BCMA chimeric antigen receptor

Abstract

Provision of BCMA chimeric antigen receptors. [Solution] This disclosure provides improved compositions for adoptive T-cell therapy for B-cell-associated conditions. This disclosure generally provides improved vectors, antibodies, antibody fragments and chimeric antigen receptors (CARs) for generating T-cell therapy and methods for using them. In particular, this disclosure provides improved anti-BCMA antibodies, antibody fragments and chimeric antigen receptors (CARs). More specifically, this disclosure provides improved human anti-BCMA antibodies, antibody fragments or CARs.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims interest under U.S. Provisional Application No. 63 / 069,784, filed on 25 August 2020, 35 U.S.C. § 119(e), and is incorporated herein by reference in its entirety.

[0002] Description regarding sequence listings The sequence listing relating to this application is provided in text format instead of as a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is BLUE-131PC_ST25.txt. The text file is 96.4KB in size, was created on August 22, 2021, and submitted electronically via EFS-Web at the same time as the specification submission.

[0003] The present invention generally relates to improved compositions and methods for treating B cell-associated conditions. More specifically, the present invention relates to improved chimeric antigen receptors (CARs) comprising human anti-B cell maturation antigen (anti-BCMA) antibodies or their antigen-binding fragments, immunoeffector cells genetically modified to express these CARs, and the use of these compositions for effectively treating B cell-associated conditions. [Background technology]

[0004] Description of related fields Several important diseases involve B lymphocytes, or B cells. Malignant transformation of B cells leads to cancers, including but not limited to lymphomas, such as multiple myeloma and non-Hodgkin lymphoma. Abnormal B cell function can also lead to the development of autoimmune diseases, including but not limited to systemic lupus erythematosus (SLE).

[0005] The majority of patients with B-cell malignancies, including non-Hodgkin lymphoma (NHL) and multiple myeloma (MM), contribute significantly to cancer mortality. The responses of B-cell malignancies to various forms of treatment are mixed. Conventional methods of treating B-cell malignancies, such as chemotherapy, radiotherapy, and antibody therapy, have yielded limited success.

[0006] Recently, attempts to treat B-cell malignancies using therapeutic antibodies or chimeric antigen receptors (CARs) targeting BCMAs have shown promising but limited success. In fact, not all patients treated with these therapies are cured or experience complete remission. One difficulty in developing such therapies is that the therapeutic effect of a given antigen-binding domain used in the therapeutic antibody or CAR is unpredictable. For example, if the antigen-binding domain is too strong, CAR T cells may be triggered to release a large amount of cytokines, potentially causing a fatal immune response known as a "cytokine storm." Conversely, if the binding of the antigen-binding domain is too weak, CAR T cells may not exhibit sufficient therapeutic effect to eliminate cancer cells. Furthermore, some CARs exhibit excessive levels of antigen-independent signaling, which can lead to T-cell dysfunction (sometimes called T-cell depletion), limiting their effectiveness.

[0007] Therefore, there remains a need to identify anti-BCMA CARs that exhibit improved efficacy while limiting antigen-independent signaling and T cell dysfunction. [Overview of the project]

[0008] This disclosure generally provides improved vectors, antibodies, antibody fragments, and chimeric antigen receptors (CARs) for generating T-cell therapies, as well as methods for using them. In particular, this disclosure provides improved anti-BCMA antibodies, antibody fragments, and chimeric antigen receptors (CARs). More specifically, this disclosure provides improved human anti-BCMA antibodies, antibody fragments, or CARs.

[0009] In one aspect of the present disclosure, a chimeric antigen receptor (CAR) is provided, comprising: a) an extracellular domain containing an anti-BCMA (B cell maturation antigen) antibody or antigen-binding fragment thereof that binds to one or more epitopes of a human BCMA polypeptide, including variable light chain CDRL1, CDRL2, and CDRL3 regions in the variable light chain amino acid sequence described in SEQ ID NOs: 7, 15, 23, 31, 39, or 47, and variable heavy chain CDRH1, CDRH2, and CDRH3 regions in the variable heavy chain amino acid sequence described in SEQ ID NOs: 8, 16, 24, 32, 40, or 48; b) a transmembrane domain; c) one or more intracellular costimulatory signaling domains; and d) a primary signaling domain.

