BCMA Chimeric Antigen Receptor and its Use

Abstract

This application provides a BCMA-targeting chimeric antigen receptor (CAR) comprising a BCMA-binding domain and an intracellular costimulatory domain derived from DAP10. Also provided are engineered immune effector cells (e.g., NK cells) containing the chimeric antigen receptor. Pharmaceutical compositions, kits, and methods for treating cancer are also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and benefits of U.S. Provisional Patent Application No. 63 / 496,823, filed on 18 April 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to sequence listings This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The sequence listing file, titled MIL-021WO1.XML, was created on January 29, 2024, and has a size of 18,368,504 bytes.

[0003] The present invention provides a BCMA-targeting chimeric antigen receptor comprising a DAP10 costimulatory domain that exhibits high efficacy in killing tumor cells. Also provided herein are compositions, cells, and methods for treating diseases associated with BCMA expression (e.g., cancer). [Background technology]

[0004] BCMA is a B-cell maturation antigen. BCMA (also known as TNFRSF17, BCM, or CD269) is a member of the tumor necrosis receptor (TNFR) family and is primarily expressed in terminally differentiated B cells, such as memory B cells and plasma cells. The BCMA protein is widely detected in multiple myeloma (MM) cells and other lymphomas, including non-Hodgkin lymphoma (NHL). Patients with B-cell malignancies, including non-Hodgkin lymphoma (NHL) and multiple myeloma (MM), are at high risk of cancer mortality.

[0005] The response of B-cell malignancies to various forms of treatment is inconsistent. Conventional methods for treating B-cell malignancies, including chemotherapy and radiotherapy, have limited efficacy due to toxic side effects. Immunotherapy with anti-CD19, anti-CD20, anti-CD22, anti-CD23, anti-CD52, anti-CD80, and anti-HLA-DR therapeutic antibodies has achieved limited success due to poor pharmacokinetic profiles, rapid elimination of antibodies by serum proteases, glomerular filtration, low penetration into tumor sites, and limited expression levels of target antigens on cancer cells.

[0006] Anti-BCMA antibody therapy and attempts to use genetically modified cells that express BCMA-targeting chimeric antigen receptors (CARs) are advancing immunotherapy for B-cell malignancies. [Overview of the project] [Means for solving the problem]

[0007] The present invention relates, in general, to improved BCMA-targeting CAR polypeptides, in particular to compositions and cells comprising improved BCMA-CAR polypeptides, and to methods of using them for the treatment of B cell-related diseases and disorders. The BCMA-targeting CAR polypeptide comprises a DAP10 costimulatory domain; the BCMA-DAP10 CAR polypeptide can enhance the proliferation and therapeutic effects of immune effector cells (e.g., NK cells and T cells) genetically engineered to express the BCMA-targeting CAR polypeptide. In particular, NK cells expressing the BCMA-targeting CAR polypeptide of the present invention are highly effective in killing tumor cells in various tumors. In this disclosure, the nucleic acid sequence encoding the BCMA-targeting CAR polypeptide comprising the DAP10 costimulatory domain is codon-optimized.

[0008] In one embodiment, the present invention provides a chimeric antigen receptor (CAR) polypeptide comprising an antigen-binding domain that specifically binds to a B cell maturation antigen (BCMA), a hinge domain, a transmembrane domain, a DAP10 costimulatory domain, and at least one intracellular signaling domain. The invention also includes polynucleotides encoding the chimeric antigen receptor polypeptide.

[0009] In some embodiments, the BCMA-binding domain of the CAR polypeptide is an anti-BCMA antibody, its antigen-binding domain, a Fab fragment, an F(ab')2 fragment, an Fv fragment, a single-chain variable fragment (scFv), a single-domain antibody, or a nanobody.

[0010] In some embodiments, the BCMA binding region is approximately 1 × 10⁻⁶ -6 Less than M, approximately 1 x 10 -7 Less than M, approximately 1 x 10 -8 Less than M, or approximately 1 × 10 -9 Less than M, or approximately 1 × 10 -10 Less than M K D Then it is combined with BCMA.

[0011] In some embodiments, the BCMA binding region includes a heavy chain variable region complementarity determination region (HCDR) 1 containing SYAIH (SEQ ID NO: 2), an HCDR 2 containing VTWHDGSNKYYAESVMG (SEQ ID NO: 3), and an HCDR 3 containing AKFGEPQYFQH (SEQ ID NO: 4).

[0012] In some embodiments, the BCMA binding region includes a light chain variable region complementary determination region (LCDR) 1 containing RASQGINNYLA (SEQ ID NO: 6), an LCDR 2 containing AASTLQS (SEQ ID NO: 7), and an LCDR 3 containing QQLKSYPFT (SEQ ID NO: 8).

[0013] In some embodiments, the BCMA binding region includes a heavy chain variable region (VH) comprising HCDR1 containing three complementarity-determining regions: SYAIH (SEQ ID NO: 2), HCDR2 containing VTWHDGSNKYYAESVMG (SEQ ID NO: 3), and HCDR3 containing AKFGEPQYFQH (SEQ ID NO: 4), and a light chain variable region (VL) comprising LCDR1 containing three complementarity-determining regions: RASQGINNYLA (SEQ ID NO: 6), LCDR2 containing AASTLQS (SEQ ID NO: 7), and LCDR3 containing QQLKSYPFT (SEQ ID NO: 8).

[0014] In some embodiments, the BCMA-binding region includes a heavy chain variable region (VH) comprising the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0015] In some embodiments, the BCMA-binding region includes a light chain variable region (VL) comprising an amino acid sequence represented by SEQ ID NO: 5, or an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97%, 98%, or 99% identical to SEQ ID NO: 5.

[0016] In some embodiments, the BCMA binding region includes a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 1 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 5.

[0017] In one preferred embodiment, the BCMA-binding domain is a single-chain variable fragment (scFv). One exemplary scFv capable of binding to BCMA includes the sequence of SEQ ID NO: 20. The BCMA-binding domain may also include an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 20.

[0018] In some embodiments, the BCMA-binding domain is encoded by the nucleic acid sequence of SEQ ID NO: 35 or SEQ ID NO: 51.

[0019] According to the present invention, a BCMA-targeting CAR polypeptide comprises an intracellular DAP10 costimulatory domain. In some embodiments, the DAP10 costimulatory domain comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 24. In some embodiments, the DAP10 costimulatory domain is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 39 or SEQ ID NO: 57.

[0020] In some embodiments, the BCMA-targeting CAR includes one or more additional intracellular costimulatory domains.

[0021] According to the present invention, a BCMA-targeting CAR polypeptide includes an intracellular signaling domain. In some embodiments, the intracellular signaling domain is a CD3ζ signaling domain. The intracellular CD3ζ signaling domain includes the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 23. In some embodiments, the CD3ζ signaling domain is encoded by a nucleic acid sequence including the sequence of SEQ ID NO: 40 or SEQ ID NO: 58.

[0022] In some embodiments, the BCMA-targeting CAR polypeptide of the present invention may include one or more additional costimulatory domains and / or one or more signaling domains. In some embodiments, the costimulatory domains may include OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD28, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell costimulator (ICOS), CDS-gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF 1.4), NKG2C, 2B4, Igα (CD79a), DAP12, Fcγ receptor, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activator molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CDSα, CDSβ, 11.2β, IL2R gamma, IL7 Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, ITGAE, CD103, ITGAL, LFA-1, ITGAM, ITGAX, ITGB1, CD29, ITGB2, ICOS, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE It is derived from ligands that specifically bind to RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGLl, CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.In some embodiments, the signaling domains are derived from CD28, CD137(4-IBB), CD134(OX40), FcRγ, FcRβ, FcεRI, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD27, CD2, CD5, CD22, CD79a, CD79b, CD66d, CD278(ICOS), ICAM-1, LFA-1(CD1la / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, DAP10, DAP12, or combinations thereof.

[0023] According to the present invention, a CAR polypeptide targeting BCMA includes a hinge domain and a transmembrane domain that link the extracellular BCMA-binding region and the cytoplasmic region (i.e., the costimulatory domain and the signaling domain) of the CAR.

[0024] In some embodiments, the hinge domain is selected from the hinge domains and / or extracellular domains of IgG, CD8a, CD4, and CD28. In some examples, the hinge domain is the hinge domain of CD28; the hinge domain of CD28 includes the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 21.

[0025] In some embodiments, the hinge domain of CD28 is encoded by a nucleic acid sequence containing the sequence of SEQ ID NO: 37 or SEQ ID NO: 54.

[0026] In some embodiments, the transmembrane domain is a transmembrane domain derived from CD8, CD16, CD27, CD28, NKG2D, NKp44, NKp46, NKp30, NKp80, DNAM-1, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD9, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, DAP10, DAP12, or variants thereof. In some examples, the transmembrane domain is the transmembrane domain of CD28; the transmembrane domain of CD28 contains the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 22.

[0027] In some embodiments, the transmembrane domain of CD28 is encoded by a nucleic acid sequence containing the sequence of SEQ ID NO: 38 or SEQ ID NO: 56.

[0028] In some embodiments, the BCMA-targeting CAR polypeptide further comprises one or more additional polypeptides. In some examples, the polypeptide is a cytokine such as IL-15. In a non-limiting example, the BCMA-targeting CAR polypeptide further comprises IL-15 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, IL-15 is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 52 or SEQ ID NO: 53.

[0029] In some embodiments, the BCMA-targeting CAR polypeptide of the present invention comprises a signal peptide and one or more linker sequences, such as a cleavable 2A peptide (e.g., E2A). For example, as shown in Figure 1, the E2A peptide may be located between the CAR polypeptide and IL-15. The E2A peptide includes, as an example, the amino acid sequence of SEQ ID NO: 27. Exemplary signal peptide sequences are presented by SEQ ID NO: 28 or SEQ ID NO: 59.

[0030] An exemplary BCMA-targeting CAR polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 29. In some examples, the BCMA-targeting CAR polypeptide comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 29.

[0031] In another aspect of the present invention, a polynucleotide encoding a BCMA-targeting CAR polypeptide is provided herein. The polynucleotide is mRNA or DNA. In some embodiments, the polynucleotide is codon-optimized. In some embodiments, the polynucleotide comprises at least one modified nucleotide. In other embodiments, the polynucleotide comprises an unmodified nucleotide.

[0032] An exemplary polynucleotide encoding a BCMA-targeting CAR polypeptide comprises the nucleotide sequence of SEQ ID NO: 44. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 44.

[0033] In one embodiment, the polynucleotide encoding a BCMA-targeting CAR polypeptide comprises the nucleotide sequence of SEQ ID NO: 60. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least 99%, or 100% identical to SEQ ID NO: 60.

[0034] Another exemplary BCMA-targeting CAR polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 13. In some examples, the BCMA-targeting CAR polypeptide comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 13.

[0035] In one embodiment, a polynucleotide encoding a BCMA-targeting CAR (e.g., signal sequence 1-BCMA-binding factor-linker-CD28(hinge)-linker-CD28(TM)-DAP10(co-stimulatory)-CD3z-E2A-signal sequence 2-sIL15) includes the nucleotide sequence of SEQ ID NO: 25. In some embodiments, the polynucleotide includes a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least 99%, or 100% identical to SEQ ID NO: 25.

[0036] In one embodiment, the polynucleotide encoding the BCMA-targeting CAR includes the codon-optimized sequence of SEQ ID NO: 55. In some embodiments, the polynucleotide includes a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least 99%, or 100% identical to SEQ ID NO: 55.

[0037] In some embodiments, the vector comprises any polynucleotide encoding a CAR targeting the BCMA of the present invention as provided. The vector may be a non-viral vector such as a plasmid, or a viral vector such as an adenovirus vector, an adenovirus-associated virus (AAV) vector, a lentiviral vector, and a retroviral vector. As a non-limiting example, the vector may comprise a polynucleotide having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 25, 44, 55, and 60.

[0038] In another aspect, the present invention provides immune effector cells genetically engineered to express at least one BCMA-targeting CAR polypeptide described herein. Immune effector cells include, but are not limited to, natural killer (NK) cells, natural killer T (NKT) cells, T cells, B cells, macrophages, mesenchymal stromal cells, dendritic cells, tumor-infiltrating lymphocytes (TILs), cytotoxic T lymphocytes (CTLs), or any combination thereof.

[0039] In some embodiments, immune effector cells are T cells such as mature T cells, T helper cells, tumor-infiltrating T cells, autologous T cells, engineered autologous T cells (eACT), allogeneic T cells, or any combination thereof.

[0040] In some embodiments, the immune effector cells are NK cells derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, hematopoietic stem cells, bone marrow, cell lines, or mixtures thereof.

[0041] In some embodiments, immune effector cells are engineered to express BCMA-targeting CARs for immunotherapy, such as cancer treatment. In some embodiments, the cells are autologous cells, allogeneic cells, or mixtures thereof.

[0042] Therefore, there is provided a pharmaceutical composition comprising a CAR polypeptide targeting BCMA, a polynucleotide encoding a CAR targeting BCMA, and an immune effector cell engineered to express the CAR polypeptide targeting BCMA. The composition may be formulated for cell-based therapy.

[0043] In another aspect of the invention, there is provided a method of treating cancer in a subject in need thereof using a composition and cells expressing a CAR targeting BCMA and a CAR targeting BCMA as contemplated herein. The CAR targeting BCMA, the composition and cells expressing the CAR targeting BCMA of the invention can be used to induce an immune response against a disorder or disease associated with BCMA expression in a subject.

[0044] In some embodiments, the immune effector cells of the invention are administered in an amount of about 1×10 6 ~10×10 8 or about 5×10 6 ~ about 9.5×10 8 or about 1×10 7 ~9×10 8 or about 5×10 7 ~8.5×10 8 or about 1×10 8 ~8.0×10 8 or about 2.0×10 8 ~8.0×10 8 or about 1.0×10 8 ~2.0×10 9 of cells.

[0045] In some embodiments, the cells are administered in an amount of 1.0×10 8 of cells.

[0046] In some embodiments, the cells are administered in an amount of 5.0×10 8 of cells.

[0047] In some embodiments, the cells are administered in an amount of 1.5×10 9 of cells.

[0048] In some embodiments, BCMA-targeting CARs, compositions, and / or manipulated immune cells are administered by infusion, injection, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, intracranially, percutaneously, subcutaneously, locally, by perfusion, or any combination thereof.

[0049] In some embodiments, BCMA-targeting CARs, compositions, and / or manipulated immune cells are administered intravenously.

[0050] In some embodiments, BCMA-targeting CARs, compositions, and / or manipulated immune cells may be used alone to treat cancer, or in combination with one or more other cancer therapies, including chemotherapy, radiation, immunotherapy, cancer vaccines, and / or targeted therapies.

[0051] In some embodiments, the subjects undergo immunodepletion chemotherapy.

