 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).

[0367] Embodiment 132. The engineered cell of any one of embodiments 121 to 131, wherein the engineered cell has reduced or knocked-out expression of CDKN2A, CDKN2B, and MTAP.

[0368] 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).

[0369] Embodiment 134. The engineered cell of any one of embodiments 121 to 133, wherein the engineered cell further comprises a transgene encoding either B-cell lymphoma-ultra-large cell (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0370] 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.

[0371] 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 (TRAC).

[0372] Embodiment 137. The engineered cells of any one of embodiments 121 to 136, wherein the engineered cells do not express cluster of differentiation 38 (CD38).

[0373] Embodiment 138. The engineered cell of any one of embodiments 121 to 137, wherein the engineered cell is an autologous cell.

[0374] Embodiment 139. The engineered cells of any one of embodiments 121 to 138, wherein the engineered cells are allogeneic cells.

[0375] 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, gamma delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells, and / or combinations thereof.

[0376] 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 lymphocytic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL).

[0377] Embodiment 142. The engineered cell of embodiment 141, wherein the cancer is multiple myeloma.

[0378] Embodiment 143. The engineered cell of any one of embodiments 121 to 142, wherein the disease is an autoimmune disease.

[0379] Embodiment 144. The engineered cell of embodiment 143, wherein the autoimmune disease is lupus. [Example]

[0380] The following examples illustrate certain embodiments of the present disclosure and various uses thereof. They are provided for illustrative purposes only and should not be construed as limiting the scope of the disclosure in any way.

[0381] Materials and Methods: Cell line: All cells were cultured in 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 Tissue Culture Collection (ATCC, Manassas, VA) (DSMZ, Braunschweig, Germany).

[0382] Lentivirus preparation Lentiviral vectors were prepared by cotransfecting suspension-adapted HEK293 cells with a proprietary lentiviral transfer vector and a commercially available packaging plasmid mix (pPACKH1, System Biosciences, Palo Alto, CA, USA). Transfected cells were cultured for 24 hours and then transferred to fresh medium. 48 hours after transfection, cells were removed by centrifugation, and the lentivirus-containing supernatant was collected.

[0383] Lentiviral particles were purified and concentrated by precipitation using PEG-it Precipitation Reagent (System Biosciences) according to the manufacturer's protocol. After precipitation from the culture supernatant, viral particles were collected by centrifugation and resuspended in 1 / 100 of the original volume of medium. Functional titers were determined by transducing HEK-293 cells with serial dilutions of the purified and concentrated virus. Five days after transduction, cells were labeled with fluorophore-conjugated rBCMA to determine the percentage, and therefore number, of cells transduced with the anti-BCMA CAR construct. Titer values ​​(tfu / mL) were calculated by linear fitting of the number of transduced cells versus the volume of virus added.

[0384] Example 1. Development and characterization of anti-BCMA antigen-binding fragments BCMA-reactive human antibodies were generated by immunization of transgenic mice (human variable domain repertoire) and hybridoma generation. Antibodies produced by individual hybridoma clones were assayed for binding to human BCMA in an ELISA assay, and antibody V-region sequences were retrieved for binding-matched clones.

[0385] Candidate antibodies from the hybridoma screen described above were converted to scFv-Fc format, recombinantly expressed, and their target binding affinities were characterized by surface plasmon resonance (SPR). Briefly, anti-human IgG Fc-specific antibodies were immobilized on a Series S CM5 sensor chip (Cytiva, Marlborough, MA, USA) to enable subsequent capture of candidate scFv-Fc molecules. To measure the kinetics of BCMA binding, recombinant human BCMA (Acro Biosystems, Newark, DE, USA) was flowed over the immobilized scFv-Fc prepared as described above at a flow rate of 30 μL / min and concentrations ranging from 0.03 nM to 30 nM. After 150 s, the injection was stopped, and dissociation was monitored for up to 400 s. A comprehensive fit across multiple concentrations was used to determine the binding kinetic parameters as well as the K DThe K values ​​were determined (Figure 1). Clones 7A8.11 and L15 had higher affinity for human BCMA than comparator I, with K D The values ​​were 0.44 nM and 0.37 nM, respectively.