[0010] In another embodiment, a chimeric antigen receptor (CAR) is provided, comprising: a) an extracellular domain comprising an anti-BCMA (B cell maturation antigen) antibody or antigen-binding fragment thereof that binds to one or more epitopes of a human BCMA polypeptide, comprising variable light chain CDRL1, CDRL2 and CDRL3 sequences described in SEQ ID NOs: 1-3, 9-11, 17-19, 25-27, 33-35 or 41-43, and variable heavy chain CDRH1, CDRH2 and CDRH3 sequences described in SEQ ID NOs: 4-6, 12-14, 20-22, 28-30, 36-38 or 44-46; b) a transmembrane domain; c) one or more intracellular costimulatory signaling domains; and d) a primary signaling domain.

[0011] In various embodiments, the variable light chain amino acid sequence is described in SEQ ID NO: 7, and / or the variable heavy chain amino acid sequence is described in SEQ ID NO: 8. In various embodiments, the variable light chain amino acid sequence is described in SEQ ID NO: 15, and / or the variable heavy chain amino acid sequence is described in SEQ ID NO: 16. In various embodiments, the variable light chain amino acid sequence is described in SEQ ID NO: 23, and / or the variable heavy chain amino acid sequence is described in SEQ ID NO: 24. In various embodiments, the variable light chain amino acid sequence is described in SEQ ID NO: 31, and / or the variable heavy chain amino acid sequence is described in SEQ ID NO: 32. In various embodiments, the variable light chain amino acid sequence is described in SEQ ID NO: 39, and / or the variable heavy chain amino acid sequence is described in SEQ ID NO: 40. In various embodiments, the variable light chain amino acid sequence is described in SEQ ID NO: 47, and / or the variable heavy chain amino acid sequence is described in SEQ ID NO: 48.

[0012] In various embodiments, the anti-BCMA antibody or antigen-binding fragment is selected from the group consisting of: camel Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody ("scFv"), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide-stabilized Fv protein ("dsFv"), and single-domain antibody (sdAb, nanobody). In some embodiments, the anti-BCMA antibody or antigen-binding fragment is scFv.

[0013] In various embodiments, the anti-BCMA antibody or its antigen-binding fragment comprises one or more light chain CDRs described in any one of SEQ ID NOs: 1-3 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs: 4-6. In some embodiments, the anti-BCMA antibody or its antigen-binding fragment comprises one or more light chain CDRs described in any one of SEQ ID NOs: 9-11 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs: 12-14. In a particular embodiment, the anti-BCMA antibody or its antigen-binding fragment comprises one or more light chain CDRs described in any one of SEQ ID NOs: 17-19 and / or one or more heavy chain CDR sequences described in any one of SEQ ID NOs: 20-22. In a particular embodiment, the anti-BCMA antibody or its antigen-binding fragment comprises one or more light chain CDRs described in any one of SEQ ID NOs: 25-27 and / or one or more heavy chain CDR sequences described in any one of SEQ ID NOs: 28-30. In certain embodiments, the anti-BCMA antibody or its antigen-binding fragment comprises one or more light chain CDRs described in any one of SEQ ID NOs. 33 to 35, and / or one or more heavy chain CDR sequences described in any one of SEQ ID NOs. 36 to 38. In certain embodiments, the anti-BCMA antibody or its antigen-binding fragment comprises one or more light chain CDRs described in any one of SEQ ID NOs. 41 to 43, and / or one or more heavy chain CDR sequences described in any one of SEQ ID NOs. 44 to 46.