[0052] In some embodiments, the methods of the present invention may be used to treat B-cell malignancies, multiple myeloma (e.g., relapsed and refractory multiple myeloma), lymphoma, and / or leukemia. [Brief explanation of the drawing]

[0053] [Figure 1] This is an illustrative diagram of a BCMA-DAP10-CAR structure. [Figure 2A] Representative images of mice inoculated with tumor cells after being administered NK cells expressing BCMA-CAR once (10M x 1) or twice (10M x 2). [Figure 2B] This study demonstrates the in vivo efficacy of BCMA-DAP10 and BCMA-CD28 CAR constructs against MM1S tumors. [Figure 3-1] This study demonstrates the in vivo efficacy of BCMA-DAP10 and BCMA-CD28 CAR constructs against multiple tumor cell lines. [Figure 3-2] This study demonstrates the in vivo efficacy of BCMA-DAP10 and BCMA-CD28 CAR constructs against multiple tumor cell lines. [Figure 4] A and B demonstrate that BCMA-DAP10-CAR and BCMA-CD28-CAR NK cells have the ability to kill tumor cells in vitro in an MM1S tumor model after multiple restimulations. [Figure 5] A and B demonstrate that BCMA-DAP10-CAR and BCMA-CD28-CAR NK cells can kill tumor cells in vitro in the RPMI-8226 model after multiple restimulations. [Figure 6] This study demonstrates the in vivo efficacy of BCMA-DAP10 CAR constructs and BCMA-CD28 CAR constructs against RPMI-8226 tumors. [Figure 7] This shows the in vivo proliferation of BCMA-DAP10 CAR NK cells and BCMA-CD28 CAR NK cells in the RPMI-8226 tumor model. [Figure 8A] This study demonstrates the in vivo efficacy of BCMA-DAP10 CAR in multiple doses in the RPMI-8226 tumor model. [Figure 8B] This study demonstrates the in vivo efficacy of BCMA-DAP10 CAR in multiple doses in the RPMI-8226 tumor model. [Figure 9] This demonstrates the in vitro efficacy of BCMA-DAP10 CAR against tumor lines expressing different levels of stress ligands (donor d). [Figure 10] This demonstrates the in vitro efficacy of BCMA-DAP10 CAR against tumor cell lines expressing different levels of stress ligands (donor e). [Figure 11A] This study demonstrates the in vitro efficacy of BCMA-DAP10-CAR against tumor cell lines and normal cells expressing different levels of stress ligands. [Figure 11B] This study demonstrates the in vitro efficacy of BCMA-DAP10-CAR against tumor cell lines and normal cells expressing different levels of stress ligands. [Figure 11C] This study demonstrates the in vitro efficacy of BCMA-DAP10-CAR against tumor cell lines and normal cells expressing different levels of stress ligands. [Figure 12] Images A and B show the expression of CAR proteins using codon-optimized nucleic acid sequences. The codon-optimized BCMA-DAP10 CAR construct (SEQ ID NO: 25) resulted in even higher transduction efficiency for BCMA-DAP10 CAR (SEQ ID NO: 55) across all four umbilical cord blood NK donors. [Figure 13] This study demonstrates the in vivo efficacy of NK cells expressing BCMA CARs (expressed using codon-optimized nucleic acid sequences) at low CAR+ doses. [Modes for carrying out the invention]

[0054] The present invention provides, in particular, a chimeric antigen receptor (CAR) targeting B cell maturation antigen (BCMA), compositions and cells expressing BCMA-targeting CARs, and methods of using them for the treatment of diseases such as cancer. The BCMA-targeting CAR constructs of the present invention incorporate a DAP10-derived costimulatory signaling domain that enhances proliferation in vivo and the functionality of immune effector cells (e.g., NK cells) expressing the BCMA-DAP10 CAR. The BCMA-DAP10 CAR polypeptide of the present invention exhibits even greater efficacy in killing tumor cells and inhibiting tumor growth. In some embodiments, the BCMA-DAP10 CAR of the present invention further comprises IL-15, which can be cleaved from the CAR polypeptide. The polynucleotide encoding the BCMA-DAP10 CAR polypeptide may have optimized codons.

[0055] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any methods and materials similar or equivalent to those described herein may be used in practice in the testing of the present invention, but preferred materials and methods are described herein. The following terms will be used in describing and claiming the present invention. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them.

[0056] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "an element" means one or more elements.

[0057] As used herein, the term “about” means, when referring to a measurable value such as a quantity or temporal duration, to include a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.01% from the specified value, such variation being appropriate for carrying out the disclosed method.

[0058] Antigen-binding domain: As used herein, the term “antigen-binding domain” refers to one or more extracellular domains of a chimeric antigen receptor that are specific to a particular antigen, such as BCMA.

[0059] Antibody: As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to an antigen. For example, in one embodiment, the antigen is B7-H6. In another embodiment, the antigen is MICA. An antibody may be an intact immunoglobulin derived from a natural or recombinant source, or it may be the immunoreactive portion of an intact immunoglobulin. The term is used in its broadest sense and includes polyclonal and monoclonal antibodies, which include intact antibodies and functional (antigen-binding) antibody fragments, which include fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments (including single-chain variable region fragments (scFv)), diabodies, and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. This term encompasses intrabody, peptide-body, chimeric antibodies, fully human antibodies, humanized antibodies, and genetically engineered and / or otherwise modified forms of immunoglobulins, such as heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, diabody, triabody, and tetrabody, tandem di-scFv, and tandem tri-scFv. Unless otherwise specified, the term “antibody” should be understood to encompass its functional antibody fragment. This term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0060] As used herein, “antibody heavy chain” refers to the larger of the two polypeptide chains present in all antibody molecules in their naturally occurring three-dimensional structure. As used herein, “antibody light chain” refers to the smaller of the two polypeptide chains present in all antibody molecules in their naturally occurring three-dimensional structure. Kappa (κ) and lambda (λ) light chains refer to the two main isotypes of antibody light chains.

[0061] The term "antibody fragment" refers to a portion of an intact antibody, specifically the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, antigen-binding fragments (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments (including single-chain variable fragments (scFv)), single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments, die bodies, and multispecific antibodies formed from antibody fragments. In specific embodiments, the antibody fragment may be an scFv.

[0062] Chimeric Antigen Receptor (CAR): As used herein, the term “chimeric antigen receptor” or “CAR” means a protein that, when expressed on the surface of a cell, enables the cell expressing the CAR to recognize its specific protein (antigen) on, for example, tumor cells, infected cells, or cells involved in autoimmune or inflammatory diseases or disorders. Such receptors are also known as chimeric T cell receptors, chimeric immune receptors, or artificial T cell receptors. Transduction of cells with a nucleic acid construct encoding a CAR allows the cells to recognize the antigen identified by the CAR. A CAR typically consists of an external domain (extracellular domain) and an internal domain (cytoplasmic domain), separated by a transmembrane domain. The external domain expressed on the cell surface includes an antigen-binding domain or receptor domain, a signal peptide that optionally directs the antigen-binding domain to the endoplasmic reticulum for processing, and optionally a spacer (or hinge) region. The antigen-binding domain (or receptor domain) contains a peptide that specifically recognizes the target antigen. In a non-limiting example, the antigen-binding domain can be a single-chain antibody, e.g., scFv. The spacer region is designed to be flexible enough to link the antigen-binding domain to the transmembrane domain and to allow the orientation of the antigen-binding domain in a manner that enables antigen recognition. Examples of spacer domains include, but are not limited to, hinge regions derived from IgG, CH2CH3 regions of immunoglobulins, hinges of CD28, hinges of DAP10, hinges of CD8, and parts of the CD3 molecule. The transmembrane domain is typically a hydrophobic alpha helix extending across the lipid bilayer of the cell membrane. The internal domain of the CAR consists of a signaling peptide that transmits an intracellular activation signal to the cytoplasm, thereby stimulating cells that express the CAR. The internal domain may include multiple such signaling domains, as described below. In some embodiments, the CAR includes at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as the “intracellular signaling domain”) containing a functional signaling domain derived from stimulating and / or co-stimulating molecules as defined below.In some embodiments, the set of polypeptides encoding the CAR are adjacent to each other. In some embodiments, the set of polypeptides includes a dimerization switch that, in the presence of a dimerization molecule, can bind the polypeptides to each other, for example, by binding an antigen-binding domain to an intracellular signaling domain. In one embodiment, the stimulating molecule is a zeta chain associated with a T cell receptor complex. In one embodiment, the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from the stimulating molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from the stimulating molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from the stimulating molecule. In one embodiment, the CAR includes an optional leader sequence at the amino terminus (N terminus) of the CAR fusion protein. In another embodiment, the CAR further includes a leader sequence at the N terminus of an extracellular antigen-binding domain, the leader sequence being optionally cleaved from the antigen-binding domain (e.g., scFv) during cell processing and localization of the CAR to the cell membrane.

[0063] Cancer: As used herein, the term “cancer” refers to a disease characterized by the rapid and uncontrolled proliferation of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers described herein include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain tumor, lymphoma, leukemia, and lung cancer.

[0064] Disease or Disorder: As used herein, the terms “disease” and “disorder” are interchangeable. “Disease” refers to a health condition in an animal in which the animal is unable to maintain homeostasis, and if the disease is not improved, the animal’s health will continue to deteriorate. In contrast, “disorder” in an animal is a health condition in which the animal is able to maintain homeostasis, but the animal’s health is less desirable than in the absence of the disorder. Leaving a disorder untreated does not necessarily lead to a further deterioration of the animal’s health.

[0065] Identical: As used herein, the term “identical” in the context of polynucleotides and nucleic acid sequences means a first sequence (e.g., an amino acid sequence or nucleic acid sequence) that contains a sufficient number or a minimum number of amino acid residues or nucleotide residues in the second amino acid sequence or nucleic acid sequence that are i) identical to or ii) conserved substitutions of aligned amino acid residues or nucleotide residues, such that the first and second amino acid sequences or nucleic acid sequences may have a common structural domain and / or common functional activity. For example, an amino acid sequence contains a common structural domain having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a reference amino acid sequence, e.g., a sequence provided herein. In another example, a nucleic acid sequence has at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a reference nucleic acid sequence, for example, a sequence provided herein.

[0066] Pharmaceutical Composition: As used herein, “pharmaceutical composition” means a composition formulated in a pharmaceutically acceptable or physiologically acceptable solution for administration to cells or animals, either alone or in combination with one or more other therapeutic agents. It is also understood that, if desired, the composition may be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various pharmaceutically active agents. Other components that may be included in the composition are not substantially limited, provided that the additional agents do not adversely affect the composition’s ability to deliver the intended therapy. In a preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, or excipient and one or more cells engineered to express a CAR as intended herein.

[0067] pharmaceutically acceptable carriers: As used herein, “pharmaceutically acceptable carriers” include any and all solvents, dispersions, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial agents, antifungal agents), isotonic agents, absorption retarders, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, similar materials and combinations thereof, as known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, pp. 1289–1329, incorporated herein by reference). Their use in pharmaceutical compositions is intended unless any conventional carrier is incompatible with the active ingredient.

[0068] As used herein, the terms “specific binding affinity,” “specifically binding,” “specifically bound,” “specifically conjugated,” “specifically binding,” or “specifically targeted” describe the binding of an anti-BCMA antibody or its antigen-binding domain (or a CAR containing the same) to BCMA (e.g., human BCMA) with a binding affinity higher than background binding. The BCMA-binding domain (or a CAR containing a BCMA-binding domain) is such that it binds to approximately 1 × 10⁻⁶-7 The dissociation constant of M (K d If it binds to or associates with BCMA, it "specifically binds" to BCMA. In some embodiments, the antigen-binding molecule is K d is approximately 1 x 10 -9 M ~ approx. 5×10 -9 When M is present, it binds specifically to the antigen with "high affinity". In some embodiments, the antigen-binding molecule is K d is 1 x 10 -10 M ~ approx. 5×10 -10 When M is present, it binds specifically to the antigen with "ultra-high affinity". In one embodiment, the antigen-binding molecule is 10 -9 M's K d It has the following characteristics: In one embodiment, the dissociation rate is approximately 1 × 10⁻⁶. -5 It is less than 1 × 10⁻⁶. In other embodiments, the antigen-binding molecule is approximately 1 × 10⁻⁶. -7 M ~ approx. 1×10 -13 M's K d In another embodiment, the antigen-binding molecule is approximately 1 × 10⁶ -10 M ~ approx. 5×10 -10 M's K d It binds to human BCMA. The affinity of the BCMA-binding domain and CAR protein according to the present invention can be easily determined using conventional techniques, for example, by competitive ELISA (enzyme-linked immunosorbent assay), or by binding association or substitution assays using labeled ligands.

[0069] Subjects: As used herein, the term “subjects” is intended to include organisms (e.g., mammals) from which an immune response may be induced. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species.

[0070] Therapeutic dose: As used herein, the term “therapeutic dose” includes an amount that is effective in “treating” a subject (e.g., a patient). Where a therapeutic dose is indicated, the exact amount of the composition to be administered may be determined by a physician, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the patient's (subject's) condition.

[0071] To treat: As used herein, the terms “to treat,” “to treat,” and “to treat” refer to a clinical intervention aimed at reversing, alleviating, delaying the onset or progression of a disease or disorder, or one or more of its symptoms, and / or preventing or delaying relapse, as described herein. For example, treatment in the form of NK cells expressing BCMA CAR as described herein may be performed on a subject after the onset of one or more symptoms and / or after the disease has been diagnosed. Treatment may be performed when there are no symptoms to, for example, prevent or delay the onset of symptoms, or to suppress the onset or progression of the disease. For example, for a susceptible individual, treatment may be performed before the onset of symptoms (for example, taking into account genetic factors or other susceptibility factors). Treatment may be continued after the symptoms have resolved to, for example, prevent or delay their relapse.

[0072] Various embodiments of the compositions and methods of the present invention are described in further detail below. Further definitions are given throughout this specification.

[0073] Chimeric antigen receptor (CAR) In one embodiment, the present invention relates to a CAR polypeptide targeting BCMA (B cell maturation antigen) and a polynucleotide encoding the same. The BCMA-targeting CAR comprises an extracellular antigen-binding domain that specifically binds to BCMA, a hinge domain, a transmembrane domain, a DAP10 costimulatory domain, and at least one intracellular activation / signaling domain. The BCMA-targeting CAR may further comprise one or more additional polypeptides, such as cytokines (e.g., IL-15). Each component within the CAR is linked by one or more linker sequences.

[0074] 1.BCMA binding region According to the present invention, the antigen-binding region of the CAR of the present invention comprises at least one BCMA-binding domain (i.e., a BCMA-binding factor). The BCMA-binding domain may be any agent that binds to BCMA or a portion of BCMA. The BCMA-binding domain may also be an antibody that specifically binds to BCMA, or an antigen-binding fragment thereof. The antibody or antigen-binding fragment that specifically binds to BCMA may be a monoclonal antibody, a single-specific antibody, a humanized antibody, a human antibody, a single-chain antibody, a domain-specific antibody, a single-domain antibody, a domain deletion antibody, an scFc fusion protein, a single-chain antibody, a chimeric antibody, a synthetic antibody, a recombinant antibody, a hybrid antibody, a mutant antibody, a CDR-transplanted antibody, an antibody fragment such as Fab, Ftab^ fragment, Fab' fragment, F(ab)2 fragment, Fv fragment, single-chain Fv(scFv) fragment, Fd fragment, dAb fragment, a diabody, a nanobody, a bivalent nanobody, a shark variable IgNAR domain, a VHH antibody, a camelid antibody, or a minibody.

[0075] A preferred BCMA-binding factor according to the present invention may be an scFv that specifically binds to BCMA. A "single-chain Fv" or "scFv" comprises a VH domain and a VL domain of an antibody, where these domains are present in a single polypeptide chain in either direction (e.g., VL-VH or VH-VL). Typically, scFv can be in the form of VH-linker-VL or VL-linker-VH.

[0076] The linker connecting the VH chain and the VL chain may include the amino acid sequence (GGGGS)n (where n is at least an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Exemplary linker sequences include, but are not limited to, GGGGSGGGGSGGGGS (SEQ ID NO: 15), GGGGSGGGGSGGGSGGGGS (SEQ ID NO: 16), GGGGSGGGGSGGGGSGGGSGGGGS (SEQ ID NO: 17), and GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 18). In some embodiments, the linker includes an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to one of SEQ ID NOs.

[0077] In one exemplary embodiment, the linker of anti-BCMA scFv contains the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 15).

[0078] In some embodiments, the linker is encoded by a nucleic acid sequence including SEQ ID NO: 19, SEQ ID NO: 9, or SEQ ID NO: 10.

[0079] GGGGGCGGAGGGTCTGGAGGAGGGGGGAGCGGGGGAGGCGGCTCT(Sequence 9)

[0080] GGCGGAGGGGGATCTGGAGGAGGAGGAAGTGGAGGCGGTGGCAGC (Sequence 10)

[0081] GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGATCC(Sequence No. 19)

[0082] In some embodiments, the BCMA binding factor includes three heavy chain complementarity-determining regions (HCDRs), namely HCDR1, HCDR2, and HCDR3, in the heavy chain variable region (VH), and / or three light chain complementarity-determining regions (LCDRs), namely LCDR1, LCDR2, and LCDR3, in the light chain variable region (VL). In some embodiments, the BCMA binding factor includes VH and / or VL.

[0083] In some embodiments, the BCMA-binding domain of the CAR of the present invention comprises HCDR1 having the amino acid sequence SYAIH (SEQ ID NO: 2), HCDR2 having the amino acid sequence VTWHDGSNKYYAESVMG (SEQ ID NO: 3), and HCDR3 having the amino acid sequence AKFGEPQYFQH (SEQ ID NO: 4).

[0084] In some embodiments, the BCMA-binding domain of the CAR of the present invention comprises LCDR1 having the amino acid sequence RASQGINNYLA (SEQ ID NO: 6), LCDR2 having the amino acid sequence AASTLQS (SEQ ID NO: 7), and LCDR3 having the amino acid sequence QQLKSYPFT (SEQ ID NO: 8).

[0085] In some embodiments, the BCMA binding region includes a heavy chain variable region (VH) comprising HCDR1 containing three complementarity-determining regions: SYAIH (SEQ ID NO: 2), HCDR2 containing VTWHDGSNKYYAESVMG (SEQ ID NO: 3), and HCDR3 containing AKFGEPQYFQH (SEQ ID NO: 4), and a light chain variable region (VL) comprising LCDR1 containing three complementarity-determining regions: RASQGINNYLA (SEQ ID NO: 6), LCDR2 containing AASTLQS (SEQ ID NO: 7), and LCDR3 containing QQLKSYPFT (SEQ ID NO: 8).