[0386] To assess the specificity of the BCMA-binding scFvs, we assessed the binding of the scFv-Fc molecules to a 6,000-member membrane proteome array (Integral Molecular, Philadelphia, PA, USA). Briefly, cDNAs encoding 6,000 unique human membrane proteins were individually transfected into HEK-293T cells, and the binding of BCMA-binding scFv-Fc molecules to these transfected cells was assessed using flow cytometry (Figure 2). Off-target binding was verified in 293T cells transfected with plasmids encoding the identified targets, 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, and ligand binding was detected using flow cytometry. Based on these results, no off-target binding was identified for the internally developed clones or the comparator Benchmark I (see U.S. Patent Application Publication No. 2017 / 0226216).

[0387] Example 2. CAR conversion of anti-BCMA antigen-binding fragments and demonstration of function in primary T cells. Of the characterized BCMA-binding antibodies, 10 were prioritized for conversion to scFv format and incorporated into CAR constructs for in vitro and in vivo evaluation. To generate lentiviral expression vectors encoding BCMA-reactive CARs, 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, sequentially fused N- to C-terminally. A signal peptide from human CD33 was used to direct CAR secretion and membrane insertion. Sequence-verified constructs were used to generate lentivirus as described above.

[0388] Alternative CAR formats were also explored, including alternative hinges (CD8, CD28), transmembrane domains (CD8), costimulatory domains (CD28), and CD3ζ domains.

[0389] CAR-T manufacturing Primary T cells were activated with Dynabeads (human T-activator CD3 / CD28) for 24 hours and then transduced with lentivirus-encoded anti-BCMA CAR. CAR-expressing cells were identified and purified using AF647-labeled recombinant BCMA protein. CAR purity was confirmed by flow cytometry. Purified CAR-T cells were used for the following studies. A total of 10 unique scFv CAR constructs were tested against three benchmark controls.

[0390] CAR-T cells were labeled with rBCMA conjugated with AF647 (BCMA-AF647) at 1 μg / mL, and fluorescence intensity was measured using flow cytometry. Figure 3 shows the surface expression of each CAR clone. Clones 33G12.1-1, 33G12.1-2, and 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 example C (see WO 2018 / 028647).

[0391] CAR-T cells were labeled with BCMA-AF647 at protein concentrations ranging from 0.3 nM to 1000 nM, and fluorescence intensity was measured by flow cytometry. Figure 4 shows the BCMA binding characteristics of each anti-BCMA CAR-T clone against soluble BCMA antigen. These binding curves demonstrate that the 7a8.11 clone has comparable BCMA binding to the comparator C benchmark.

[0392] Using a representative T cell donor, we tracked the growth kinetics of different BCMA CAR-T clones when cultured in AIM-V medium containing human serum and IL2. Figure 5 shows the viable cell counts of cells harvested every 2-3 days for 80 days to track growth. Figure 5 demonstrates the differences in growth kinetics and length of growth of anti-BCMA CAR-T clones in primary T cells, highlighting the superior growth profile of the 7a8.11 CAR-T clone relative to comparator C and comparator I.

[0393] In vitro CAR-T activity was assessed using multiple myeloma cell lines (JJN3, RPMI8226, U226B1, KMS11, KMS12, KMS34, NCIH929, and MM1S) engineered to stably express luciferase. Briefly, cells were transduced with mCherry / luciferase-expressing lentivirus. mCherry-positive cells were selected by FACS sorting to derive a pure population of mCherry-positive cells expressing luciferase. The cells were then co-cultured with BCMA CAR-T cells from several donors at various E:T ratios. 24 hours after co-culture, cytotoxicity of target multiple myeloma cells was assessed by adding luciferin substrate and measuring luminescence using a plate reader assay. Figures 6A and 6B show representative data showing the cytotoxicity of target lines when co-cultured with each CAR-T clone and compared to two BCMA CAR-T benchmarks (Comparator I and Comparator C). Figure 6A is a heat map of the mean % cytotoxicity of anti-BCMA CAR-T clones across four donors in eight multiple myeloma cell lines after 24 hours at an E:T ratio of 1:2. Figure 6B shows a bar graph of these same data of the mean % cytotoxicity and standard deviation from the four donors. All endogenous BCMA CAR-T clones demonstrated killing of multiple myeloma cells, with the 7a8.11 BCMA CAR-T having comparable cytotoxic effects across all multiple myeloma lines for both Comparator I and Comparator C. More specifically, clone 7a8.11 had greater cytotoxicity than Comparator I in three cell lines and only slightly reduced cytotoxicity compared to Comparator C.