[0014] In various embodiments, the anti-BCMA antibody or its antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in any one of SEQ ID NOs: 7, 15, 23, 31, 39, or 47, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in any one of SEQ ID NOs: 8, 16, 24, 32, 40, or 48. In some embodiments, the anti-BCMA antibody or its antigen-binding fragment includes a variable light chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 7, and / or a variable heavy chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 8. In some embodiments, the anti-BCMA antibody or its antigen-binding fragment includes a variable light chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 15, and / or a variable heavy chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 16. In some embodiments, the anti-BCMA antibody or its antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 23, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 24.In some embodiments, the anti-BCMA antibody or its antigen-binding fragment includes a variable light chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 31, and / or a variable heavy chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 32. In some embodiments, the anti-BCMA antibody or its antigen-binding fragment includes a variable light chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 39, and / or a variable heavy chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 40. In some embodiments, the anti-BCMA antibody or its antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 31, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 48.

[0015] In various embodiments, the anti-BCMA antibody or its antigen-binding fragment includes a variable light chain sequence described in any one of SEQ ID NOs: 7, 15, 23, 31, 39, or 47 and / or a variable heavy chain sequence described in any one of SEQ ID NOs: 8, 16, 24, 32, 40, or 48. In some embodiments, the anti-BCMA antibody or its antigen-binding fragment includes a variable light chain sequence described in SEQ ID NOs: 7 and / or a variable heavy chain sequence described in SEQ ID NOs: 8. In some embodiments, the anti-BCMA antibody or its antigen-binding fragment includes a variable light chain sequence described in SEQ ID NOs: 15 and / or a variable heavy chain sequence described in SEQ ID NOs: 16. In some embodiments, the anti-BCMA antibody or its antigen-binding fragment includes a variable light chain sequence described in SEQ ID NOs: 23 and / or a variable heavy chain sequence described in SEQ ID NOs: 24. In some embodiments, the anti-BCMA antibody or its antigen-binding fragment includes a variable light chain sequence described in SEQ ID NOs: 31 and / or a variable heavy chain sequence described in SEQ ID NOs: 32. In some embodiments, the anti-BCMA antibody or its antigen-binding fragment includes a variable light chain sequence described in SEQ ID NO: 39 and / or a variable heavy chain sequence described in SEQ ID NO: 40. In some embodiments, the anti-BCMA antibody or its antigen-binding fragment includes a variable light chain sequence described in SEQ ID NO: 47 and / or a variable heavy chain sequence described in SEQ ID NO: 48.

[0016] In certain embodiments, the antibody or its antigen-binding fragment is an scFv, and the variable light chain is c-terminal to that of the variable heavy chain.

[0017] In various embodiments, the CAR transmembrane domain is isolated from a polypeptide selected from the group consisting of the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. In some embodiments, the transmembrane domain is isolated from a polypeptide selected from the group consisting of: CD8α; CD4, CD45, PD1, and CD152. In some embodiments, the transmembrane domain is isolated from CD8α. In some embodiments, the transmembrane domain is isolated from PD1. In some embodiments, the transmembrane domain is isolated from CD152.

[0018] In various embodiments, one or more CAR co-stimulatory signaling domains are isolated from a co-stimulatory molecule selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. In some embodiments, one or more co-stimulatory signaling domains are isolated from a co-stimulatory molecule selected from the group consisting of CD28, CD134, and CD137. In some embodiments, one or more co-stimulatory signaling domains are isolated from CD28. In some embodiments, one or more co-stimulatory signaling domains are isolated from CD134. In some embodiments, one or more co-stimulatory signaling domains are isolated from CD137.

[0019] In various embodiments, the primary signaling domain is isolated from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In some embodiments, the primary signaling domain is isolated from CD3ζ.

[0020] In various embodiments, CAR further comprises a hinge region polypeptide. In some embodiments, the hinge region polypeptide comprises the hinge region of CD8α. In some embodiments, the hinge region polypeptide comprises the hinge region of PD1. In some embodiments, the hinge region polypeptide comprises the hinge region of CD152.