[0086] In some embodiments, the BCMA-binding domain includes a VH chain having the amino acid sequence represented by SEQ ID NO: 1. QITLRESGGDVVQPGRSLRLSCAASGFTFSSYAIHWVRQAPGKGLEWVAVTWHDGSNKYYAESVMGRFTISRDNSKNTLYLHMNSLRAEDTGVYYCARAKFGEPQYFQHWGQGTLVTVSS(Sequence ID 1).

[0087] It is assumed that amino acid substitutions at any position other than the CDR sequence can be changed to other amino acids, for example, as conservative amino acid substitutions (as defined herein). In some embodiments, VH includes a sequence that is 70% identical to SEQ ID NO: 1. In some embodiments, VH includes a sequence that is 75% identical to SEQ ID NO: 1. In some embodiments, VH includes a sequence that is 80% identical to SEQ ID NO: 1. In some embodiments, VH includes a sequence that is 85% identical to SEQ ID NO: 1. In some embodiments, VH includes a sequence that is 90% identical to SEQ ID NO: 1. In some embodiments, VH includes a sequence that is 95% identical to SEQ ID NO: 1. In some embodiments, VH includes a sequence that is 96% identical to SEQ ID NO: 1. In some embodiments, VH includes a sequence that is 97% identical to SEQ ID NO: 1. In some embodiments, VH includes a sequence that is 98% identical to SEQ ID NO: 1. In some embodiments, VH includes a sequence that is 99% identical to SEQ ID NO: 1.

[0088] In some embodiments, the BCMA-binding domain includes a VL chain having the amino acid sequence of SEQ ID NO: 5. DIVMTQSPSFLSASVGDRVTITCRASQGINNYLAWYQQKPGIAPKLLIYAASTLQSGVPSRFGGSGSGTEFTLTISSLQPEDFATYYCQQLKSYPFTFGPGTKVEIK(Sequence ID 5).

[0089] It is assumed that amino acid substitutions at any position other than the CDR sequence can be changed to other amino acids, for example, as a conservative amino acid substitution (as defined herein). In some embodiments, VL includes a sequence that is 70% identical to SEQ ID NO: 5. In some embodiments, VL includes a sequence that is 75% identical to SEQ ID NO: 5. In some embodiments, VL includes a sequence that is 80% identical to SEQ ID NO: 5. In some embodiments, VL includes a sequence that is 85% identical to SEQ ID NO: 5. In some embodiments, VL includes a sequence that is 90% identical to SEQ ID NO: 5. In some embodiments, VL includes a sequence that is 95% identical to SEQ ID NO: 5. In some embodiments, VL includes a sequence that is 96% identical to SEQ ID NO: 5. In some embodiments, VL includes a sequence that is 97% identical to SEQ ID NO: 5. In some embodiments, VL includes a sequence that is 98% identical to SEQ ID NO: 5. In some embodiments, VL includes a sequence that is 99% identical to SEQ ID NO: 5.

[0090] In some embodiments, the BCMA-binding domain of the chimeric antigen receptor of the present invention comprises the VH chain of SEQ ID NO: 1 and the VL chain of SEQ ID NO: 5.

[0091] In some embodiments, the BCMA-binding domain of the chimeric antigen receptor of the present invention comprises an scFv including the VH chain of SEQ ID NO: 1 and the VL chain of SEQ ID NO: 5, wherein the VH chain and the VL chain are linked by one of the linker sequences described herein (e.g., SEQ ID NOs: 15-18).

[0092] In some embodiments, the BCMA-binding domain in the chimeric antigen receptor of the present invention includes an scFv comprising the VH chain of SEQ ID NO: 1 and the VL chain of SEQ ID NO: 5.

[0093] As a non-limiting example, the BCMA-binding domain of the chimeric antigen receptor of the present invention includes the amino acid sequence represented by SEQ ID NO: 20. QITLRESGGDVVQPGRSLRLSCAASGFTFSSYAIHWVRQAPGKGLEWVAVTWHDGSNKYYAESVMGRFTISRDNSKNTLYLHMNSLRAEDTGVYYCARAKFGEPQYFQHWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSFLSASVGDRVTITCRASQGINNYLAWYQQKPGIAPKLLIYAASTLQSGVPSRFGGSGSGTEFTLTISSLQPEDFATYYCQQLKSYPFTFGPGTKVEIK (Sequence ID 20)

[0094] In some embodiments, the BCMA-binding domain includes an scFv having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 20.

[0095] In some embodiments, the BCMA-bound scFv is encoded by the nucleic acid sequence of SEQ ID NO: 35 or SEQ ID NO: 51.

[0096] In some embodiments, the molecule that specifically binds to the antigen is approximately 1 × 10⁻⁶ -7 The dissociation constant of M (K d ) binds. In some embodiments, the antigen-binding molecule is K d is approximately 1 x 10 -9 M ~ approx. 5×10 -9 When M is present, it specifically binds the antigen with "high affinity". In some embodiments, the antigen-binding molecule is K d is 1 x 10 -10 M ~ approx. 5×10 -10 When M is present, it specifically binds the antigen with "very high affinity". In one embodiment, the antigen-binding molecule is 10 -9 M's K d It has the following characteristics: In one embodiment, the dissociation rate is approximately 1 × 10⁻⁶. -5 It is less than 1 × 10⁻⁶. In other embodiments, the antigen-binding molecule is approximately 1 × 10⁻⁶. -7M ~ approx. 1×10 -13 M's K d In another embodiment, the antigen-binding molecule is approximately 1 × 10⁶ -10 M ~ approx. 5×10 -10 M's K d It binds to human BCMA.

[0097] In another specific embodiment, a molecule that specifically binds to BCMA does not cross-react with other proteins under similar binding conditions. In another specific embodiment, a molecule that specifically binds to BCMA does not cross-react with other non-BCMA proteins. In a specific embodiment, provided herein are antibodies or fragments thereof that bind to BCMA with a higher affinity than another unrelated antigen. In a particular embodiment, provided herein are antibodies or fragments thereof that bind to BCMA (e.g., human BCMA) with an affinity 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher than another unrelated antigen, as measured, for example, by radioimmunoassay, surface plasmon resonance, or binding equilibrium exclusion. In specific embodiments, the degree of binding of the anti-BCMA antibody or its antigen-binding fragment described herein to an unrelated non-BCMA protein is less than 10%, 15%, or 20% of the antibody's binding to the BCMA protein, as measured, for example, by radioimmunoassay.

[0098] In specific embodiments, provided herein are chimeric antigen receptors that bind to human BCMA with higher affinity than to BCMA of another species. In particular embodiments, provided herein are antibodies or fragments thereof that bind to human BCMA with 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher affinity than to BCMA of another species, as measured, for example, by radioimmunoassay, surface plasmon resonance, or binding equilibrium exclusion. In particular embodiments, a chimeric antigen receptor containing a BCMA-binding factor that binds to human BCMA binds to BCMA of another species with less than 10%, 15%, or 20% of the binding of the antibody or fragment to the human BCMA protein, as measured, for example, by radioimmunoassay, surface plasmon resonance, or binding equilibrium exclusion.

[0099] 2. Hinge area In certain embodiments, the BCMA-targeting CAR polypeptide of the present invention may include a hinge domain located between the extracellular antigen-binding domain and the transmembrane domain. The hinge domain may be included in the CAR polypeptide to provide an appropriate distance between the antigen-binding domain and the cell surface, or to mitigate steric hindrance that may adversely affect the antigen-binding or effector function of CAR gene-modified immune cells. For example, the hinge domain can position the antigen-binding domain away from the effector cell surface, enabling appropriate intercellular contact, antigen binding, and activation.

[0100] Hinge domains are of specific lengths, such as 10-50, 10-40, 10-30, 10-20, 10-15, 20-50, 20-40, 20-30, 15-50, 15-45, 15-30, 15-20, 12-20, 12-15, or 15-20 amino acid lengths.

[0101] In some embodiments, the hinge domain is derived from the human CD28 hinge domain. As a non-limiting example, the hinge domain of a BCMA-targeting CAR polypeptide includes a CD28 hinge domain having the amino acid sequence SEQ ID NO: 21: RAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPKDPK. In certain specific embodiments, the hinge domain includes an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least 98%, or at least about 99% identical to SEQ ID NO: 21. In some embodiments, the CD28 hinge domain may be encoded by the nucleic acid sequence of SEQ ID NO: 37 or SEQ ID NO: 54.

[0102] In some embodiments, the hinge domain is derived from the human CD28 hinge domain. As a non-limiting example, the hinge domain of a BCMA-targeting CAR polypeptide includes a CD28 hinge domain having the amino acid sequence of SEQ ID NO: 61: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP. In certain specific embodiments, the hinge domain includes an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least 98%, or at least about 99% identical to SEQ ID NO: 61. In some embodiments, the CD28 hinge domain may be encoded by the nucleic acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63.

[0103] Alternatively and optionally, one or more hinge domains or spacer domains derived from other proteins may be used. As used herein, the term “spacer domain” refers to a region that moves the antigen-binding domain away from the effector cell surface, enabling proper intercellular contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). Spacer domains may be derived from natural, synthetic, semi-synthetic, or recombinant sources. For example, hinge domains and spacer domains may be derived from human IgG hinge domains, CD8a hinge domains, or Fc domains of human immunoglobulins that bind to Fc receptors (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgD, or IgE).

[0104] In some embodiments, the BCMA-targeting CARs contemplated herein may include modified hinge domains and / or spacer domains. The modified hinge domains and / or spacer domains may include a portion of a naturally occurring hinge region having up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions) or up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions) in length of at least 10 amino acids (e.g., at least 12, 13, 14, or 15 amino acids), or a portion of a naturally occurring hinge region containing a core hinge region (which may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length).

[0105] 3. Transmembrane domain In various embodiments, the extracellular antigen-binding domain and intracellular signaling domain of the BCMA-targeting CAR of the present invention may be fused by a transmembrane domain. The transmembrane domain may also immobilize the CAR on the cell membrane of immunoeffector cells. The transmembrane domain may be derived from any membrane-bound or transmembrane protein. The transmembrane domain may be derived from a native, synthetic, semi-synthetic, or recombinant source. In some embodiments, the amino acid sequence of the transmembrane domain may be modified or substituted to minimize interaction with the binding domain of a native binding partner present in the same CAR-expressing cell, for example.

[0106] In some embodiments, the transmembrane domain of the BCMA-targeting CAR of the present invention is derived from the transmembrane domain of human CD28. For example, the CD28 transmembrane domain of the BCMA-targeting CAR polypeptide may include the amino acid sequence of SEQ ID NO: 22:FWVLVVVGGVLACYSLLVTVAFIIFWV. In some embodiments, for example, the transmembrane domain includes a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least 98%, or at least about 99% identical to SEQ ID NO: 22. In some embodiments, the CD28 transmembrane domain may be encoded by the nucleic acid sequence of SEQ ID NO: 38 or SEQ ID NO: 56.

[0107] Alternatively, the transmembrane domains of CARs may include the alpha, beta, or zeta chains of the T cell receptor, or the transmembrane regions of CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0108] 4. Cytoplasmic domain The cytoplasmic region of a CAR polypeptide contains an intracellular activation signaling domain. The intracellular signaling domain of a chimeric antigen receptor is involved in the activation of at least one of the normal effector functions of immune cells engineered to express the chimeric antigen receptor. The term “effector function” generally refers to a specialized function of a differentiated cell. The effector function of an immune cell (e.g., NK cells or T cells) can be helper activity, including cytolytic activity for killing tumor cells, cytotoxic activity, or cytokine secretion. In this context, the term “intracellular signaling domain” refers to the portion of a protein that transmits effector function signals, causing the cell to perform a specialized function. In some embodiments, the intracellular signaling domain is derived from the intracellular signaling domain of a native activating protein. Examples of such naturally occurring activated proteins include native receptors containing the zeta chain of the T cell receptor or any of its homologs (e.g., eta, delta, gamma, or epsilon), MB1 chain, B29, FcRIII, FcRI, signaling molecules such as CD3ζ, CD28, CD27, 4-IBB, DAP10, OX40, and other similar molecules. While the entire intracellular signaling domain is usually employed, it is often not necessary to use the entire intracellular polypeptide. To the extent that a truncated portion of the intracellular signaling domain may be used, such a truncated portion may be used in place of the intact chain, as long as it transmits the effector functional signal. Therefore, the term “intracellular signaling domain” means that it contains a truncated portion of the intracellular signaling domain sufficient to transmit the effector functional signal during CAR binding to a target.

[0109] In one preferred embodiment, the human CD3ζ intracellular signaling domain is used as the intracellular signaling domain of the BCMA-targeting CAR polypeptide of the present invention. An example of the CD3ζ intracellular signaling domain includes the amino acid sequence of SEQ ID NO: 23. RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (Sequence ID 23).

[0110] In certain specific embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least 98%, or at least about 99% identical to SEQ ID NO: 23. In some embodiments, the CD3ζ intracellular signaling domain may be encoded by the nucleic acid sequence of SEQ ID NO: 40 or SEQ ID NO: 58.

[0111] In some embodiments, the BCMA-targeting CAR polypeptide may include one or more co-stimulatory signaling domains. The intracellular co-stimulatory signaling domain refers to the intracellular portion of the co-stimulatory molecule. The term "co-stimulatory molecule" refers to a co-binding partner of an immune cell that mediates a co-stimulatory response by immune cells, such as proliferation, by specifically binding to a co-stimulatory ligand.

[0112] In some embodiments, the BCMA-targeting CAR polypeptide of the present invention includes a co-stimulatory domain derived from DAP10 (hematopoietic cell signaling precursor (DNAX-activated protein 10)). An example of a DAP10-derived co-stimulatory domain includes the amino acid sequence LCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO: 24). In certain embodiments, the DAP10 co-stimulatory domain includes an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least 98%, or at least about 99% identical to SEQ ID NO: 24. In some embodiments, the DAP10 co-stimulatory domain may be encoded by the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 57.

[0113] CD28-derived co-stimulatory domains may be used in the BCMA-targeting CAR polypeptide of the present invention. An example of a CD28 co-stimulatory domain includes the amino acid sequence of SEQ ID NO: 64. RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(Sequence ID 64)

[0114] In addition to the DAP10-derived co-stimulatory domain and CD3ζ intracellular signaling domain, the BCMA-targeting CAR polypeptides described herein may also contain one or more co-stimulatory domains and / or one or more intracellular activation signaling domains.The co-stimulatory domain and intracellular activation signaling domain include, for example, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD28, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell costimulatory factor (ICOS), CDS gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (a member of the tumor necrosis factor superfamily; TNFSF1,4), NKG2C, 2B4, Ig alpha (CD79a), DAP12, Fcγ receptor, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins Proteins, cytokine receptors, integrins, signal transduction lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CDS Alpha, CDS Beta, 11.2 Beta, IL2R Gamma, IL7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, ITGAE, CD103, ITGAL, LFA-1, ITGAM, ITGAX, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE The ligands may be derived from RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGLl, CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0115] 5. Other components In some embodiments, one or more other polypeptides and / or proteins may be incorporated into the BCMA-targeting CAR construct as described herein. Additional proteins and polypeptides may be utilized for any function, e.g., the activation of any cell expressing the CAR polypeptide and / or CAR.

[0116] In some embodiments, the BCMA-targeting CAR polypeptide described herein may further comprise one or more cytokines. The CAR and other proteins may be separated, for example, by a cleavable 2A sequence.

[0117] In one exemplary embodiment, the cytokine IL-15 is incorporated into a CAR construct targeting BCMA. IL-15 is a pro-inflammatory cytokine important for the differentiation and proliferation of T cells and NK cells, and for the development of dendritic cells. An example of IL-15 is the amino acid sequence of SEQ ID NO: 26. GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(Sequence ID 26)

[0118] In some embodiments, the IL15 protein incorporated into the BCMA-targeting CAR construct contains an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least 98%, or at least about 99% identical to SEQ ID NO: 26.

[0119] In one embodiment, the IL-15 peptide is encoded by the nucleic acid sequence of SEQ ID NO: 52 or SEQ ID NO: 53.

[0120] In some embodiments, CAR polypeptides and other proteins (e.g., IL-15) within the same construct are intended to be generated into two distinct polypeptides, and cleavable 2A sequences (e.g., T2A, F2A, and E2A) may be utilized. 2A self-cleaving peptides (i.e., 2A peptides) are a class of peptides with AA lengths of 18–22 that can induce ribosome skipping during protein translation in cells. These peptides share the core sequence motif of DxExNPGP and are found in a wide range of viral families. They assist in the generation of polyproteins by preventing ribosomes from forming peptide bonds. Members of 2A peptides are named after the viruses in which they were first described. For example, F2A, the first 2A peptide to be described, originates from the foot-and-mouth disease virus. The name "2A" itself derives from the genetic numbering system of this virus.