[0394] To measure effector cytokine production, Huh7 target cells were engineered to express the BCMA antigen, and parental cells were used as a negative control. Briefly, Huh7 cells were transfected with a lentivirus containing TNFRSF17 and a puromycin resistance cassette, and cells successfully integrating the gene were selected based on puromycin resistance. Huh7BCMA target cells were then co-cultured with anti-BCMA CAR-T clones at various E:T ratios, and supernatants were collected at 24 hours to quantify IFNy and IL2 production using the Meso Scale Discovery assay (MSD). Figure 7 shows representative effector cytokine production from anti-BCMA CAR-T cells when co-cultured with a BCMA-expressing huh7 engineered cell line at an E:T ratio of 1:2. All anti-BCMA CAR-T cells demonstrate 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 levels of IFNy and IL2 produced by comparator example I under the same conditions.

[0395] To determine the effect of soluble BCMA on CAR-T killing, Huh7 BCMA-expressing cells were seeded onto RTCA E plates (Agilent) for impedance-based measurements of cytotoxicity by xCELLigence. Soluble recombinant BCMA protein (10 μg / mL) was added to the cells prior to co-culture with BCMA CAR-T cells. Figure 8 shows the difference in % cell lysis observed after 40 hours of co-culture with BCMA-expressing huh7 cells in the presence or absence of soluble BCMA protein. These data demonstrate that 7A8.11 BCMA CAR-T cells had the highest level of cell lysis across all BCMA CAR-T clones and also showed comparable levels of cell lysis to comparator example C in the presence and absence of soluble protein. Supernatants were collected from these co-cultures 24 hours after the addition of CAR-T cells, and IFNy 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 cultures. Figure 9 demonstrates the varying effects of soluble BCMA protein on effector cytokine production. All BCMA CAR-T cells evaluated showed lower levels of IFNy and IL-2 production in the presence of soluble antigen; however, the 7A8.11 CAR-T clone was least adversely affected by the presence of soluble protein compared to other endogenously developed CAR clones. To assess differences in antigen-driven CAR-T expansion and cell persistence, we performed an in vitro serial killing experiment in which CAR-T cells were repeatedly co-cultured with the multiple myeloma target cell line JJN3 at a 1:1 E:T ratio. Every 2–3 days, co-cultures were sampled and assessed for T cell and tumor cell numbers and viability by flow cytometry. Following this determination, the CAR-T / tumor cell co-cultures were then "fed" with viable JJN3 cells to return the co-cultures to an E:T ratio of 1:1. Figure 10A shows the CAR-T expansion of each clone over the 12-day serial killing experiment (top panel), and the % cytolysis of target JJN3 cells after each co-culture round (bottom panel) as a measure of CAR-T cell function and persistence.Figure 10A shows that 7A8.11 CAR-T cells have superior antigen-driven expansion and persistence, based on the duration of target expansion and prolonged cytolysis, over other BCMA CAR-T clones. Furthermore, 7A8.11 CAR-T cells demonstrated proliferation and functional persistence comparable to Comparator C and superior to Comparator I. Figure 10B shows CAR-T cell persistence and proliferation (upper panel) and target cell cytolysis (lower panel) after repeated antigen stimulation in the presence of soluble BCMA. Figure 10B also evaluates antigen-dependent CAR-T cell expansion and functional persistence by JJN3 coculture in the presence of soluble recombinant BCMA protein. Compared to expansion in the absence of soluble BCMA (Figure 9A), expansion of all CAR-Ts was reduced and duration was shortened. However, 7A8.11 CAR-T cells remained superior in proliferation compared to other internal clones and Comparator I. Based on both the Figure 10A and Figure 10B datasets, 7A8.11 CAR-T cells demonstrate favorable functional persistence and proliferation in response to antigen, and functionality comparable to comparator benchmark, comparator C, and comparator I.