[0021] In various embodiments, the CAR further comprises a spacer region. In some embodiments, the spacer region polypeptide comprises the CH2 and CH3 regions of IgG1, IgG2, IgG4, or IgD.

[0022] In various embodiments, the CAR further comprises a signal peptide.

[0023] In various embodiments, CAR includes an amino acid sequence described in any one of SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, and 68. In some embodiments, CAR includes an amino acid sequence described in SEQ ID NO: 50. In some embodiments, CAR includes an amino acid sequence described in SEQ ID NO: 52. In some embodiments, CAR includes an amino acid sequence described in SEQ ID NO: 54. In some embodiments, CAR includes an amino acid sequence described in SEQ ID NO: 56. In some embodiments, CAR includes an amino acid sequence described in SEQ ID NO: 58. In some embodiments, CAR includes an amino acid sequence described in SEQ ID NO: 60. In some embodiments, CAR includes an amino acid sequence described in SEQ ID NO: 62. In some embodiments, CAR includes an amino acid sequence described in SEQ ID NO: 64. In some embodiments, CAR includes an amino acid sequence described in SEQ ID NO: 66. In some embodiments, CAR includes an amino acid sequence described in SEQ ID NO: 68.

[0024] In various embodiments, the CAR comprises a polypeptide containing any one amino acid sequence of the CARs envisioned herein.

[0025] In another embodiment of this disclosure, polynucleotides encoding either CARs or polypeptides as contemplated herein are provided. In some embodiments, the polynucleotide comprises a polynucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of the polynucleotide sequences described in SEQ ID NOs. 49, 51, 53, 55, 57, 59, 61, 63, 65, and 67. In some embodiments, the polynucleotide comprises a polynucleotide sequence described in any one of the SEQ ID NOs. 49, 51, 53, 55, 57, 59, 61, 63, 65, and 67.

[0026] In another aspect of this disclosure, a vector comprising any one of the polynucleotides contemplated herein is provided. In some embodiments, the vector is an expression vector. In some embodiments, the vector is an episomal vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the lentiviral vector is selected from the group consisting essentially of: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), bisnamaedi virus (VMV) virus, caprin arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and Simian immunodeficiency virus (SIV).

[0027] In certain embodiments, the vector further comprises a left (5') retroviral LTR, a Psi(Ψ) packaging signal, a central polypurine tube / DNA flap (cPPT / FLAP), a retroviral export element, a promoter operably bound to the polynucleotide described in claim 45, and a right (3') retroviral LTR. In some embodiments, the vector further comprises a heterologous polyadenylated sequence. In some embodiments, the vector further comprises a hepatitis B virus posttranscriptional regulatory element (HPRE) or a woodchuck posttranscriptional regulatory element (WPRE). In some embodiments, the promoter of the 5' LTR is replaced with a heterologous promoter. In some embodiments, the heterologous promoter is a cytomegalovirus (CMV) promoter, a Roussarcoma virus (RSV) promoter, or a Simian virus 40 (SV40) promoter. In some embodiments, the 5' LTR or 3' LTR is a lentiviral LTR. In some embodiments, the 3' LTR comprises one or more modifications. In some embodiments, the 3' LTR comprises one or more deletions. In some embodiments, the 3'LTR is a self-inactivating (SIN)LTR. In some embodiments, the polyadenylated sequence is a bovine growth hormone polyadenylated or signaling rabbit β-globin polyadenylated sequence. In some embodiments, the polynucleotide comprises an optimized Kozak sequence.In some embodiments, the promoters operably linked to the polynucleotide are selected from the group consisting of: cytomegalovirus immediate early gene promoter (CMV), elongation factor 1 alpha promoter (EF1-α), phosphoglycerate kinase-1 promoter (PGK), ubiquitin-C promoter (UBQ-C), cytomegalovirus enhancer / chicken beta-actin promoter (CAG), polyoma enhancer / herpes simplex thymidine kinase promoter (MC1), beta-actin promoter (β-ACT), Simian virus 40 promoter (SV40), and myeloproliferative sarcoma virus enhancer (MND)U3 promoter, which has a deleted negative control region and a substituted DL587REV primer binding site.