[0121] In some embodiments, the cleavable peptide is E2A. As a non-limiting example, E2A includes the sequence GPQCTNYALLKLAGDVESNPGP (SEQ ID NO: 27). In some embodiments, the cleavable peptide is positioned between the CAR polypeptide and IL-15.

[0122] The BCMA-targeting CARs described herein may further include a signal peptide. The signal peptide may contain 3-30, 3-20, 3-15, 5-30, 5-20, 5-15, 10-30, 10-20, or 10-15 amino acid residues. An example of a signal peptide is the sequence MEFGLSWLFLVAILKGVQC (SEQ ID NO: 28). An example of a signal peptide is the sequence MRISKPHLRSISIQCYLCLLLNSHFLTEA (SEQ ID NO: 59).

[0123] In one exemplary embodiment, a BCMA-targeting CAR comprises a signal peptide, a BCMA-binding domain, a hinge and transmembrane domains, a DAP10 costimulatory domain, and at least one intracellular signaling domain.

[0124] In another exemplary embodiment, a construct for expressing a BCMA-targeting CAR comprises a signal peptide, an extracellular BCMA-binding domain, hinge and transmembrane domains, a DAP10 costimulatory domain, at least one intracellular signaling domain, and a cytokine.

[0125] In one exemplary embodiment, a suicide gene product such as caspase 9 (e.g., inducible caspase 9) is used in combination with CAR.

[0126] Example amino acid sequence of caspase 9: MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS (Sequence ID 14)

[0127] Any polypeptide included in this disclosure may contain an amino acid sequence identical to SEQ ID NO: 14, or at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of SEQ ID NO: 14.

[0128] 6. Linker The BCMA-targeting CARs intended herein may contain linker residues between various domains. In some embodiments, the BCMA-targeting CARs intended herein may contain 1, 2, 3, 4, or 5 or more linkers. In certain embodiments, the length of the linkers is about 1 to about 30 amino acids, about 1 to about 25 amino acids, about 5 to about 30 amino acids, about 5 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or the length of any intervening amino acids. In some embodiments, the linkers are the length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids. Those skilled in the art will recognize that the design of a CAR in a particular embodiment may include a linker that is all or partially flexible, and that the linker may include one or more parts that give a less flexible structure to provide the desired CAR structure, as well as a flexible linker.

[0129] In one exemplary embodiment, the BCMA-targeting CAR polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 29. QITLRESGGDVVQPGRSLRLSCAASGFTFSSYAIHWVRQAPGKGLEWVAVTWHDGSNKYYAESVMGRFTISRDNSKNTLYLHMNSLRAEDTGVYYCARAKFGEPQYFQHWGQGTL VTVSSGGGGSGGGGSGGGGSDIVMTQSPSFLSASVGDRVTITCRASQGINNYLAWYQQKPGIAPKLLIYAASTLQSGVPSRFGGSGSGTEFTLTISSLQPEDFATYYCQQLKSYP FTFGPGTKVEIKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (Sequence ID 29)

[0130] In some embodiments, the BCMA-targeting CAR polypeptide may contain an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 29.

[0131] In one aspect of the present invention, a CAR polypeptide targeting BCMA comprises the following, from the N-terminus to the C-terminus: Signal sequence 1 - BCMA binding factor - linker - CD28 (hinge) - linker - CD28 (TM) - DAP10 (co-stimulus) - CD3z - E2A - Signal sequence 2 - sIL-15.

[0132] In one exemplary embodiment, the BCMA-targeting CAR polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 13. (Sequence ID 13)

[0133] BCMA-targeting CAR polypeptides may contain amino acid sequences that are at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identical to SEQ ID NO: 13.

[0134] Polynucleotides encoding CARs that target BCMA In another embodiment, the present invention provides nucleic acid molecules encoding one or more CAR polypeptides as described herein. As used herein, the terms “nucleic acid molecule” and “polynucleotide” are interchangeable. In some embodiments, the nucleic acid molecule is provided as a messenger RNA (mRNA) molecule. In other embodiments, the nucleic acid molecule is provided as a DNA construct. In some embodiments, the DNA construct is a non-viral vector such as a plasmid, cosmid, or artificial chromosome. In other embodiments, the DNA construct is a viral vector such as AAV, lentivirus, and retrovirus.

[0135] Accordingly, the present invention provides a polynucleotide encoding a BCMA-targeting CAR polypeptide, wherein the CAR comprises an anti-BCMA binding domain (e.g., a human anti-BCMA binding domain), a hinge domain, a transmembrane domain, a DAP10 costimulatory domain, and an intracellular signaling domain comprising a major CD3ζ signaling domain. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding one or more polypeptides incorporated into the CAR construct. For example, the polynucleotide further comprises a nucleic acid sequence encoding a cytokine such as IL-15. In some embodiments, the polynucleotide further comprises a nucleic acid sequence of a single peptide and / or linker sequence (e.g., E2A).

[0136] In some embodiments, the BCMA-binding domain is an anti-BCMA-binding domain as described herein. In some examples, the BCMA-binding domain is encoded by one of the nucleic acid sequences of SEQ ID NOs: 30, 35-36, and 47-51. In some embodiments, the nucleic acid sequence encoding the BCMA-binding domain of CAR includes a nucleic acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to one of the sequences of SEQ ID NOs: 30, 35-36, and 47-51.

[0137] In some embodiments, the polynucleotide encoding the CAR targeting BCMA includes the nucleic acid sequence of SEQ ID NO: 30 and the nucleic acid sequence of SEQ ID NO: 32.

[0138] In some embodiments, the polynucleotide encoding the CAR targeting BCMA includes the nucleic acid sequence of SEQ ID NO: 30 and the nucleic acid sequence of SEQ ID NO: 49.

[0139] In some embodiments, the polynucleotide encoding the CAR targeting BCMA includes the nucleic acid sequence of SEQ ID NO: 30 and the nucleic acid sequence of SEQ ID NO: 50.

[0140] In some embodiments, the polynucleotide encoding the CAR targeting BCMA includes the nucleic acid sequence of SEQ ID NO: 32 and the nucleic acid sequence of SEQ ID NO: 47.

[0141] In some embodiments, the polynucleotide encoding the CAR targeting BCMA includes the nucleic acid sequence of SEQ ID NO: 32 and the nucleic acid sequence of SEQ ID NO: 48.

[0142] In some embodiments, the polynucleotide encoding the CAR targeting BCMA includes the nucleic acid sequence of SEQ ID NO: 47 and the nucleic acid sequence of SEQ ID NO: 49.

[0143] In some embodiments, the polynucleotide encoding the CAR targeting BCMA includes the nucleic acid sequence of SEQ ID NO: 48 and the nucleic acid sequence of SEQ ID NO: 50.

[0144] Exemplary nucleic acid sequences encoding the BCMA-binding domain include sequence number 35, or sequences that are approximately 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 35.

[0145] Exemplary nucleic acid sequences encoding the BCMA-binding domain include sequence number 36, or sequences that are approximately 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 36.

[0146] The nucleic acid sequence encoding the BCMA-binding domain may be codon-optimized. In one embodiment, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes SEQ ID NO: 51. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes a nucleic acid sequence that is approximately 75% identical to SEQ ID NO: 51. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes a nucleic acid sequence that is approximately 80% identical to SEQ ID NO: 51. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes a nucleic acid sequence that is approximately 85% identical to SEQ ID NO: 51. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes a nucleic acid sequence that is approximately 90% identical to SEQ ID NO: 51. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes a nucleic acid sequence that is approximately 91% identical to SEQ ID NO: 51. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes a nucleic acid sequence that is approximately 92% identical to SEQ ID NO: 51. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes a nucleic acid sequence that is approximately 93% identical to SEQ ID NO: 51. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain contains a nucleic acid sequence that is approximately 94% identical to SEQ ID NO: 51. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain contains a nucleic acid sequence that is approximately 95% identical to SEQ ID NO: 51. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain contains a nucleic acid sequence that is approximately 96% identical to SEQ ID NO: 51. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain contains a nucleic acid sequence that is approximately 97% identical to SEQ ID NO: 51. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain contains a nucleic acid sequence that is approximately 98% identical to SEQ ID NO: 51. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain contains a nucleic acid sequence that is approximately 99% identical to SEQ ID NO: 51.

[0147] In another embodiment, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes SEQ ID NO: 35. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes a nucleic acid sequence that is approximately 75% identical to SEQ ID NO: 35. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes a nucleic acid sequence that is approximately 80% identical to SEQ ID NO: 35. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes a nucleic acid sequence that is approximately 85% identical to SEQ ID NO: 35. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes a nucleic acid sequence that is approximately 90% identical to SEQ ID NO: 35. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes a nucleic acid sequence that is approximately 91% identical to SEQ ID NO: 35. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes a nucleic acid sequence that is approximately 92% identical to SEQ ID NO: 35. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes a nucleic acid sequence that is approximately 93% identical to SEQ ID NO: 35. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain includes a nucleic acid sequence that is approximately 94% identical to SEQ ID NO: 35. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain contains a nucleic acid sequence that is approximately 95% identical to SEQ ID NO: 35. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain contains a nucleic acid sequence that is approximately 96% identical to SEQ ID NO: 35. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain contains a nucleic acid sequence that is approximately 97% identical to SEQ ID NO: 35. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain contains a nucleic acid sequence that is approximately 98% identical to SEQ ID NO: 35. In some embodiments, the codon-optimized nucleic acid sequence encoding the BCMA-binding domain contains a nucleic acid sequence that is approximately 99% identical to SEQ ID NO: 35. [Table 1-1] [Table 1-2]

[0148] In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide includes a nucleic acid sequence encoding a DAP10 costimulatory domain having the sequence of SEQ ID NO: 24. In some embodiments, the nucleic acid sequence encoding the DAP10 costimulatory domain is codon-optimized. As a non-limiting example, the sequence encoding the codon-optimized DAP10 costimulatory domain includes the sequence of SEQ ID NO: 57. In some embodiments, the nucleic acid sequence encoding the DAP10 costimulatory domain includes the sequence of SEQ ID NO: 39.

[0149] In some embodiments, the polynucleotide encoding a BCMA-targeting CAR polypeptide includes a nucleic acid sequence encoding a CD28 hinge domain having the sequence of SEQ ID NO: 21. In other embodiments, the nucleic acid sequence encoding the CD28 hinge domain is codon-optimized. As a non-limiting example, the codon-optimized sequence encoding the CD28 hinge domain includes the sequence of SEQ ID NO: 37 or SEQ ID NO: 54. In other embodiments, the polynucleotide encoding a BCMA-targeting CAR polypeptide includes a nucleic acid sequence encoding a CD28 hinge domain having the sequence of SEQ ID NO: 61; the nucleic acid sequence encoding the CD28 hinge domain is codon-optimized. As a non-limiting example, the codon-optimized sequence encoding the CD28 hinge domain includes the sequence of SEQ ID NO: 62 or SEQ ID NO: 63.

[0150] Table 2 includes exemplary nucleic acid sequences for each of the other components of the CAR that target the BCMA of the present invention. [Table 2-1] [Table 2-2]

[0151] An exemplary polynucleotide encoding a BCMA-targeting CAR containing a DAP10-derived co-stimulatory domain comprises the nucleic acid sequence presented by SEQ ID NO: 44. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may comprise a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 44. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may comprise a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 44. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may comprise a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 44. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may comprise a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 44. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may comprise a nucleic acid sequence that is at least 91% identical to SEQ ID NO: 44. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may comprise a nucleic acid sequence that is at least 92% identical to SEQ ID NO: 44. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may comprise a nucleic acid sequence that is at least 93% identical to SEQ ID NO: 44. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 94% identical to SEQ ID NO: 44. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 44. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 96% identical to SEQ ID NO: 44. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 97% identical to SEQ ID NO: 44.In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may include a nucleic acid sequence that is at least 98% identical to SEQ ID NO: 44. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may include a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 44.

[0152] The polynucleotide encoding the BCMA-targeting CAR polypeptide may be codon-optimized.

[0153] In one embodiment, the polynucleotide encoding the BCMA-targeting CAR polypeptide contains the nucleic acid sequence of SEQ ID NO: 60. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 60. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 60. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 60. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 60. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 91% identical to SEQ ID NO: 60. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 92% identical to SEQ ID NO: 60. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 93% identical to SEQ ID NO: 60. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 94% identical to SEQ ID NO: 60. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 60. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 96% identical to SEQ ID NO: 60. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 97% identical to SEQ ID NO: 60.In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may include a nucleic acid sequence that is at least 98% identical to SEQ ID NO: 60. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may include a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 60.

[0154] In one embodiment, the polynucleotide encoding the BCMA-targeting CAR polypeptide contains the nucleic acid sequence of SEQ ID NO: 25. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 25. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 25. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 25. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 25. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 91% identical to SEQ ID NO: 25. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 92% identical to SEQ ID NO: 25. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 93% identical to SEQ ID NO: 25. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 94% identical to SEQ ID NO: 25. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 25. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 96% identical to SEQ ID NO: 25. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 97% identical to SEQ ID NO: 25.In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may include a nucleic acid sequence that is at least 98% identical to SEQ ID NO: 25. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may include a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 25.

[0155] In one embodiment, the polynucleotide encoding the BCMA-targeting CAR polypeptide contains the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 55. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 55. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 55. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 55. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 91% identical to SEQ ID NO: 55. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 92% identical to SEQ ID NO: 55. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 93% identical to SEQ ID NO: 55. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 94% identical to SEQ ID NO: 55. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 55. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 96% identical to SEQ ID NO: 55. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may contain a nucleic acid sequence that is at least 97% identical to SEQ ID NO: 55.In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may include a nucleic acid sequence that is at least 98% identical to SEQ ID NO: 55. In some embodiments, the polynucleotide encoding the BCMA-targeting CAR polypeptide may include a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 55. [Table 3-1] [Table 3-2]

[0156] In some embodiments, the polynucleotide comprises at least one modified nucleotide. In other embodiments, the polynucleotide comprises only unmodified nucleotides.

[0157] The polynucleotides encoding the CAR polypeptide of the present invention can be obtained using recombinant methods known in the art, for example, by screening a library from cells expressing the CAR construct, by deriving the CAR construct from a vector known to contain it, or by directly isolating it from cells and tissues containing it using standard techniques. Alternatively, the polynucleotides can also be produced by in vitro synthesis.

[0158] In alternative embodiments, polynucleotide sequences encoding various components of a BCMA-targeting CAR may be located on different nucleic acid molecules, e.g., different plasmids or vectors, e.g., viral vectors, e.g., lentiviral vectors. For example, (i) the sequence encoding the antigen-binding domain may be located on a first nucleic acid, e.g., a first vector, and (ii) the sequence encoding the intracellular signaling domain may be located on a second nucleic acid, e.g., a second vector.

[0159] In some embodiments, the polynucleotide encoding the CAR of the present invention is an mRNA molecule. The mRNA may further include poly(A) sequences, for example, sequences containing adenines of 50-5000, 100-5000, 50-2000, 100-2000, 50-1000, or 100-1000.

[0160] cell In one embodiment, the present invention provides cells genetically engineered to express BCMA-targeting CAR polypeptides as described herein. The cells may be stem cells, immune effector cells, or a mixture thereof. "Immune effector cells" means any cell of the immune system having one or more effector functions (e.g., cytotoxic cell-killing activity, cytokine secretion, ADCC and / or CDC induction). Specific immune cells include, but are not limited to, natural killer (NK) cells, T cells, gamma delta T cells, alpha beta T cells, invariant NKT (iNKT) cells, B cells, macrophages, mesenchymal stromal cells, dendritic cells, or a mixture thereof. In one embodiment, the immune effector cells are T cells. In another embodiment, the immune effector cells are NK cells.

[0161] As used herein, the terms “genetically engineered” or “recombinant” refer to adding additional genetic material in the form of DNA or RNA, such as DNA or RNA encoding a CAR polypeptide that targets the BCMA of the present invention, to the total genetic material within a cell. The terms “genetically engineered cell,” “engineered cell,” and “genetically engineered or engineered cell to express” are interchangeable.

[0162] In some embodiments, immune effector cells (e.g., T cells or NK cells) are transformed with polynucleotides encoding a CAR construct, and the CAR is expressed on the cell surface. CART cells or CAR-NK cells exhibit antitumor properties.