[0396] In vivo animal studies: All animal experiments were performed in a facility certified by the Assessment of Laboratory Animal Care (AALAC) under Institutional Animal Care and Use Committee (IACUC) guidelines and appropriate animal research approvals. To evaluate in vivo BCMA CAR-T function in an in vivo disseminated model of multiple myeloma, we intravenously injected 10e6 multiple myeloma 1S-luc cells into NSG mice. Four days after tumor cell injection, mice were imaged and then intravenously injected with either 0.3e6 or 3e6 CAR-T cells. Animals were weighed and imaged (via intraperitoneal injection of luciferin) every 3–4 days to look for signs of disease and track tumor cell growth. Figure 11 shows the in vivo tumor control and clinical benchmarks of anti-BCMA CAR-T clones at both high and low doses of CAR-T cells in a disseminated multiple myeloma model (MM1.S). At both high and low doses, 7A8.11 CAR-T cells demonstrated better tumor control than either comparator benchmark (Comparator C and Comparator I) and were functionally equivalent to the L15 endogenous clone. Furthermore, administration of CAR-T cells did not result in any treatment-related toxicity, such as cytokine release syndrome (CRS), at these dose levels. Three days after CAR-T infusion, animals were bled, and serum cytokine levels were quantified using MSD. Figure 12 demonstrates dose-responsive effector cytokine production by the BCMA CAR-T clone in an in vivo MM1.S tumor model.

[0397] Example 3. BCMA CAR-T REX cell T REX To characterize BCMA binding on the surface of the chassis-expressed 7A8.11 CAR, CAR-T cells were labeled with BCMA-AF647 at concentrations ranging from 0.3 nM to 1000 nM of protein, and fluorescence intensity was measured by flow cytometry. Figure 13 shows the fluorescence intensity of 7A8.11 CAR-T cells compared to primary 7A8.11 CAR-T cells and benchmarked against comparators I and C. REXThe BCMA binding curves of T REX The 7A8.11 CAR expressed in T cells demonstrates the highest capacity for BCMA binding at the cell surface compared to the 7A8.11 CAR expressed in primary T cells and comparators I and C. Furthermore, T REX The EC50 of 7A8.11 in IL-14 is higher than when expressed in primary T cells, higher than comparator factor I, and similar to comparator factor C.

[0398] Example 4. BCMA CAR-T in multiple myeloma cell lines REX In vitro cytotoxicity of cells. CAR-Ts were seeded at four different E:T ratios with each of the following luciferase-expressing target lines: KMS12, U226B1, JJN3, and RPMI8226. Cytotoxicity was measured 24 hours after CAR-T addition, and viability was measured after the addition of luciferin substrate. Luminescence from live cells was measured using a plate reader. 7A8.11 CAR-T REX The cells exhibited comparable cytotoxic function to primary T cells expressing the 7A8.11 CAR and comparators I and C across all E:T ratios and for all target lines (see Figure 14).

[0399] Example 5. BCMA CAR-T REX Cytokine profile of cells. Figure 15 shows the performance of 7A8.11 CAR-T cells when co-cultured with the target cell line JJN3 and compared to primary T cells expressing 7A8.11 and comparator benchmarks I and C. REX An example of an effector cytokine profile from 7A8.11 CAR-T cells was plated with JJN3 at a 1:1 E:T ratio, and supernatants were sampled after 24 hours of co-culture to measure cytokine production of IFNy and IL2 by MSD. REX The cells demonstrated 2-15% effector cytokine production compared to 7A8.11 primary T cells and less than 5% of the effector cytokine production of clinical comparator cells, demonstrating the efficacy of 7A8.11 CAR-T REXThis suggests a potentially safer cytokine profile by the cells.