[0028] In another aspect of this disclosure, cells expressing either a CAR or a polypeptide as contemplated herein are provided. In some embodiments, the cells comprise either a polynucleotide or a vector as contemplated herein. In some embodiments, the cells are genetically engineered host cells. In some embodiments, the cells are hematopoietic cells. In some embodiments, the cells are hematopoietic stem cells or progenitor cells. In some embodiments, the cells are CD34+ hematopoietic stem or progenitor cells. In some embodiments, the cells are immune effector cells. In some embodiments, the cells are T cells. In some embodiments, the cells are CD3 + CD4 + and / or CD8 +The host cell is a cell. In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell. In some embodiments, the cell is an αβ-T cell. In some embodiments, the cell is a γδ-T cell. In some embodiments, the host cell is a natural killer (NK) cell. In some embodiments, the natural killer cell is a natural killer T (NKT) cell. In some embodiments, the host cell is a macrophage.

[0029] In various embodiments, immunoeffector cells are transduced with one of the vectors intended herein, activated and stimulated in the presence of a PI3K pathway inhibitor, thereby maintaining the proliferation of transduced immunoeffector cells compared to the proliferation of activated and stimulated transduced immunoeffector cells in the absence of a PI3K pathway inhibitor. In some embodiments, activated and stimulated immunoeffector cells in the presence of a PI3K pathway inhibitor either i) increased the expression of one or more markers selected from the group consisting of CD62L, CD127, CD197, and CD38, or ii) increased the expression of all markers CD62L, CD127, CD197, and CD38 compared to activated and stimulated immunoeffector cells in the absence of a PI3K pathway inhibitor. In some embodiments, activated and stimulated immune effector cells in the presence of a PI3K pathway inhibitor either i) increased the expression of one or more markers selected from the group consisting of CD62L, CD127, CD27, and CD8, or ii) increased all of the CD62L, CD127, CD27, and CD8 markers compared to activated and stimulated immune effector cells in the absence of a PI3K pathway inhibitor. In some embodiments, the PI3K inhibitor is ZSTK474.

[0030] In various embodiments, cells or their progeny exhibit high IFNγ release in co-culture with BCMA-expressing cells. In some embodiments, cells or their progeny exhibit similar or higher IFNγ release in co-culture with BCMA-expressing cells compared to the same cells, except that the CAR contains an extracellular domain containing mouse-derived anti-BCMA scFv. In some embodiments, the co-cultured BCMA-expressing cells are Daudi cells, HT1080.BCMA cells, and / or RPMI-8226 cells. In some embodiments, cells or their progeny exhibit high IFNγ release in co-culture with low-BCMA-expressing cells. In some embodiments, low-BCMA-expressing cells have at least 5-fold less surface BCMA expression compared to Daudi, HT1080.BCMA, and / or RPMI-8226 cells. In some embodiments, low-BCMA-expressing cells have at least 10-fold less surface BCMA expression compared to HT1080.BCMA cells. In some embodiments, low-BCMA-expressing cells have at least 10-fold less surface BCMA expression compared to RPMI-8226 cells. In some embodiments, low BCMA-expressing cells are RL and / or Toledo cells. In some embodiments, CAR T cells exhibit higher IFNγ release in co-culture with low antigen-density cells compared to the same CAR T cells, except that the CAR contains an extracellular domain containing mouse-derived anti-BCMA scFv. In some embodiments, the cells exhibit low antigen-independent signaling. In some embodiments, the cells exhibit low antigen-independent signaling compared to the same CAR T cells, except that the CAR contains an extracellular domain containing mouse-derived anti-BCMA scFv.

[0031] In another aspect of this disclosure, a composition is provided comprising any one of the cells contemplated herein and a physiologically acceptable excipient.