[0163] A method is provided for producing immunoeffector cells expressing CARs as envisioned herein. In one embodiment, the method comprises transfecting or transducing immunoeffector cells isolated from an organism so that the immunoeffector cells express one or more CARs as envisioned herein. In certain embodiments, immunoeffector cells are isolated from an organism and genetically engineered without further in vitro manipulation. Such cells can then be directly re-administered to an organism. In further embodiments, immunoeffector cells are first activated and stimulated to proliferate in vitro before being genetically engineered to express CARs. In this regard, immunoeffector cells may be cultured before and / or after genetic engineering (i.e., transfected or transfused to express CARs targeting BCMA as envisioned herein).

[0164] For example, immune effector cells (e.g., T cells or NK cells) are transduced with a viral vector encoding the BCMA-targeting CAR polypeptide of the present invention. The viral vector is a retroviral vector such as a lentiviral vector or an AAV vector. In another example, immune effector cells (e.g., T cells or NK cells) are transfused with a nucleic acid molecule encoding the BCMA-targeting CAR polypeptide of the present invention, such as mRNA, cDNA, or DNA.

[0165] Cell supply source According to the present invention, immune effector cells (e.g., T cells and NK cells) are obtained from a subject for proliferation and genetic engineering to express the CAR polypeptide of the present invention.

[0166] Immune effector cells can be autogeneic ("self") or non-self ("non-self," e.g., allogeneic, syngeneic, or heterogeneous). As used herein, "autogeneic" refers to cells of the same origin as the subject. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the cells being compared. As used herein, "synogeneic" refers to cells of a different subject that are genetically identical to the cells being compared. As used herein, "heterogeneous" refers to cells of a different species than the cells being compared. In preferred embodiments, the cells are autogeneic.

[0167] In some embodiments, the immune effector cells engineered to express BCMA-targeting CARs as intended herein are T cells (also called T lymphocytes). T cells can be immature T cells, mature T cells, resting T cells, or activated T cells, T helper (Th) cells (e.g., T helper 1 (Th1) or T helper 2 (Th2) cells), or any other subset of T cells. Other exemplary populations of T cells suitable for use in particular embodiments include naive T cells (TN), T memory stem cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), and effector T cells (TEFF). T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural fluid, and splenic tissue. In some embodiments, T cells can be obtained from units of blood collected from a subject using any number of techniques known to those skilled in the art. For example, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes.

[0168] In some embodiments, immune effector cells include NK cells. NK cells may be derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, hematopoietic stem cells, or bone marrow. NK cells may be derived from umbilical cord blood mononuclear cells. NK cells may also be CD56+ NK cells.

[0169] In some embodiments, any immunoeffector cell line (e.g., T cells or NK cells) available in the art may be used. For example, NK cells may be derived from the NK-92 cell line.

[0170] In some embodiments, cells transduced with polynucleotides encoding CARs targeting BCMA as described herein are grown. In some embodiments, the cells are grown in culture for a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days).

[0171] In some embodiments, cells are grown in a suitable medium containing one or more interleukins, which, when measured by methods such as flow cytometry, result in a cell increase of at least 100-fold, at least 150-fold, and at least 200-fold (e.g., 200-fold, 250-fold, 300-fold, 350-fold) over a 14-day growth period.

[0172] In some embodiments, the manipulated immune effector cell population for cancer treatment includes CARs targeting BCMA as intended herein. For example, the manipulated immune effector cell population is a population of T cells and / or NK cells.

[0173] Pharmaceutical compositions and preparations The present invention provides compositions comprising BCMA-targeting CARs, nucleic acid molecules encoding them, and cells expressing BCMA-targeting CARs. The compositions contemplated herein may include, but are not limited to, one or more BCMA-targeting CAR polypeptides, polynucleotides, vectors containing them, or genetically engineered immune effector cells. The compositions include, but are not limited to, pharmaceutical compositions. In light of this disclosure, the preparation of pharmaceutical compositions comprising such compositions will be known to those skilled in the art. In preferred embodiments, the composition comprises one or more cells engineered to express one or more BCMA-targeting CAR polypeptides as described herein. In some embodiments, pharmaceutical compositions comprising effective amounts of cells, and compositions comprising immune effector cells (e.g., NK cells) as described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, or excipient and cells expressing BCMA-targeting CAR polypeptides as contemplated herein.

[0174] Pharmacopoecially acceptable carriers, diluents, or excipients include, but are not limited to, any adjuvants, carriers, excipients, flow enhancers, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, surfactants, or emulsifiers that are approved by the U.S. Food and Drug Administration for use in humans or livestock. Examples of pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose, and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; water free of pyrogens; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and any other suitable substances used in pharmaceutical formulations.

[0175] In certain embodiments, the pharmaceutical composition comprises an effective amount of immune effector cells expressing the CAR intended herein. As used herein, the term “effective amount” refers to an amount of genetically engineered therapeutic cells, such as NK cells, that is effective in achieving a beneficial or desirable preventive or therapeutic outcome, including clinical outcomes (e.g., anti-cancer).

[0176] The pharmaceutical composition comprising an immunoeffector cell population (e.g., NK cells) engineered to express a BCMA-targeting CAR of the present invention may also comprise a buffer, e.g., neutral buffered saline, phosphate-buffered saline; carbohydrates, e.g., glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, e.g., glycine; antioxidants; chelating agents, e.g., EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The composition is preferably formulated for parenteral administration, e.g., intravascular (intravenous or intra-arterial), intraperitoneal, or intramuscular administration.

[0177] The composition may be a liquid composition. The liquid pharmaceutical composition, whether in solution, suspension or other similar form, may contain one or more of the following: sterile diluents, e.g., water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride, fixing oils such as synthetic mono or diglycerides which may be solvents or suspension media, polyethylene glycol, glycerin, propylene glycol or other solvents; antimicrobial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetates, citrates or phosphates; and tonicity modifiers, e.g., sodium chloride or dextrose. Parenteral formulations may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. The pharmaceutical composition for injection is preferably sterile.

[0178] In some embodiments, the immune effector cells (e.g., NK cells) discussed herein, and the compositions intended herein, are formulated in pharmaceutically acceptable cell culture media. Such compositions are suitable for administration to human subjects. In certain embodiments, the pharmaceutically acceptable cell culture media is a serum-free medium. Serum-free media offer several advantages over serum-containing media, including a simpler and more clearly defined composition, lower levels of contaminants, elimination of potentially infectious substances, and lower cost. In various embodiments, the serum-free medium may be free of animal-derived components and optionally free of proteins. Optionally, the medium may contain recombinant proteins that are biopharmaceutically acceptable. A “animal-derived component-free” medium refers to a medium in which the components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-derived component-free media, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, a “protein-free” medium is defined as substantially protein-free.

[0179] In other embodiments, the compositions comprising immunoeffector cells described herein are formulated in the form of a solution comprising a cryopreservation medium. For example, a cryopreservation medium comprising a cryopreservative may be used to maintain high cell viability after thawing. For example, the compositions comprising immunoeffector cells described herein may be cryopreserved in a medium discussed in PCT application publication WO2022173866A1 (the contents of which are incorporated herein in their entirety by reference). In some embodiments, the cryopreservation medium comprises a nonpyrogenic and isotonic crystalloid solution, disaccharides, a cryoprotective agent, and albumin. In some embodiments, the nonpyrogenic and isotonic crystalloid solution is present at a concentration of 25% v / v to 50% v / v. In some embodiments, the nonpyrogenic and isotonic crystalloid solution is present at a concentration of about 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, the nonpyrogenic and isotonic crystalloid solution is present at a concentration of 30% v / v to 40% v / v.

[0180] In some embodiments, the non-exothermic and isotonic crystalloid liquid is present at concentrations of 37.1% v / v, 37.3% v / v, 37.5% v / v, 37.7% v / v, or 37.9% v / v.

[0181] In some embodiments, the non-exothermic and isotonic crystalloid liquid is present at a concentration of 37.7 v / v.

[0182] In some embodiments, non-exothermic and isotonic crystalloid solutions are present at concentrations of 38.0% v / v, 38.2% v / v, 38.4% v / v, 38.6% v / v, 38.8% v / v, or 39.0% v / v.

[0183] In some embodiments, a non-exothermic and isotonic crystalloid solution is present at a concentration of 38.6% v / v.

[0184] In some embodiments, the disaccharide is selected from the group consisting of sucrose, lactose, maltose, trehalose, cellobiose, and chitobiose. Therefore, in some embodiments, the disaccharide is sucrose. In some embodiments, the disaccharide is lactose. In some embodiments, the disaccharide is maltose. In some embodiments, the disaccharide is trehalose. In some embodiments, the disaccharide is cellobiose. In some embodiments, the disaccharide is chitobiose.

[0185] In some embodiments, the cryopreservation media provided herein include one or more of the following: sodium chloride, potassium chloride, magnesium chloride hexahydrate, sodium acetate trihydrate, sodium gluconate, adenosine, dextran-40, lactobionic acid, HEPES, sodium hydroxide, L-glutathione, potassium chloride, potassium bicarbonate, potassium phosphate, dextrose, sucrose, mannitol, calcium chloride dihydrate, magnesium chloride, sodium hydroxide, potassium hydroxide, DMSO, human serum albumin, and trehalose.

[0186] In some embodiments, the cryopreservation medium provided herein contains about 2.35% w / v human serum albumin (HSA). In some embodiments, the cryopreservation medium contains human serum albumin (HSA) between about 2.0% w / v and 5.0% w / v. In some embodiments, the cryopreservation medium contains human serum albumin (HSA) between about 2.0% w / v and 3.0% w / v. In some embodiments, the cryopreservation medium provided herein contains about 1% v / v and 10% v / v human serum albumin (HSA). In some embodiments, the cryopreservation medium provided herein contains about 5% v / v and 10% v / v human serum albumin (HSA). In some embodiments, the cryopreservation medium contains 9.4% v / v human serum albumin.

[0187] In some embodiments, the cryopreservation medium contains trehalose between approximately 10 mM and 100 mM. In some embodiments, the cryopreservation medium contains trehalose between approximately 10 mM and 50 mM. In some embodiments, the cryopreservation medium contains trehalose between approximately 20 mM and 40 mM. In some embodiments, the cryopreservation medium contains trehalose between approximately 30 mM.

[0188] In some embodiments, a cryopreservation medium is provided comprising human serum albumin (HSA), sodium chloride, sodium gluconate, sodium acetate trihydrate, potassium chloride, magnesium chloride, dimethyl sulfoxide (DMSO), and trehalose.

[0189] In some embodiments, a cryopreservation medium is provided: the medium comprises a non-pyrogenic and isotonic crystalloid solution, a cryoprotective agent, albumin, and disaccharides. In some embodiments, a cryopreservation medium is provided: the medium comprises PLASMA-LYTE A, a cryoprotective agent, human serum albumin (HSA), and trehalose. In some embodiments, the cryoprotective agent is DMSO. In some embodiments, a cryopreservation medium is provided: the medium comprises 37.7% PLASMA-LYTE A, 50% DEMO, 2.35% w / v HSA, and 30 mM trehalose.

[0190] In some embodiments, effector cells engineered to express BCMA-targeting CAR polypeptides as described herein are formulated in an equilibrium crystalloid solution such as Plasma-Lyte. In some embodiments, effector cells are formulated in Plasma-Lyte A. In some embodiments, the concentration of Plasma-Lyte A is 20–60%. In some embodiments, the concentration of Plasma-Lyte A is 40%. In some embodiments, effector cells are further formulated in a stabilizer such as serum albumin. In exemplary embodiments, the serum albumin is human serum albumin (HSA) at a concentration of 5–20%. In some embodiments, the concentration of HSA is 10%. In some embodiments, effector cells are further formulated in a stabilizer such as trehalose. In exemplary embodiments, the concentration of trehalose is 5 mM–50 mM. In some embodiments, the concentration of trehalose is 30 mM. In some embodiments, effector cells are formulated in a frozen medium. In some embodiments, the freezing medium is CS10. In some embodiments, the concentration of CS10 is 40% to 60%. In some embodiments, the concentration of CS10 is 50%.

[0191] As a non-limiting example, the freezing medium consists of 50% CS10, 40% (v / v) Plasma-Lyte A, 10% HSA, and 30 mM trehalose dihydrate.

[0192] In some embodiments, the composition comprises an effective amount of immunoeffector cells engineered to express BCMA-targeting CAR polypeptides as described herein, either alone or in combination with one or more therapeutic agents. Thus, the CAR-expressing immunoeffector cell composition may be administered alone or in combination with other known cancer treatments such as radiotherapy, chemotherapy, transplantation, immunotherapy, hormone therapy, or photodynamic therapy.

[0193] Packages and kits Any of the compositions described herein may be included in a package and / or kit for clinical use. In non-limiting examples, cells, reagents for producing cells, vectors, and reagents and / or components for producing vectors may be included in the kit. In certain embodiments, NK cells may be included in the kit and may or may not express BCMA-targeting CARs including the DAP10 costimulatory domain, optionally cytokines, or optionally suicide genes. Such a kit may or may not have one or more reagents for manipulating the cells. Such reagents include, for example, small molecules, proteins, nucleic acids, antibodies, buffers, primers, nucleotides, salts, and / or combinations thereof. One or more CARs, suicide gene products, and / or nucleotides encoding cytokines may be included in the kit. Cytokines, or proteins such as antibodies including monoclonal antibodies, may be included in the kit. Nucleotides encoding components of the manipulated CAR receptor may be included in the kit, along with reagents for producing them.

[0194] How to use In another aspect of the present invention, methods for using BCMA-targeting CARs, cells containing the same, and compositions are provided.

[0195] The BCMA-targeting CARs and genetically engineered immune effector cells expressing BCMA-targeting CARs described herein provide an improved method of adoptive immunotherapy for use in the prevention, treatment, and improvement of B-cell-related conditions. The BCMA-targeting CARs and genetically engineered immune effector cells expressing BCMA-targeting CARs described herein provide an improved method of immunotherapy for use in increasing the cytotoxicity of target cancer cells or for use in reducing the number of target cancer cells.

[0196] In some embodiments, T cells or NK cells are genetically engineered to express the CAR polypeptides of the invention that target cancer cells expressing BCMA, and a type of cell therapy is provided in which the T cells and / or NK cells are infused into a recipient who needs them. The infused cells can kill cells that cause disease in the recipient, such as tumor cells. The T cells and NK cells expressing the CAR targeting BCMA of the invention can undergo robust in vivo cell proliferation and can survive for a long period of time.

[0197] In some embodiments, a method of treating cancer, such as BCMA-related cancer, in a subject in need of treatment is provided; the method includes administering to the subject in need of treatment a therapeutically effective amount of a composition as described in this disclosure. The therapeutically effective amount of a composition containing genetically engineered therapeutic cells may vary depending on factors such as the disease state, age, gender, and weight of the individual, as well as the ability of the stem cells and progenitor cells to induce a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects exceed the toxic or detrimental effects of the virus or transduced therapeutic cells.

[0198] According to the present invention, a pharmaceutical composition containing immune effector cells (e.g., NK cells) contemplated herein is generally described as being administered at a dosage of 10 4 ~10 10 cells per kg of body weight, or 10 6 ~10 8 cells per kg of body weight, preferably 10 5 ~10 6 cells per kg of body weight (including all integer values within those ranges). For example, in some embodiments, a pharmaceutical composition containing NK cells may be administered at a dosage of 1×10 8 cells. In some embodiments, a pharmaceutical composition containing NK cells may be administered at a dosage of 2×10 8 cells. In some embodiments, a pharmaceutical composition containing NK cells may be administered at a dosage of 3×10 8It may be administered in cellular doses. In some embodiments, the pharmaceutical composition containing NK cells is 4 × 10 8 It may be administered in cellular doses. In some embodiments, the pharmaceutical composition containing NK cells is 5 × 10 8 It may be administered in cellular doses. In some embodiments, the pharmaceutical composition containing NK cells is 6 × 10 8 It may be administered in cellular doses. In some embodiments, the pharmaceutical composition containing NK cells is 7 × 10 8 It may be administered in cellular doses. In some embodiments, the pharmaceutical composition containing NK cells is 8 × 10 8 It may be administered in cellular doses. In some embodiments, the pharmaceutical composition containing NK cells is 9 × 10 8 It may be administered in cellular doses. In some embodiments, the pharmaceutical composition containing NK cells is 1.0 × 10⁻⁶ 9 It may be administered in cellular doses. In some embodiments, the pharmaceutical composition containing NK cells is 1.1 × 10⁻⁶ 9 It may be administered in cellular doses. In some embodiments, the pharmaceutical composition containing NK cells is 1.2 × 10⁻¹⁶ 9 It may be administered in doses of cells. In some embodiments, the pharmaceutical composition containing NK cells is 1.3 × 10 9 It may be administered in cellular doses. In some embodiments, the pharmaceutical composition containing NK cells is 1.4 × 10⁻⁶ 9 It may be administered in cellular doses. In some embodiments, the pharmaceutical composition containing NK cells is 1.5 × 10⁻⁶ 9 It may be administered in cellular doses. In some embodiments, the pharmaceutical composition containing NK cells is 1.6 × 10⁻⁶ 9 It may be administered in cellular doses. In some embodiments, the pharmaceutical composition containing NK cells is 1.7 × 10⁻⁶ 9 It may be administered in cellular doses. In some embodiments, the pharmaceutical composition containing NK cells is 1.8 × 10⁻⁶ 9 It may be administered in cellular doses. In some embodiments, the pharmaceutical composition containing NK cells is 1.9 × 10⁻⁶ 9 It may be administered in doses of cells. In some embodiments, the pharmaceutical composition containing NK cells is 2.0 × 109 It may be administered at a dosage of cells. The number of cells will depend on the ultimate use for which the composition is intended, such as the type of cells included in the composition. For the uses provided herein, the cells are generally at a volume of 1 liter or less, 950 mL or less, 900 mL or less, 850 mL or less, 800 mL or less, 750 mL or less, 700 mL or less, 650 mL or less, 600 mL or less, 500 mL or less, and even 250 mL or 100 mL or less. Thus, the desired cell density is usually greater than 10 6 cells / ml, and generally greater than 10 7 cells / ml, and generally greater than 10 8 cells / ml, generally greater than 10 9 or more. The clinically appropriate number of immune cells can be distributed over multiple injections that cumulatively reach 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 cells or more. In some embodiments, particularly where all of the injected cells are redirected to a specific target antigen, fewer numbers of cells in the range of 10 6 to 10 9 / kilogram (from about 6×10 7 to 6×10 10 per patient) may be administered. The composition may be administered multiple times at dosages within these ranges.