[0400] Example 6. BCMA CAR-T REX In vivo tumor control of multiple myeloma cells by . Figure 16 shows the 7A8.11 CAR-T compared to clinical benchmarks, comparators I and C. REX Figure 1 shows in vivo tumor control of MM1.S cells by 7A8.11 CAR-T cells. Non-targeting HER2 CAR-T cells were used as a control. In this in vivo dissemination model of multiple myeloma, 10e6 MM1S-luc cells were intravenously injected into NSG mice. Four days after tumor cell injection, the mice were imaged and then treated with 7A8.11 CAR-T cells. REX CAR-T cells or 7A8.11 primary T cells, or comparators I and C, were intravenously injected. Animals were weighed and imaged (via intraperitoneal injection of luciferin) every 3-4 days to look for signs of disease and track tumor cell growth. Figure 16 shows the results of CAR-T cell transplantation with 7A8.11 primary T cells. REX The cells show identical tumor clearance kinetics and the same or better tumor clearance kinetics compared to comparators I and C.

[0401] Example 7. Daratumumab treatment induces anti-BCMA T cell proliferation from NK cells REX Protects cells. Figure 17 shows that daratumumab (Dara) treatment inhibits anti-BCMA-T REX protects cells from NK cells and the remaining T REX Briefly, purified NK cells were cultured overnight (NK-Xpander medium, 500 IU / mL IL-2) in the presence or absence of Dara (10 μg / mL). The next day, NK cells were washed and stained with anti-BCMA-T. REX cells and NK:T at a ratio of 1:1 or 0.5:1 REX After 5 hours, the co-cultures were assessed by flow cytometry to detect NK cells and anti-BCMA T cells. REX Quantify cell number and measure anti-BCMA-T REXDara-mediated protection of cell numbers was demonstrated (Figure 17A). Cells further proceeded to two sequential killings of JJN3 target cells at the indicated E:T ratios (Figure 17B), or one killing by the BCMA-ectopically expressing SNU-182 adherent cell line (Figure 17C), with tumor cell killing assessed by Xcelligence, as measured by % tumor cell lysis by luciferase assay. Data are representative of studies performed with NK cells from three different donors. As seen in Figure 17, Dara treatment significantly reduced the number of anti-BCMA T cells. REX This allows for the preservation of the number of these cells while maintaining the cytotoxic potential of the cells.

[0402] Example 8. Cryo-recovered BCMA CAR-T REX The cells exhibit tumor clearance in vivo. NSG mice were inoculated with 10E6 MM1S-luciferase tumor cells. Three days later, primary BCMA CAR-T cells (7A8.11 CAR-T cells), cryo-recovered, and cultured for optimal activity (fresh). REX Cells (7A8.11), or BCMA CAR-T administered immediately after recovery from hypothermia REX MM1S cells (7A8.11) were administered at 10E6 cells per mouse. 25 days after MM1S cell administration, bone marrow was collected from the mice and analyzed for the presence of MM1S tumor cells, healthy bone marrow cells, or other populations. BCMA CAR-T cells were cryo-recovered and immediately administered. REX The cells demonstrated comparable tumor clearance and mouse bone marrow recovery to other groups (see Figure 18).

[0403] NSG mice were inoculated with 10E6 MM1S-luciferase tumor cells. Three days later, primary BCMA CAR-T cells from two donors (Benchmark C CAR-T cells) or BCMA CAR-T REX Cells (7A8.11) were administered at the indicated dose immediately after cryo-recovery. Tumor burden was monitored twice weekly using IVIS imaging. BCMA CAR-T REX The cells demonstrated profound tumor clearance in vivo when administered immediately after cryo-recovery (see Figure 19).

[0404] Example 9. BCMA antigen density and cellular composition between SLE and healthy donors. The ability of BCMA-targeted cells to deplete BCMA+ cells is thought to depend on the antigen density on the cell surface. Therefore, we compared the BCMA antigen density and cellular composition of systemic lupus erythematosus (SLE) patients with those of healthy donors. PBMCs were isolated from fresh whole blood from SLE patients and healthy donors, and the PBMCs were evaluated for BCMA receptor density and B-cell subset percentages. As shown in Figure 20, the cellular composition of the B-cell compartment was broadly similar between healthy donors and SLE patients. Furthermore, target expression (BCMA receptor density) was also similar between healthy donors and SLE patients (or slightly higher in SLE patients, particularly in plasmablasts).