[0032] In another aspect of this disclosure, a method is provided for generating immune effector cells comprising a CAR or polypeptide as intended herein, comprising introducing one of the vectors as intended herein into the immune effector cells. In some embodiments, the method further comprises stimulating the immune effector cells and inducing them to proliferate by contacting the cells with antibodies that bind to CD3 and antibodies that bind to CD28, thereby generating a population of immune effector cells. In some embodiments, the immune effector cells are stimulated and induced to proliferate before the introduction of the vectors. In some embodiments, the immune effector cells comprise T lymphocytes. In various embodiments, the immune effector cells comprise NK cells. In some embodiments, in the presence of a PI3K pathway inhibitor, immune effector cells were activated and stimulated to either i) increase the expression of one or more markers selected from the group consisting of CD62L, CD127, CD197, and CD38, or ii) increase the expression of all markers CD62L, CD127, CD197, and CD38 compared to activated and stimulated immune effector cells in the absence of a PI3K pathway inhibitor. In some embodiments, in the presence of a PI3K pathway inhibitor, immune effector cells were activated and stimulated to either i) increase the expression of one or more markers selected from the group consisting of CD62L, CD127, CD27, and CD8, or ii) increase the expression of all markers CD62L, CD127, CD27, and CD8 compared to activated and stimulated immune effector cells in the absence of a PI3K pathway inhibitor. In some embodiments, the PI3K inhibitor is ZSTK474.

[0033] Another aspect of this disclosure provides a method for treating a B-cell-associated condition in a subject requiring such treatment, comprising administering a therapeutic effect amount of any one of the compositions provided herein to the subject. In some embodiments, the B-cell-associated condition is cancer. In some embodiments, the cancer is solid cancer. In some embodiments, the cancer is humoral cancer. In some embodiments, the cancer is hematological malignancy. In some embodiments, B-cell-associated conditions include multiple myeloma (MM), non-Hodgkin lymphoma (NHL), B-cell proliferation of uncertain malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, immunomodulatory disorder, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas disease, Graves' disease, Wegener's granulomatosis, polyarteritis nodosa, Sjögren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis, Goodpasture disease, Kawasaki disease, autoimmune hemolytic anemia, and rapidly progressing glomerulonephritis, heavy chain disease, primary or immune cell-associated amyloidosis, or monoclonal ganmopathies of unknown significance. In some embodiments, B-cell-associated conditions include B-cell malignancies. In some embodiments, the B-cell malignancy is multiple myeloma (MM) or non-Hodgkin lymphoma (NHL). In some embodiments, MM is selected from the group consisting of overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerosing myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma. In some embodiments, NHL is selected from the group consisting of Burkitt lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, progenitor B-lymphoblastic lymphoma, and mantle cell lymphoma. In some embodiments, the B-cell-associated condition is a plasma cell malignancy. In some embodiments, the B-cell-associated condition is an autoimmune disease. In some embodiments, the autoimmune disease is systemic lupus erythematosus. In some embodiments, the B cell-associated condition is rheumatoid arthritis.In some embodiments, the B-cell associated condition is idiopathic thrombocytopenic purpura, myasthenia gravis, or autoimmune hemolytic anemia.

[0034] Another aspect of this disclosure provides a method for improving one or more symptoms associated with cancer expressing BCMA in a subject, comprising administering to a subject an amount sufficient to improve at least one symptom associated with cancer cells expressing BCMA. In some embodiments, the one or more symptoms to be improved are selected from the group consisting of weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, lymph node dilation, abdominal distension or abdominal pain, bone or joint pain, fractures, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination.

[0035] Another aspect of the present disclosure provides a method for reducing the number of cells expressing BCMA in a subject, comprising administering to the subject an amount of the composition according to claim 116 sufficient to reduce the number of cells expressing BCMA compared to the number of cells expressing BCMA before administration.