[0199] In some embodiments, the immune effector cells expressing a CAR targeting BCMA of the present invention, such as T cells, are at least 0.1×10 5 cells, at least 0.5×10 5 cells, at least 1×10 5 cells, at least 5×10 5 cells, at least 1×10 6 cells, at least 0.5×10 7 cells, at least 1×10 7 cells, at least 0.5×10 8 cells, at least 1×108 cells, at least 0.5×10 9 cells, at least 1×10 9 cells, at least 2×10 9 cells, at least 3×10 9 cells, at least 4×10 9 cells, at least 5×10 9 cells, or at least 1×10 10 cells, and is administered to a subject in need thereof.

[0200] In some embodiments, immune effector cells expressing a CAR targeting BCMA of the present invention, such as NK cells, are at least 0.1×10 5 cells, at least 0.5×10 5 cells, at least 1×10 5 cells, at least 5×10 5 cells, at least 1×10 6 cells, at least 0.5×10 7 cells, at least 1×10 7 cells, at least 0.5×10 8 cells, at least 1×10 8 cells, at least 0.5×10 9 cells, at least 1×10 9 cells, at least 2×10 9 cells, at least 3×10 9 cells, at least 4×10 9 cells, at least 5×10 9 cells, or at least 1×10 10 cells, and is administered to a subject in need thereof.

[0201] As a non-limiting example, NK cells engineered to express a CAR targeting BCMA of the present invention are about 1×10 6 ~1×10 10 or about 5×10 6 ~about 5×10 9 or about 1×10 7 ~3×10 9 or about 2×10 7 ~5×10 8 or about 4×10 7~1.0×10 9 , or approximately 1 x 10 8 ~1.5×10 9 It is administered in a dose of 1 × 10⁶ living cells. In some embodiments, NK cells engineered to express BCMA-targeting CARs as described herein are administered in a dose of 1 × 10⁶. 8 In the dose of live cells, or 5 × 10 8 In the dose of live cells, or 1.5 × 10 9 It is administered in doses of living cells.

[0202] In some embodiments, the amount of immune effector cells (e.g., T cells and NK cells) expressed in the BCMA-targeting CAR of the present invention and administered to the target is at least 0.1 × 10¹⁶ per kg of body weight. 4 Cells, at least 0.5 × 10⁶ per kg of body weight 4 Cells, at least 1 × 10⁶ cells per kg of body weight 4 Cells, at least 5 × 10⁶ cells per kg of body weight 4 Cells, at least 1 × 10⁶ cells per kg of body weight 5 Cells, at least 0.5 × 10⁶ per kg of body weight 6 Cells, at least 1 × 10⁶ cells per kg of body weight 6 Cells, at least 1.5 × 10⁶ cells per kg of body weight. 6 Cells, at least 2.0 × 10⁶ per kg of body weight 6 Cells, at least 3.0 × 10⁶ per kg of body weight 6 Cells, at least 4.0 × 10⁶ per kg of body weight 6 Cells, at least 5.0 × 10⁶ per kg of body weight 6 Cells, at least 6.0 × 10⁶ per kg of body weight 6 Cells, at least 7.0 × 10⁶ per kg of body weight 6 cells, 8.0×10 6 Cells, at least 9.0 × 10⁶ per kg of body weight 6 Cells, at least 6.0 × 10⁶ per kg of body weight 6 Cells, at least 7.0 × 10⁶ per kg of body weight 6 Cells, 8.0 × 10⁶ per kg of body weight 6 cells, 9.0×10 6Cells, at least 0.5 × 10⁶ per kg of body weight 7 Cells, at least 1 × 10⁶ cells per kg of body weight 7 Cells, at least 0.5 × 10⁶ per kg of body weight 8 Cells, at least 1 × 10⁶ cells per kg of body weight 8 Cells, at least 2 × 10⁶ cells per kg of body weight 8 Cells, at least 3 × 10⁶ cells per kg of body weight 8 Cells, at least 4 × 10⁶ cells per kg of body weight 8 Cells, at least 5 × 10⁶ cells per kg of body weight 8 Cells, at least 1 × 10⁶ per kg 9 Cells, or at least 1 × 10⁶ cells per kg of body weight. 10 These are cells. In some embodiments, the amount of immune effector cells (e.g., T cells and NK cells) expressed in the BCMA-targeting CAR of the present invention and administered to the target is at least 1.5 × 10¹⁶ cells per kg of body weight. 6 These are cells. In some embodiments, the amount of immune effector cells (e.g., T cells and NK cells) expressed in the CAR that targets the BCMA of the present invention and administered to the target is at least 8.0 × 10¹⁶ cells per kg of body weight. 6 These are cells. In some embodiments, the amount of immune effector cells (e.g., T cells and NK cells) expressed in the BCMA-targeting CAR of the present invention and administered to the target is at least 2.5 × 10¹⁶ cells per kg of body weight. 7 These are cells. In certain embodiments, there are approximately 1 × 10⁶ cells per kg of body weight. 6 NK cells per kg of body weight: approximately 1 x 10⁶ 8 Approximately 2 x 10⁶ NK cells per kg of body weight 6 NK cells per kg of body weight: approximately 0.9 x 10⁶ 8 Approximately 3 x 10¹⁶ NK cells per kg of body weight 6 NK cells per kg of body weight: approximately 0.8 x 10⁶ 8 Approximately 4 x 10⁶ NK cells per kg of body weight 6 NK cells per kg of body weight: approximately 0.7 × 10⁶ 8 Approximately 5 x 10⁶ NK cells per kg of body weight 6 NK cells per kg of body weight: approximately 0.6 × 10⁶8 NK cells, or approximately 5 x 10⁶ cells per kg of body weight. 6 Each NK cell is approximately 0.5 × 10⁻⁶ 8 Individual NK cells are administered to the target.

[0203] It is recognized that multiple doses of the compositions intended herein may be required to influence the desired therapeutic effect. For example, the compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more, over periods of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years, or over the lifetime of the subject requiring treatment.

[0204] The compositions of the present invention may be administered by any convenient method, including by aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. In preferred embodiments, the compositions are administered parenterally. When used herein, the terms “parenteral administration” and “administered parenterally” refer to, but are not limited to, methods of administration other than enteral and topical administration, usually by injection, including, but not limited to, injections and infusions of intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intratracheal, subcutaneous, subepidermal, intraarticular, subarachnoid, intraspinal, and intrasternal methods. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection. Intravenous administration is preferred for the compositions of the present invention.

[0205] Therefore, subjects administered an effective amount of the composition enhance the cellular immune response to B cell-related conditions in the subjects. The immune response may include cytotoxic T cell-mediated cellular immune responses and helper T cell responses, which are capable of killing infected cells. A humoral immune response, primarily mediated by helper T cells that can activate B cells and lead to antibody production, may also be induced.

[0206] Therapeutic applications In some embodiments, BCMA-targeting CAR polypeptides, BCMA-CAR-containing cells, and compositions of the present invention may be used to treat diseases associated with BCMA expression. Diseases associated with BCMA expression include, but are not limited to, proliferative disorders such as cancer or malignant tumors, or precancerous conditions such as myelodysplasia, myelodysplastic syndromes, or preleukemia; or diseases or conditions associated with cells expressing BCMA (e.g., wild-type or mutant BCMA), including non-cancer-related indications associated with cells expressing BCMA (e.g., wild-type or mutant BCMA). For example, diseases associated with BCMA expression may include conditions associated with cells that previously expressed BCMA but do not currently express BCMA due to downregulation of BCMA expression resulting from treatment with BCMA-targeting molecules, such as BCMA inhibitors described herein. In one embodiment, cancer associated with BCMA (e.g., wild-type or mutant BCMA) expression is hematological cancer. In one embodiment, hematological cancer is leukemia or lymphoma. In one embodiment, cancers associated with the expression of BCMA (e.g., wild-type or mutant BCMA) are malignancies of differentiated plasma B cells. In one embodiment, cancers associated with the expression of BCMA (e.g., wild-type or mutant BCMA) include, but are not limited to, one or more acute leukemias, including, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), and acute lymphoblastic leukemia (ALL); and cancers and malignancies including, but are not limited to, one or more chronic leukemias, including, but are not limited to, chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL).Additional cancers or hematological disorders associated with the expression of BMCA (e.g., wild-type or variant BCMA) include, but are not limited to, B-cell prelymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasms, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative states, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, Waldenström macroglobulinemia, and “preleukemia,” a diverse group of hematological disorders linked by inefficient production (or dysplasia) of myeloid blood cells.

[0207] In some embodiments, the cancer is multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, or glioblastoma. In some embodiments, diseases associated with the expression of BCMA include plasma cell proliferation disorders, e.g., asymptomatic myeloma (sacral multiple myeloma or slowly progressive myeloma), monoclonal immunoglobulinemia of unknown significance (MGUS), Waldenström macroglobulinemia, plasmacytoma (e.g., plasma cell disease, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloid light chain amyloidosis, and POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome). Further diseases associated with the expression of BCMA (e.g., wild-type or mutant BCMA) include, for example, atypical and / or nonclassical cancers, malignancies, precancerous conditions or proliferative disorders associated with the expression of BCMA (e.g., wild-type or mutant BCMA), such as, but not limited to, the cancers described herein, such as prostate cancer (e.g., castration-resistant or treatment-resistant prostate cancer, or metastatic prostate cancer), pancreatic cancer, or lung cancer.

[0208] In some embodiments, the NK cells and compositions containing the BCMA-targeting CAR(s) of the present invention may be administered to subjects with multiple myeloma who have not previously received anti-BCMA therapy. In some embodiments, the NK cells and compositions containing the BCMA-targeting CAR(s) of the present invention may be administered to subjects with multiple myeloma who have previously received anti-BCMA therapy. In some embodiments, the NK cells and compositions containing the BCMA-targeting CAR(s) of the present invention may be administered to subjects who have had at least one previous treatment for multiple myeloma. In some embodiments, the NK cells and compositions containing the BCMA-targeting CAR(s) of the present invention may be administered to subjects who have had at least two previous treatments for multiple myeloma. In some embodiments, the NK cells and compositions containing the BCMA-targeting CAR(s) of the present invention may be administered to subjects who have had at least three previous treatments for multiple myeloma. For example, subjects with multiple myeloma may be refractory to immunomodulators. For example, subjects with multiple myeloma may be refractory to proteasome inhibitors. For example, subjects with multiple myeloma may be refractory to anti-CD38 antibodies. In some embodiments, the prior anti-BCMA therapy is a therapy that administers to an individual BCMA-targeting chimeric antigen receptor (CAR) T cells, BCMA-targeting antibody-drug conjugates, and / or BCMA-targeting antibodies. In one embodiment, the prior anti-BCMA therapy is BCMA-targeting chimeric antigen receptor (CAR) T cell therapy.

[0209] Non-cancer-related conditions associated with BCMA (e.g., wild-type or variant BCMA) include viral infections; e.g., HIV; fungal infections; e.g., C. neof ormans; autoimmune diseases; e.g., rheumatoid arthritis, systemic lupus erythematosus (SLE or lupus), pemphigus vulgaris, and Sjögren's syndrome; inflammatory bowel disease, ulcerative colitis; graft-associated allospecific immune disorders related to mucosal immunity; and undesirable immune responses to humoral biological agents (e.g., factor VIII). In various embodiments, non-cancer-related indications associated with BCMA expression include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation.

[0210] Preferred cancers to be treated by the methods described herein include B-cell malignancies, such as multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma. In some embodiments, multiple myeloma may be treated using the BCMA-targeting CAR polypeptide, BCMA-CAR-containing cells, and compositions of the present invention. In some embodiments, relapsed multiple myeloma may be treated using the BCMA-targeting CAR polypeptide, BCMA-CAR-containing cells, and compositions of the present invention. In some embodiments, refractory multiple myeloma may be treated using the BCMA-targeting CAR polypeptide, BCMA-CAR-containing cells, and compositions of the present invention.

[0211] Combination therapy BCMA-targeting CARs and immunoeffector cells expressing them may be used in combination with other known drugs and therapies. BCMA-targeting CAR therapy and at least one additional therapeutic agent may be administered simultaneously, in the same or different compositions, or sequentially. In the case of sequential administration, cells expressing the CARs described herein may be administered first, followed by the additional agent, or the order of administration may be reversed. Administration may occur at intervals ranging from simultaneous to several minutes, several days, several weeks, or several months.

[0212] In some embodiments, additional therapy involves the administration of side effect limiting agents (e.g., drugs intended to reduce the occurrence and / or severity of side effects of the treatment, such as antiemetics).

[0213] In some embodiments, the additional therapy may be another specific anti-cancer therapy such as radiotherapy, surgery (e.g., tumor resection and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, hormone therapy, oncolytic viruses, or a combination of the foregoing.

[0214] A wide variety of chemotherapeutic agents can be used in combination with the composition of the present invention. "Chemotherapeutic agent" is used to refer to a compound or composition administered in the treatment of cancer.

[0215] Additional immunotherapies may be used in combination with or in conjunction with the compositions and methods described herein. Examples of immunotherapeutic agents include antibodies, antibody-drug conjugates, cancer vaccines, immune effector cells, and immune checkpoint inhibitors.

[0216] The compositions and methods of the present invention may be used in combination with surgery. Approximately 60% of individuals with cancer undergo some form of surgery, including preventive, diagnostic, or staging, curative, and palliative surgeries. Post- and / or pre-operatively, patients who require it may be treated with immune effector cells.

[0217] In some embodiments, other agents may be used in combination with specific embodiments of the present invention to improve the therapeutic effect of the treatment. Exemplary therapeutic agents that can be combined with the compositions of the present invention include small molecule enzyme inhibitors, antitransfer agents, cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapy agents, therapeutic antibodies, or other activators and adjuvants.

[0218] In some embodiments, known agents and treatments may be administered prior to the administration of BCMA-targeting CARs and immune effector cells expressing them (e.g., NK cells). In some exemplary embodiments, lymphocyte depletion chemotherapy is performed prior to BCMA-targeting CAR therapy. Fludarabine is an exemplary lymphocyte depletion chemotherapy agent. Exemplary lymphocyte depletion agents are disclosed, among others, in WO2010046917A2 and WO2003099007A1, which are incorporated in whole by reference. Typically, fludarabine phosphate is administered at a dose of 25 mg / m² per day. 2 The single dose is administered over 5, 4, 3, or 2 consecutive days. Maximum 30 mg / m² per day over 5, 4, 3, and / or 2 consecutive days. 2 Doses up to the following may be used. Before administering BCMA-targeting CARs and immunoeffector cells expressing them (e.g., NK cells), fludarabine phosphate may be administered for 5 consecutive days at a rate of 25 mg / m² per day. 2 It may be administered as a single dose. Before administering BCMA-targeting CARs and immunoeffector cells expressing them (e.g., NK cells), fludarabine phosphate may be administered for four consecutive days at a rate of 25 mg / m² per day. 2 It may be administered as a single dose. Before administering BCMA-targeting CARs and immunoeffector cells expressing them (e.g., NK cells), fludarabine phosphate may be administered for three consecutive days at a rate of 25 mg / m² per day. 2 It may be administered as a single dose.