[0405] Example 10. BCMA CAR-T cells deplete target-expressing healthy human plasma cells to the same extent as MM1S (BCMA+) tumor cells BCMA CAR-T cell research lots were produced from fresh peripheral blood of individual healthy donors. Briefly, PBMCs from healthy donors were collected by Ficoll gradient centrifugation, and CD4 and CD8 T cells were enriched from the white blood cell fraction. 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.

[0406] Primary human plasma cells (CD138+ selected) were isolated from fresh whole blood of healthy donors. The plasma cells were then co-cultured with 7A8.11 BCMA CAR-T cells or untransduced T cells (non-targeting) at various effector:target ratios. As a control, MM1S cells (a multiple myeloma cell line expressing BCMA) were also co-cultured with the product or untransduced T cells at various effector:target ratios. As shown in Figure 21, BCMA CAR-T cells were able to deplete primary human plasma cells or MM1S tumor cells to a similar extent (89% vs. 95% at a 1:1 effector:target ratio and 93% vs. 86% at a 1:2 effector:target ratio, respectively), while untransduced T cells mediated only modest depletion of either primary human plasma cells or the MM1S tumor cell line.

[0407] Example 11. In vitro differentiated plasmablasts from SLE and healthy donors show dose-dependent depletion by BCMA CAR-T cells (E:T) Primary human naive B cells (IgD+CD27-selected) were isolated from fresh or frozen PBMCs from healthy or SLE donors. The naive B cells were then differentiated using a proprietary cytokine mixture to drive plasmablast differentiation (BCMA+ B cells). After 5 days of differentiation, the cells were then co-cultured with 7A8.11-transduced CAR-T cells or untransduced T cells (non-targeting) at various effector:target ratios. 7A8.11-transduced CAR-T cells were able to deplete healthy or SLE primary human differentiated plasmablasts to a similar extent (see Figure 22).

[0408] Example 12. BCMA targeting of CAR-T cells reduced BCMA+ cells in a xenogeneic model of graft-versus-host disease To evaluate the ability of BCMA-targeted CAR-T cells to deplete BCMA-expressing primary B cells in vivo, a pharmacodynamic (PD) study was performed in a xenogeneic model of graft-versus-host disease (XenoGvHD). To prepare for this XenoGvHD PD study, healthy human donor PBMCs were isolated from fresh leukopacks (StemExpress) and cryopreserved until engraftment. Autologous untransformed T cells (UTT) or autologous BCMA-targeted CAR-T cells (BCMA CAR-T) were produced from the same donor's PBMCs. Briefly, isolated T cells were activated, transduced with the 16C6 BCMA lentiviral vector, and expanded in culture flasks. The cells were then washed, harvested, and frozen in cryopreservation medium.

[0409] On day -1, 8-10 week-old female NOD scid gamma mice (NSG; Jackson Laboratories) were preconditioned with sublethal total body irradiation (1 Gy). On day 0, 15 million total PBMCs from a healthy human donor were intravenously injected. Four hours later, mice were intravenously injected with either phosphate-buffered saline (PBS), 3 million autologous UTT cells, or 3 million autologous BCMA CAR-T cells (n = 5 mice / group). On day 12, mice were euthanized, and tissues were collected for FACS analysis to determine whether these BCMA CAR-T cells depleted the BCMA+ cells found to be present in this in vivo model.

[0410] Mice treated with BCMA CAR-T products demonstrated significantly reduced percentages 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 multiplex ELISAs from Meso-scale Discovery and were found to be significantly elevated in mice treated with BCMA targeting CAR-T cells compared to mice treated with PBS and UTT (Figure 24).