[0036] In another aspect of this disclosure, an antibody or antigen-binding fragment thereof that binds to one or more epitopes of a human BCMA polypeptide is provided, comprising: variable light chain CDRL1, CDRL2, and CDRL3 regions within a variable light chain amino acid sequence described in SEQ ID NOs: 7, 15, 23, 31, 39, or 47, and / or variable heavy chain CDRH1, CDRH2, and CDRH3 regions within a variable heavy chain amino acid sequence described in SEQ ID NOs: 8, 16, 24, 32, 40, or 48. In some embodiments, the antibody or antigen-binding fragment comprises a variable light chain CDRL1, CDRL2, and CDRL3 sequence described in any one of SEQ ID NOs: 1-3, 9-11, 17-19, 25-27, 33-35, or 41-43, and / or variable heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs: 4-6, 12-14, 20-22, 28-30, 36-38, or 44-46.

[0037] In certain embodiments, the antibody or antigen-binding fragment is selected from the group consisting of: camel Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody ("scFv"), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide-stabilized Fv protein ("dsFv"), and single-domain antibody (sdAb, nanobody). In some embodiments, the antibody or its antigen-binding fragment is scFv.

[0038] In some embodiments, the antibody or its antigen-binding fragment comprises one or more light chain CDRs described in any one of SEQ ID NOs: 1-3 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs: 4-6. In some embodiments, the antibody or its antigen-binding fragment comprises one or more light chain CDRs described in any one of SEQ ID NOs: 9-11 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs: 12-14. In some embodiments, the antibody or its antigen-binding fragment comprises one or more light chain CDRs described in any one of SEQ ID NOs: 17-19 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs: 20-22. In some embodiments, the antibody or its antigen-binding fragment comprises one or more light chain CDRs described in any one of SEQ ID NOs: 25-27 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs: 28-30. In some embodiments, the antibody or its antigen-binding fragment comprises one or more light chain CDRs described in any one of SEQ ID NOs: 33-35 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs: 36-38. In some embodiments, the antibody or its antigen-binding fragment comprises one or more light chain CDRs described in any one of SEQ ID NOs: 41-43 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs: 44-46.

[0039] In various embodiments, the antibody or its antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in any one of SEQ ID NOs: 7, 15, 23, 31, 39, or 47, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in any one of SEQ ID NOs: 8, 16, 24, 32, 40, or 48. In some embodiments, the antibody or its antigen-binding fragment includes a variable light chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 7, and / or a variable heavy chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 8. In some embodiments, the antibody or its antigen-binding fragment includes a variable light chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 15, and / or a variable heavy chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 16. In some embodiments, the antibody or its antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 23, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 24.In some embodiments, the antibody or its antigen-binding fragment includes a variable light chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 31, and / or a variable heavy chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 32. In some embodiments, the antibody or its antigen-binding fragment includes a variable light chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 39, and / or a variable heavy chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 40. In some embodiments, the antibody or its antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 47, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 48.

[0040] In various embodiments, the antibody or its antigen-binding fragment includes a variable light chain sequence described in any one of SEQ ID NOs: 7, 15, 23, 31, 39, or 47 and / or a variable heavy chain sequence described in any one of SEQ ID NOs: 8, 16, 24, 32, 40, or 48. In some embodiments, the antibody or its antigen-binding fragment includes a variable light chain sequence described in SEQ ID NOs: 7 and / or a variable heavy chain sequence described in SEQ ID NOs: 8. In some embodiments, the antibody or its antigen-binding fragment includes a variable light chain sequence described in SEQ ID NOs: 15 and / or a variable heavy chain sequence described in SEQ ID NOs: 16. In some embodiments, the antibody or its antigen-binding fragment includes a variable light chain sequence described in SEQ ID NOs: 23 and / or a variable heavy chain sequence described in SEQ ID NOs: 24. In some embodiments, the antibody or its antigen-binding fragment includes a variable light chain sequence described in SEQ ID NOs: 31 and / or a variable heavy chain sequence described in SEQ ID NOs: 32. In some embodiments, the antibody or its antigen-binding fragment includes a variable light chain sequence described in SEQ ID NOs: 39 and / or a variable heavy chain sequence described in SEQ ID NOs: 40. In some embodiments, the antibody or its antigen-binding fragment comprises a variable light chain sequence described in SEQ ID NO: 47 and / or a variable heavy chain sequence described in SEQ ID NO: 48.