[0219] In some embodiments, subjects are treated with cyclophosphamide before receiving BCMA-targeted CAR therapy. Subjects may be administered cyclophosphamide in a single dose of 300 mg / m2 of BSA per day five, four, or three days before receiving BCMA-targeted CAR therapy. For example, subjects may be administered cyclophosphamide in a single dose of 300 mg / m2 of BSA per day five, four, or three days before being administered NK cells expressing the anti-BCMA CAR described herein. [Examples]

[0220] The following examples are included to demonstrate specific embodiments of the present invention. Those skilled in the art should understand that the technologies disclosed in the following examples represent technologies that have been discovered by the inventors to function well in the implementation of the present invention, and thus can be considered to constitute specific modes for their implementation. However, those skilled in the art should fully understand that, in light of this disclosure, many changes can be made to the disclosed specific embodiments, and still obtain the same or similar results without departing from the spirit and scope of the present invention.

[0221] Example 1: Construction of Chimeric Antigen Receptor This example shows a BCMA CAR construct used to reduce tumor burden. As described in Figure 1, in Examples 1-9, constructs containing nucleotides encoding a BCMA DAP10 CAR, which includes an anti-BCMA specific binding factor, a CD28 hinge, a CD28 transmembrane domain, a DAP10 co-stimulatory domain, an IL-15 cytokine (such as soluble IL-15), etc., were used. The amino acid sequences expressed from the constructs, and the nucleic acid sequences encoding the amino acid sequences are listed in Table 4. These sequences were used in Examples 2-9. [Table 4-1] [Table 4-2]

[0222] Example 2: In Vivo Efficacy of BCMA CAR Construct Against MM1S Tumor This example shows the efficacy of BCMA-DAP10-CAR contained in SEQ ID NO: 29 and BCMA-CD28-CAR contained in SEQ ID NO: 45, which were expressed in CB-NK cells, against MM1S tumor.

[0223] Female NSG mice aged 10-12 weeks were whole-body irradiated with 150 cGy 24 hours before tumor inoculation. MM.1S-ffluc-MDA cells were prepared in PBS suspension at a concentration of 2.5 × 10^6 cells / ml, and 0.5 × 10^6 cells / animal were intravenously inoculated. Bioluminescence imaging was taken 1 day before administration and 6 days after tumor inoculation, and animals were randomized into groups of 4 animals each based on total flux. The tumor was administered to the animals 7 days after inoculation. CAR-expressing NK cells (i.e., CAR NK cells) at relevant concentrations were resuspended in PBS and transported to the animal housing facility in small portions on ice to ensure timely infusion to the animals while maintaining the viability of CAR-expressing NK cells. Bioluminescence imaging was performed weekly with Xenogen IVIS to monitor tumor progression. Body weight was obtained three times a week, and any signs of toxicity were monitored along with clinical observation. Microsampling (via submandibular blood collection) was performed weekly for cellular dynamics analysis to quantify in vivo CAR NK cell proliferation by either ddPCR or flow cytometry. Autopsies were performed on animals from the study of interest to obtain various tissues for toxicological / pathological evaluation, based on humanitarian or experimental evaluation criteria.

[0224] For a single 10M dose of CAR-expressing NK cells, both BCMA-DAP10-CAR and BCMA-CD28-CAR functioned equally well. Of the two 10M doses of CAR-expressing NK cells, BCMA-DAP10-CAR outperformed BCMA-CD28-CAR, with three mice surviving at day 49 compared to only one with BCMA-CD28-CAR (Figure 2A). All CAR-transduced NK cells showed benefit compared to tumor alone (Figure 2B).

[0225] CAR-NK cell production For CAR-NK cell production, research-grade umbilical cord blood (CB) units were obtained from the MD Anderson Cancer Center Cord Blood Bank. CB mononuclear cells were isolated from frozen CB units by Ficoll density gradient centrifugation. In vitro proliferation of umbilical cord blood-derived NK cells (CB-NK cells) was performed using uAPC stimulation on day 0, in addition to supplying IL-2 every two days. On day 6, cells were transduced by spinoculation with RD114 virus. Cells were stimulated with a second dose of uAPC on day 8 or 9, supplied with IL-2 every two days until use in in vivo or in vitro testing on day 15, or cryopreserved on day 21 for later use.

[0226] Example 3: In vitro efficacy of BCMA CAR construct against multiple tumor lines This example demonstrates the efficacy of BCMA-DAP10-CAR, included in SEQ ID NO: 29, and BCMA-CD28-CAR, included in SEQ ID NO: 45, expressed in CB-NK cells, against various tumor lines.

[0227] MM1S-Luc, RPMI-8226-Luc, JJN3-Luc, and JJN3-Luc BCMA KO cells were washed once with PBS and incubated at 37°C for 20 minutes with cell tracer deep red dye (Invitrogen#C34565) in PBS at a cell density of 2.5 million / ml. At the end of incubation, 20 ml of cell culture medium was added to the cells, and the cells were centrifuged at 500 g for 5 minutes, the supernatant was removed, and the cells were washed again with the corresponding cell culture medium. The cells were then resuspended in culture medium at 250,000 / ml, and 30 μl of cells were added to each well of a v-bottom 384-well assay plate (Greiner, catalog: 781280). The cells were incubated in a cell culture incubator at 37°C with 5% CO2 for 1-2 hours. Fresh effector cells were harvested on day 15, washed once in cytokine-free NK cell medium (CellGenix GMP SCGM containing 10% HI-FBS and 2 mM glutamine), and then 10 μl of effector cells were added to assay plates at various E:T ratios. Target cells and effector wells were co-cultured for 20 hours, then the cells were centrifuged and the supernatant was collected for cytokine release assays. Cells were incubated at 37°C for 1 hour in a cell culture incubator with 10 μl of caspase 3 / 7 reagent (Intellicyt, catalog: 91035) diluted 1:500 in the corresponding target cell medium, and then subjected to FACS analysis using either Sartorius iQue3 or Sartorius iQue Screener Plus. Cytotoxicity of effector cells was reported using the percentage of caspase 3 / 7-positive target cells.

[0228] In vitro killing activity was generally superior with the BCMA-DAP10-CAR construct across multiple tumor cell lines (shown in Figure 3).

[0229] Example 4: Ability of BCMA CAR NK cells to kill tumor cells in vitro after multiple restimulations. This example demonstrates the efficacy of BCMA-DAP10-CAR, included in SEQ ID NO: 29, and BCMA-CD28-CAR, included in SEQ ID NO: 45, when expressed in CB-NK cells.

[0230] Effector cells were harvested and resuspended in SCGM (CellGenix, catalog number: 20802-0500) supplemented with 10% heat-inactivated FBS (Sigma, catalog number: F4135-500mL), 1% L-glutamine (Gibco, catalog number: 25030-081), 1% Penn Strep (Gibco, catalog number: 15140-122), and 100 IU / ml human IL-2 (Miltenyi, catalog number: 130-097-748). Effector cells were seeded in triplets in 48-well flat-bottom non-tissue culture treated plates (Corning, catalog number: 3548) at a density of 2e5 cells / well (MM1S model, Figures 4A, 4B) or 5e4 cells / well (RPMI-8226 model, Figures 5A, 5B). Target cells were pre-transduced with NucLight Red lentivirus (Sartorius, catalog number: 4476), selected using 1 μg / mL puromycin (Sigma, catalog number: P8833-10MG), resuspended in the complete medium described above, and seeded at a density of 5e4 cells / well. The plates were placed in an IncuCyte S3 (Sartorius Inc.), and quadruple readings per well were measured every 30 minutes using a 10x objective lens in both bright-field and red channels. Target cells were prepared as described above and seeded again at a density of 5e4 cells / well every 48–72 hours (MM1S model, Figures 4A, 4B) or every 48 hours (RPMI-8226, Figures 5A, 5B) for a total of nine tumor stimulation / re-antigen stimulation cycles. Cell lysis of target cells is expressed as the average number of red objects per image (Figures 4, 4A) and the area under the overall mean curve of the number of red objects (Figures 5A, 5B).

[0231] BCMA-DAP10-CAR showed superior efficacy compared to BCMA-CD28-CAR against repeated stimulation by MM1S, and also demonstrated significant control of RPMI-8226 tumor cells during repeated stimulation.

[0232] Example 5: In vivo efficacy of BCMA CAR construct against RPMI-8226 tumors This example demonstrates the in vivo efficacy of BCMA-DAP10-CAR, included in SEQ ID NO: 29, and BCMA-CD28-CAR, included in SEQ ID NO: 45, against RPMI-8226 tumors in CB-NK cells.

[0233] Female NSG mice aged 10–12 weeks were whole-body irradiated with 150 cGy 24 hours prior to tumor inoculation. RPMI-8226-luc cells were prepared in PBS suspension at a concentration of 2.5 × 10^6 cells / ml, and 0.5 × 10^6 cells / animal were intravenously inoculated. Bioluminescence imaging was taken 1 day before administration and 6 days after tumor inoculation, and animals were randomized into groups of 4 animals each based on total flux. The tumor was administered to the animals 7 days after inoculation. CAR-expressing NK cells at relevant concentrations were resuspended in PBS and transported to the animal housing facility in small portions on ice to ensure timely infusion while maintaining the viability of CAR-expressing NK cells. Bioluminescence imaging was performed weekly with Xenogen IVIS to monitor tumor progression. Body weight was obtained three times a week, and any signs of toxicity were monitored along with clinical observation. Microsampling (via submandibular blood collection) was performed weekly for cellular dynamics analysis to quantify in vivo CAR NK cell proliferation by either ddPCR or flow cytometry. Autopsies were performed on animals from the study of interest to obtain various tissues for toxicological / pathological evaluation, based on humanitarian or experimental evaluation criteria.

[0234] BCMA-DAP10-CAR demonstrated superior efficacy, with twice as many mice surviving at day 63 and one mouse still alive at day 75, compared to the control group where all mice died from tumor growth by day 63 (Figure 6).

[0235] Example 6: In vivo proliferation of BCMA CAR NK cells in the RPMI-8226 tumor model This example demonstrates the proliferative efficacy of CB-NK cells that have been engineered to express BCMA-DAP10-CAR (SEQ ID NO: 29) and BCMA-CD28-CAR (SEQ ID NO: 45) in response to a tumor in the RPMI-8226 tumor model.

[0236] Female NSG mice aged 10–12 weeks were whole-body irradiated with 150 cGy 24 hours prior to tumor inoculation. RPMI-8226-luc cells were prepared in PBS suspension at a concentration of 2.5 × 10^6 cells / ml, and 0.5 × 10^6 cells / animal were intravenously inoculated. Bioluminescence imaging was taken 1 day before administration and 6 days after tumor inoculation, and animals were randomized into groups of 4 animals each based on total flux. The tumor was administered to the animals 7 days after inoculation. CAR NK cells at relevant concentrations were resuspended in PBS and transported to the animal housing facility in small portions on ice to ensure timely infusion to the animals while maintaining CAR NK cell viability. Bioluminescence imaging was performed weekly with Xenogen IVIS to monitor tumor progression. Body weight was obtained three times a week, and any signs of toxicity were monitored along with clinical observation. Microsampling (via submandibular blood collection) was performed weekly for cellular dynamics analysis to quantify in vivo CAR NK cell proliferation by either ddPCR or flow cytometry. Autopsies were performed on animals from the study of interest to obtain various tissues for toxicological / pathological evaluation, based on humanitarian or experimental evaluation criteria.

[0237] As shown in Figure 7, both NK cells expressing the BCMA-DAP10-CAR construct and NK cells expressing the BCMA-CD28-CAR construct proliferate in vivo in response to RPMI-8226 tumors. Furthermore, NK cells expressing the BCMA-DAP10-CAR construct proliferate with even greater efficiency than NK cells expressing the BCMA-CD28-CAR construct (Figure 7).

[0238] Example 7: In vivo efficacy of BCMA CAR in multiple doses in the RPMI-8226 tumor model This example demonstrates the efficacy of BCMA-DAP10-CAR, included in Sequence ID No. 29 and expressed in CB-NK cells, against RPMI-8226 tumors; NK cells expressing the CAR were administered in multiple doses.

[0239] Female NSG mice aged 10–12 weeks were whole-body irradiated with 150 cGy 24 hours prior to tumor inoculation. RPMI-8226-luc cells were prepared in PBS suspension at a concentration of 2.5 × 10^6 cells / ml, and 0.5 × 10^6 cells / animal were intravenously inoculated. Bioluminescence imaging was taken 1 day before administration and 6 days after tumor inoculation, and animals were randomized into groups of 4 animals each based on total flux. The tumor was administered to the animals 7 days after inoculation. CAR-expressing NK cells at relevant concentrations were resuspended in PBS and transported to the animal housing facility in small portions on ice to ensure timely infusion while maintaining the viability of CAR-expressing NK cells. Bioluminescence imaging was performed weekly with Xenogen IVIS to monitor tumor progression. Body weight was obtained three times a week, and any signs of toxicity were monitored along with clinical observation. Microsampling (via submandibular blood collection) was performed weekly for cellular dynamics analysis to quantify in vivo CAR NK cell proliferation by either ddPCR or flow cytometry. Autopsies were performed on animals from the study of interest to obtain various tissues for toxicological / pathological evaluation, based on humanitarian or experimental evaluation criteria.

[0240] BCMA-DAP10-CAR showed the highest efficacy in vivo at a dose of 3M CAR-NK cells per mouse (Figures 8A and 8B).

[0241] Example 8: In vitro efficacy of BCMA DAP10 CAR against tumor lines expressing different levels of stress ligands This example demonstrates the efficacy of BCMA-DAP10-CAR, included in SEQ ID NO: 29, and BCMA-CD28-CAR, included in SEQ ID NO: 45, when expressed in CB-NK cells.

[0242] Target cells were prepared as JJN3 (BCMA knockout), JJN3 (BCMA knockout) + MICA / MICB, JJN3 (BCMA wild-type), and JJN3 (BCMA wild-type) + MICA / MICB. They were seeded in 96-well plates at a volume of 100 μL, yielding 20,000 target cells / well. NK cells expressing CAR and control NK cells (untransduced, UTD) were prepared, yielding 1.8 × 10⁶ cells. 6 The cells were resuspended in assay medium at a concentration of cells / mL. The effector cell suspension was then diluted, and 100 μL of the dilution was added to a plate containing target cells to achieve the effector:target (E:T) ratio shown in the plots in Figures 9-11. The cells were incubated in a 37°C / 5% CO2 incubator for 20-24 hours.

[0243] After incubation, the cells were transferred to another round-bottom 96-well plate and stained. After pelletizing by centrifugation, the cells were stained for 15 minutes in the dark with eFluor-780 (diluted 1:1000 in PBS), a viability indicator that can be fixed on ice. After washing and pelletizing again, the cells were resuspended in 50 μL of human Fc block (diluted 1:10 in staining buffer) and incubated in the dark at room temperature for 10 minutes. Next, the cells were stained for 30 minutes in the dark on ice with 50 μL of fluorescence-conjugated mouse anti-human CD138 antibody. The stained cells were washed, resuspended, and recorded using an Attune flow cytometer.

[0244] The data was sent out and analyzed using FlowJo v10.6.2. CD138 was analyzed after excluding the survival indicator dye. + Target viable cells were quantified. The average number of target viable cells was determined for all control wells (target cells only, untreated). Cytotoxicity was calculated using the following formula: Cytotoxicity = 100 - (Number of target cells in test well / Average number of target cells in control well) × 100

[0245] CellTiter Glo-based cytotoxic assay MC38 parental target cells, MC38-ULBP2 High, and primary bronchial epithelial target cells were sampled in assay medium (SCGM with 10% FBS) in 2 × 10⁶ cells. 5 The solution was prepared at a concentration of cells / mL. Target cells were seeded in a volume of 100 μL, and 20,000 target cells were obtained per well in a 96-well flat-bottom plate. The cells were allowed to adhere for 12-14 hours before adding effector cells. CAR-expressing NK cells and control NK cells (untransduced, UTD) were prepared and measured at 1.8 × 10⁶. 6 The cells were resuspended in assay medium at a concentration of cells / mL. The effector cell suspension was then diluted, and 100 μL of the dilution was added to a plate containing target cells to achieve the effector:target (E:T) ratio shown in the plots in Figures 9–11. The cells were incubated in a 37°C / 5% CO2 incubator for 20–24 hours.