[0411] [Table 1-1]

[0412] [Table 1-2]

[0413] [Table 1-3]

[0414] [Table 1-4]

[0415] [Table 1-5]

[0416] The embodiments described herein can be practiced in the absence of any one or more elements or limitations not specifically disclosed herein. The terms and expressions used are used as terms of description and not of limitation, and the use of such terms and expressions is not intended to exclude the features shown and described or equivalents thereof, but it is recognized that various modifications are possible within the scope of the claimed embodiments. Thus, while the present specification is specifically disclosed by embodiments, it should be understood that any features, modifications, and variations of the concepts disclosed herein may be employed by those skilled in the art, and such modifications and variations are considered to be within the scope of these embodiments as defined by the description and the appended claims. While several aspects of the present disclosure may be identified herein as particularly advantageous, it is intended that the present disclosure is not limited to these particular aspects of the disclosure.

[0417] A claim or description including "or" between one or more members of a group is considered to be satisfied when one, more than one, or all group members are present in, used in, or relevant to a given product or process, unless stated to the contrary or clear from context. The present disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which two or more or all group members are present in, used in, or relevant to a given product or process.

[0418] Furthermore, this disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as a list, for example, in Markush group format, each subgroup of elements is also disclosed, and any element(s) can be removed from the group.

[0419] Generally, when the present disclosure or aspects of the present disclosure are referred to as including particular elements and / or features, it is understood that particular embodiments of the present disclosure or aspects of the present disclosure consist of or consist essentially of such elements and / or features, and for simplicity, these embodiments are not specifically set forth herein.

[0420] 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 section of this application shall not be construed as an admission that such reference is available as prior art to the present disclosure.

Claims

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

2. The antigen-binding domain may be an antibody or an antigen-binding fragment thereof, Fab, Fab', F(ab')2, Fd, Fv, single-chain fragment variable (scFv), single-chain antibody, V Ｈ 2. The isolated nucleic acid sequence of claim 1, comprising a H, a vNAR, a nanobody (single domain antibody), or any combination thereof.

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

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

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

9.

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

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

8. 8. The isolated nucleic acid sequence of claim 1, wherein the one or more intracellular domains comprise a costimulatory domain or a portion thereof.

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

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

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

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

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

14. 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. 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 the S241P mutation.

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

96.

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

97.

18. 16. The isolated nucleic acid sequence of any one of claims 1 to 15, wherein the nucleic acid sequence encodes a CAR having the amino acid sequence set forth 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); the VH comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4, 31, and 85; An anti-BCMA chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the VL comprises: CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 6, 33, and 87; CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 7, 34, and 88; and CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 8, 35, and 89.

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

21. The anti-BCMA CAR of claim 19 or 28, wherein the VL comprises an amino acid sequence selected from 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); 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; An anti-BCMA chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the VL comprises: CDR1 comprising the amino acid sequence of SEQ ID NO: 6; CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and CDR3 comprising the amino acid sequence of SEQ ID NO:

8.

23. The anti-BCMA CAR 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-BCMA CAR according to claims 19 to 23, wherein the CAR comprises a transmembrane domain and one or more intracellular domains.

25. The anti-BCMA CAR 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, and CD28.

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

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

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

29. The anti-BCMA CAR according to any one of claims 24 to 28, wherein the intracellular domain comprises a CD3z costimulatory domain and a CD28 costimulatory domain.

30. The anti-BCMA CAR according to any one of claims 24 to 28, wherein the intracellular domain comprises a CD3z costimulatory domain and a 4-1BB costimulatory domain.

31. The anti-BCMA CAR according to any one of claims 24 to 28, wherein the intracellular domain comprises a CD3z costimulatory domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain.

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

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

96.

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

97.

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

98.

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

39. A cell comprising the vector of claim 38.

40. A cell comprising a nucleic acid sequence encoding the chimeric antigen receptor (CAR) of any one of claims 19 to 37, further comprising reduced or knocked out expression 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. 42. The cell of any one of claims 39 to 41, wherein the cell has reduced or knocked out expression 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. 44. The cell of any one of claims 39 to 43, further comprising a transgene encoding either B-cell lymphoma-extra-large cell (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. 46. ​​The cell of claim 45, wherein the endogenous immune-related gene is beta 2 microglobulin (B2M) and / or T cell receptor alpha constant (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); the VH comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4, 31, and 85; A cell comprising a BCMA-specific antigen-binding domain, wherein the VL comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 7, 34, and 88; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 8, 35, and 89.

49. The cell of claim 48, wherein the VH comprises an amino acid sequence selected from SEQ ID NOs: 1, 28, and 82.

50. 50. The cell of claim 48 or 49, wherein the VL comprises an amino acid sequence selected from 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); 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; A cell comprising a BCMA-specific antigen-binding domain, wherein the VL comprises: CDR1 comprising the amino acid sequence of SEQ ID NO: 6; CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and 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. 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. 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. 55. The cell of any one of claims 48 to 54, having reduced or knocked out expression of CDKN2A, CDKN2B and MTAP.

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

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

58. 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. 59. The cell of claim 58, wherein the endogenous immune-related gene is beta 2 microglobulin (B2M) and / or T cell receptor alpha constant (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); 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; 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; The cells comprise a BCMA-specific antigen-binding domain, including reduced or knocked-out expression 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. 63. The cell of claim 61 or claim 62, wherein the BCMA-specific antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:

96.

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

65. 1. A method of 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); the VH comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 2, 29, and 83; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 3, 30, and 84; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4, 31, and 85; A method for treating a disease, wherein the VL comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 6, 33, and 87; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 7, 34, and 88; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 8, 35, and 89.

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

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

68. 1. A method of 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); 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; A method for treating a disease, 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. 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. 70. The method of any one of claims 65 to 69, further comprising inhibiting cancer growth, inducing cancer regression, and / or prolonging survival in said subject.

71. 71. The method of any one of claims 65 to 70, wherein the cells further comprise a reduction in or knockout of expression of one or more endogenous regulatory factors.

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

73. 73. The method of any one of claims 65 to 72, comprising reducing or knocking out expression of CDKN2A, CDKN2B and MTAP.

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

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

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

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

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

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

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

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

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

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

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

85. 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-BCMA CAR 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 the isolated nucleic acid of any one of claims 1 to 18, the anti-BCMA CAR of any one of claims 19 to 37, the vector of claim 38, the cell of any one of claims 39 to 64, or the pharmaceutical composition of claim 78.

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

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

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

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

92. Use of the isolated nucleic acid of any one of claims 1 to 18, the anti-BCMA CAR of any one of claims 19 to 37, the vector of claim 38, the cell of any one of claims 39 to 64, or the pharmaceutical composition of claim 78 in treating a disease in a subject in need thereof.

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

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

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

96. 94. The use of claim 93, wherein the autoimmune disease is lupus.

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

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

8.

99. 98. The use of claim 97, wherein the VH comprises an amino acid sequence selected from SEQ ID NOs: 1, 28, and 82.

100. 98. The use of claim 97, wherein the VL comprises an amino acid sequence selected from SEQ ID NOs: 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 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 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 shown in SEQ ID NO:

98.

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

106. 106. The use of any one of claims 97 to 105, wherein the engineered cells further comprise a reduction in or knockout of expression of one or more endogenous regulatory factors.

107. 107. The use of 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. 108. The use of any one of claims 97 to 107, wherein the engineered cells have reduced or knocked out expression of CDKN2A, CDKN2B and MTAP.

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

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

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

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

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

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

118. 118. The use of 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. 120. The use of claim 119, wherein the autoimmune disease is lupus.

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

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

8.

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

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

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

96.

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

97.

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

98.

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

130. 130. The engineered cell of any one of claims 121-129, wherein the engineered cell further comprises a reduction in or knockout of expression of one or more endogenous regulatory factors.

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

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

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

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

136. 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 (TRAC).

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

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

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

140. The engineered cells may be T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), tumor-infiltrating lymphocytes, regulatory T cells, CD8 ＋ T cells, CD4 ＋ 140. The engineered cell of any one of claims 121 to 139, selected from T cells, gamma delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer T (NKT) cells and / or combinations thereof.

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

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

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

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

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