[0246] After incubation, the cell supernatant was aspirated. After washing with 200 μL of assay medium, each well was refilled with 100 μL of assay medium and 100 μL of CellTiter Glo solution. The cells were incubated at room temperature for approximately 7 minutes with gentle shaking. Luminescence was measured using a Tecan spark analyzer.

[0247] The average luminescence (relative luminescence units, RLU) was determined for all control wells (target cells only; untreated). Furthermore, RLU was determined for each effector cell only in the absence of target cell stimulation. Cytotoxicity was calculated using the following formula: Cytotoxicity = 100 - (RLU (test well) - RLU (effector cells only) / average RLU control) × 100

[0248] Example 9: Improvement of BCMA CAR surface expression after codon optimization This example demonstrates the surface expression of BCMA-DAP10-CAR on NK cells from four different CBU donors using constructs containing SEQ ID NOs. 25 and 55 in Table 5. In this example, the construct containing the sequence represented by SEQ ID NO. 25 enhanced the surface expression of BCMA-CAR in all four CBU donors.

[0249] A total of eight serial dilutions of the virus were performed using a 2-fold dilution. 24-well plates were pre-coated overnight with 40 μg / mL retronectin, and then blocked in complete medium at 37°C for 10 minutes before use. 1 ml of the virus dilution was added to the plate, and then centrifuged at 2000 × g at 32°C for 90 minutes. After centrifugation, the plate was aspirated and 1 × 10⁶ particles were extracted per 1 mL of medium. 6 The NK was added along with 1 ml of an appropriate virus dilution. The plate was then centrifuged at 400 x g for 5 minutes at 32°C and placed in an incubator at 37°C for 48 hours.

[0250] After incubation, cells were transferred to a v-bottom 96-well plate and stained. The cells were pelleted by centrifugation, washed with flow buffer, and then stained for 30 minutes in 1:10 Fc block with fluorescence-conjugated mouse anti-human CD3, mouse anti-human CD56, mouse anti-human NKG2D, and recombinantly generated human BCMA on ice in the dark. The stained cells were washed, resuspended in Sytox Viability Dye, and recorded using a MACSquant flow cytometer. The data was transmitted and analyzed using FlowJo v10.6.2. CD3 was analyzed after excluding viability dyes. - CD56 + BCMA + Target living cells were quantified.

[0251] Figures 12A and 12B show the surface expression of BCMA CAR using constructs containing SEQ ID NO: 55 or SEQ ID NO: 25 in four CBU donors. Cell surface expression of BCMA CAR using the construct containing SEQ ID NO: 25 resulted in higher transduction efficiency in all four umbilical cord blood NK donors compared to the BCMA-DAP10 CAR construct containing SEQ ID NO: 55. [Table 5-1] [Table 5-2]

[0252] Example 10: In vivo efficacy of codon-optimized BCMA CAR NK at low doses Female NSG mice aged 10-12 weeks were whole-body irradiated with 150 cGy 24 hours before tumor inoculation. MM.1S-ffluc-MDA cells were then administered in 2.5 × 10⁶ cells. 6 Prepare a PBS suspension at a concentration of cells / ml, then 0.5 × 10⁶ 6 Cells were intravenously inoculated into cells / animals. Bioluminescence imaging was taken one day before administration and six days after tumor inoculation, and animals were randomized into groups of five based on total flux. The cells were administered to animals seven days after tumor inoculation. NK cells expressing CAR (encoded by the nucleic acid sequence represented by SEQ ID NO: 25) at relevant concentrations (i.e., CAR NK cells) were resuspended in PBS and transported to animal housing facilities in small portions on ice to ensure timely infusion into animals while maintaining the viability of CAR-expressing NK cells. Bioluminescence imaging was performed weekly with Xenogen IVIS to monitor tumor progression. Body weight was obtained three times a week, and any signs of toxicity were monitored along with clinical observation. Microsampling (via submandibular blood collection) was performed weekly for cellular dynamics analysis to quantify in vivo CAR NK proliferation by either ddPCR or flow cytometry. Autopsies were performed on animals from the study of interest for humanitarian or experimental endpoints to obtain various tissues for toxicological / pathological evaluation.

[0253] As shown in Figure 13, CAR + NK cells were able to suppress tumor growth in two of the three groups tested.

[0254] Equal parts Any patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. While the present invention is disclosed with reference to specific embodiments, it will be apparent to those skilled in the art that other embodiments and variations of the invention can be devised without departing from the true spirit and scope of the invention. The appended claims are intended to be construed as encompassing all such embodiments and equivalent variations.

Claims

1. A polynucleotide encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a B cell maturation antigen (BCMA) binding domain, a hinge domain, a transmembrane domain, a DAP10 costimulatory domain, and at least one intracellular signaling domain.

2. The polynucleotide according to claim 1, wherein the BCMA binding region comprises a heavy chain variable region complementarity determining region (HCDR) 1 containing SYAIH (SEQ ID NO: 2), an HCDR 2 containing VTWHDGSNKYYAESVMG (SEQ ID NO: 3), and an HCDR 3 containing AKFGEPQYFQH (SEQ ID NO: 4).

3. The polynucleotide according to claim 1 or 2, wherein the BCMA binding region comprises a light chain variable region complementarity determination region (LCDR) 1 containing RASQGINNYLA (SEQ ID NO: 6), an LCDR 2 containing AASTLQS (SEQ ID NO: 7), and an LCDR 3 containing QQLKSYPFT (SEQ ID NO: 8).

4. The polynucleotide according to any one of the prior claims, wherein the BCMA-binding region includes a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97%, 98%, or 99% identical to SEQ ID NO:

1.

5. The polynucleotide according to any one of the prior claims, wherein the BCMA-binding region includes a light chain variable region (VL) comprising an amino acid sequence selected from SEQ ID NO: 5, or an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 97%, 98%, or 99% identical to SEQ ID NO:

5.

6. The BCMA binding region is i) The heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 1 and ii) The light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 5, A polynucleotide according to any one of the prior claims, comprising:

7. The BCMA binding region is the Fab fragment, F(ab') ２ A polynucleotide according to any one of the prior claims, which is a fragment, an Fv fragment, a single-chain variable fragment (scFv), a single-domain antibody, or a nanobody.

8. The polynucleotide according to claim 7, wherein the BCMA-binding region is a single-chain variable fragment (scFv).

9. The polynucleotide according to claim 8, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

20.

10. The polynucleotide according to any one of the prior claims, wherein the BCMA-binding region comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:

20.

11. The BCMA binding region is approximately 1 × 10 －６ Less than M, approximately 1 x 10 －７ Less than M, approximately 1 x 10 －８ Less than M, or approximately 1 x 10 －９ Less than M, or approximately 1 x 10 －１０ K less than M Ｄ A polynucleotide according to any one of the prior claims, which binds to BCMA.

12. The polynucleotide according to any one of the prior claims, wherein the DAP10 costimulatory domain includes the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:

24.

13. The polynucleotide according to any one of the prior claims, wherein the CAR comprises one or more additional costimulatory domains.

14. The one or more additional costimulatory domains mentioned above include OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD28, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell costimulatory molecule (ICOS), CDS gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF1.4), NKG2C, 2B4, Igα (CD79a), DAP12, Fcγ receptor, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signal transduction lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CDSα, CDSβ, 11.2β, IL2R gamma, IL7 Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, ITGAE, CD103, ITGA L, LFA-1, ITGAM, ITGAX, ITGB1, CD29, ITGB2, ICOS, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE The polynucleotide according to claim 13, selected from ligands that specifically bind to RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL, CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof, of co-stimulatory domains.

15. The polynucleotide according to any one of the prior claims, wherein the hinge domain is selected from the hinge domains and / or extracellular domains of IgG, CD8a, CD4, and CD28.

16. The polynucleotide according to claim 15, wherein the hinge domain is the hinge domain of CD28.

17. The polynucleotide according to claim 16, wherein the CD28 hinge domain comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:

21.

18. The polynucleotide according to claim 16, wherein the CD28 hinge domain comprises the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:

61.

19. The polynucleotide according to any one of the prior claims, wherein the transmembrane domain is the transmembrane domain of CD8, CD16, CD27, CD28, NKG2D, NKp44, NKp46, NKp30, NKp80, DNAM-1, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD9, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, DAP10, DAP12, or a variant thereof.

20. The polynucleotide according to claim 19, wherein the transmembrane domain is the transmembrane domain of CD28.

21. The polynucleotide according to claim 20, wherein the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:

22.

22. The polynucleotide according to any one of the prior claims, wherein the intracellular signaling domain is selected from intracellular signaling domains of CD28, CD137 (4-IBB), CD134 (OX40), FcRγ, FcRβ, FcεRI, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD27, CD2, CD5, CD22, CD79a, CD79b, CD66d, CD278 (ICOS), ICAM-1, LFA-1 (CD1la / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, DAP10, DAP12, or combinations thereof.

23. The polynucleotide according to claim 22, wherein the intracellular signaling domain is a CD3ζ signaling domain.

24. The polynucleotide according to claim 23, wherein the intracellular CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:

23.

25. The polynucleotide according to any one of the prior claims, wherein the CAR further comprises a signal peptide.

26. The polynucleotide according to claim 25, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO:

59.

27. The polynucleotide according to any one of the prior claims, wherein the polynucleotide further comprises a polynucleotide encoding one or more additional polypeptides of interest.

28. The polynucleotide according to claim 27, comprising a further polynucleotide or a variant thereof that encodes IL-15.

29. The polynucleotide according to claim 28, wherein IL-15 comprises the amino acid sequence of SEQ ID NO:

26.

30. The polynucleotide according to any one of claims 27 to 29, wherein the polynucleotide further comprises a cleavable peptide located between the CAR polypeptide and the one or more additional polypeptides.

31. The polynucleotide according to claim 30, wherein the cleavable peptide is an E2A peptide containing the amino acid sequence of SEQ ID NO:

27.

32. The polynucleotide according to any one of the prior claims, wherein the polynucleotide encodes the CAR having an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:

29.

33. The polynucleotide according to any one of claims 1 to 31, wherein the polynucleotide encodes the CAR having an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:

13.

34. The polynucleotide according to any one of claims 1 to 32, wherein the polynucleotide comprises a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least 99%, or 100% identical to SEQ ID NO:

60.

35. The polynucleotide according to any one of claims 1 to 32, wherein the polynucleotide comprises a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least 99%, or 100% identical to SEQ ID NO:

44.

36. The polynucleotide according to claim 33, wherein the polynucleotide comprises a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least 99%, or 100% identical to SEQ ID NO:

25.

37. The polynucleotide according to claim 33, wherein the polynucleotide comprises a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least 99%, or 100% identical to sequence number 55.

38. The polynucleotide according to any one of the prior claims, wherein the polynucleotide further comprises a nucleic acid sequence encoding IL-15.

39. The polynucleotide according to claim 38, wherein IL-15 comprises the amino acid sequence of SEQ ID NO:

26.

40. A polypeptide containing the amino acid sequence of SEQ ID NO:

29.

41. The polypeptide according to claim 40, wherein the polypeptide comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:

29.

42. The polypeptide according to claim 40, wherein the polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least 99%, or 100% identical to SEQ ID NO: 60 or SEQ ID NO:

44.

43. A polypeptide containing the amino acid sequence of SEQ ID NO:

13.

44. The polypeptide according to claim 43, wherein the polypeptide comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:

13.

45. The polypeptide according to claim 43, wherein the polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least 99%, or 100% identical to SEQ ID NO: 25 or SEQ ID NO:

55.

46. A vector comprising a polynucleotide according to any one of claims 1 to 39.

47. The vector according to claim 46, wherein the vector is a viral vector or a non-viral vector.

48. The vector according to claim 47, wherein the viral vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a lentiviral vector, or a retrovirus vector.

49. The vector according to claim 47, wherein the non-viral vector is a plasmid.

50. A cell comprising a polynucleotide according to any one of claims 1 to 39, a polypeptide according to any one of claims 40 to 45, or a vector according to any one of claims 46 to 49.

51. The cell according to claim 50, wherein the cell is an immune cell.

52. The cells according to claim 51, wherein the immune cells are natural killer (NK) cells, natural killer T (NKT) cells, T cells, B cells, macrophages, mesenchymal stromal cells, dendritic cells, tumor-infiltrating lymphocytes (TILs), cytotoxic T lymphocytes (CTLs), or any combination thereof.

53. The cell according to claim 52, wherein the immune cell is an NK cell.

54. The cells according to claim 53, wherein the NK cells are derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, hematopoietic stem cells, bone marrow, cell lines, or mixtures thereof.

55. The cell according to claim 52, wherein the immune cell is a T cell.

56. The cell according to claim 55, wherein the T cell is a mature T cell, a T helper cell, a tumor-infiltrating T cell, an autologous T cell, an engineered autologous T cell (eACT), an allogeneic T cell, or any combination thereof.

57. The cells according to any one of claims 50 to 56, wherein the cells are prepared for the treatment of cancer.

58. A composition comprising a polynucleotide according to any one of claims 1 to 39, a polypeptide according to any one of claims 40 to 45, a vector according to any one of claims 46 to 49, or a cell according to any one of claims 50 to 57.

59. A method for treating cancer in a subject requiring cancer treatment, comprising administering to the subject a therapeutically effective amount of a composition comprising a polynucleotide according to any one of claims 1 to 39, a polypeptide according to any one of claims 40 to 45, a vector according to any one of claims 46 to 49, cells according to any one of claims 50 to 57, or the composition according to claim 58.

60. A method for inducing an immune response to BCMA-positive cancer in a subject requiring such induction, the method comprising administering to the subject an effective amount of a composition comprising a polynucleotide according to any one of claims 1 to 39, a polypeptide according to any one of claims 40 to 45, a vector according to any one of claims 46 to 49, cells according to any one of claims 50 to 57, or the composition according to claim 58.

61. The method according to claim 59 or 60, wherein the cells are autologous, allogeneic, or a mixture thereof.

62. The cells are about 1.0×10 ６ to 1.0×10 １０ , or about 5.0×10 ６ to about 9.5×10 ８ , or about 1.0×10 ７ to 9.0×10 ８ , or about 5×10 ７ to 8.5×10 ８ , or about 1.0×10 ８ to 8.0×10 ８ , or about 2.0×10 ８ to 8.0×10 ８ , or about 1.0×10 ８ to 2.0×10 ９ and are administered in an amount of, the method according to claim 61.

63. The aforementioned cells are 1.0 × 10 ８ The method according to claim 62, administered in an amount.

64. The aforementioned cells are 5.0 × 10 ８ The method according to claim 62, administered in an amount.

65. The aforementioned cells are 1.5 × 10 ９ The method according to claim 62, administered in an amount.

66. The method according to any one of claims 59 to 65, wherein the cells are administered by injection, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, intracranially, percutaneously, subcutaneously, locally, by perfusion, or any combination thereof.

67. The method according to claim 66, wherein the cells are administered intravenously.

68. The method according to any one of claims 59 to 67, wherein the subject receives one or more additional cancer therapies, including chemotherapy, radiation, immunotherapy, cancer vaccines, and / or targeted therapy.

69. The method according to claim 68, wherein the subject receives lymphocyte depletion chemotherapy.

70. The method according to any one of claims 59 to 69, wherein the cancer is a B-cell malignant tumor, multiple myeloma, lymphoma, and / or leukemia.

71. The method according to claim 70, wherein the subject has recurrent multiple myeloma.

72. The method according to claim 70, wherein the subject has refractory multiple myeloma.

73. Use of a composition for the treatment of cancer in a subject, wherein the composition comprises a polynucleotide according to any one of claims 1 to 39, a polypeptide according to any one of claims 40 to 45, a vector according to any one of claims 46 to 49, or a cell according to any one of claims 50 to 57.

74. A composition for use in a method of treating cancer in a subject requiring cancer treatment, comprising a polynucleotide according to any one of claims 1 to 39, a polypeptide according to any one of claims 40 to 45, a vector according to any one of claims 46 to 49, or a cell according to any one of claims 50 to 57.

75. A polynucleotide encoding a chimeric antigen receptor comprising a sequence encoding a BCMA-specific binding domain, wherein the sequence encoding the BCMA-binding domain comprises the sequence of SEQ ID NO: 35 or SEQ ID NO:

51.

76. The polynucleotide according to claim 75, wherein the chimeric antigen receptor further comprises a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

77. The polynucleotide according to claim 76, wherein the co-stimulatory domain is derived from DAP10, and the DAP10 co-stimulatory domain comprises an amino acid sequence that is at least 85%, 95%, 99%, or 100% identical to SEQ ID NO:

24.

78. The polynucleotide according to any one of claims 76 to 77, wherein the transmembrane portion is derived from CD28.

79. The polynucleotide according to any one of claims 75 to 78, wherein the polynucleotide further comprises a sequence or variant thereof encoding IL-15.

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