[0041] In certain embodiments, the antibody or its antigen-binding fragment is an scFv, and the variable light chain is c-terminal to that of the variable heavy chain. [Brief explanation of the drawing]

[0042] [Figure 1] Figure 1 shows an illustrative schematic diagram of an anti-BCMA CAR structure. [Figure 2-1] Figures 2A–2D show vector copies per cell (Figures 2A and 2C) and CAR construct expression on T cells as evaluated by FACS (Figures 2B and 2D). [Figure 2-2] Same as above. [Figure 3-1]Figures 3A–3I show the amount of IFNγ released from anti-BCMA CAR T cells in 24-hour co-culture with BCMA-negative rhabdomyosarcoma (RD) cells (Figure 3B) or HT1080 cells (Figures 3E and 3H), compared to BCMA-expressing Daudi cells (Figure 3C) or HT1080 BCMA cells (Figures 3F and 3I). [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Figures 3J and 3K show the amount of IL2 released from anti-BCMA CAR T cells in co-culture with BCMA low-Jeko1 cells (Figure 3J) or BCMA high-RPMI8226 cells (Figure 3K). [Figure 4] Figures 4A–4C show the amount of IFNγ released from anti-BCMA CAR T cells expressing comparator CAR, CAR1, or CAR5 alone (Figure 4A) or in co-culture with cancer cells (Figures 4B and 4C). The cells were co-cultured for 24 hours with antigen-low cell lines RL and Toledo (Figure 4B), and antigen-high cell lines Daudi and HT1080.BCMA (Figure 4C). [Figure 5] Figure 5 shows the time course of cytotoxicity of T cells expressing comparators CAR, CAR1, or CAR5 against BCMA expressing HT.1080 cells. [Figure 6] Figure 6 shows the expression / density of BCMA antigens in HT.1080, RL, Toledo, Daudi, RPMI-8226, and HT.1080.BCMA cancer cells. [Figure 7] Figures 7A and 7B show the proliferation of CAR T cells co-cultured with HT1080-nucRed cells that do not express BCMA (antigen-independent proliferation; Figure 7A) or HT1080-nucRed.BCMA cells that express BCMA (antigen-dependent proliferation; Figure 7B). [Modes for carrying out the invention]

[0043] A brief explanation of array keys Sequence IDs 1-48 describe exemplary light chain CDR sequences, heavy chain CDR sequences, variable light chain, and variable heavy chain amino acid sequences for anti-BCMA CARs as intended herein.

[0044] Sequence IDs 49–68 describe the polynucleotide and amino acid sequences of exemplary BCMA CAR constructs intended herein.

[0045] Sequence ID 69 describes the amino acid sequence of human BCMA.

[0046] Sequence IDs 71-81 describe the amino acid sequences of various linkers.

[0047] Sequence IDs 82-106 describe the amino acid sequences of the protease cleavage site and the self-cleaving polypeptide cleavage site.

[0048] In the sequence described above, X refers to any amino acid if present, or to the absence of an amino acid.

[0049] Detailed explanation A. Overview The present invention generally relates to improved compositions and methods for treating B cell-associated conditions. In particular, the present invention relates to improved human anti-BCMA antibodies, CARs, and CAR T cells for treating B cell-associated conditions (e.g., cancer).

[0050] As used herein, the term “B-cell-associated state” refers to a state characterized by inadequate B-cell activity and B-cell malignancy. In certain embodiments, this disclosure relates to improved adoptive cell therapy for B-cell-associated states using genetically modified immune effector cells. Genetic methods offer a means to enhance immune recognition and the elimination of cancer cells.

[0051] One promising strategy involves genetically engineering immune effector cells to express chimeric antigen receptors (CARs) that redirect cytotoxicity towards cancer cells. However, the potential therapeutic effect of any given CAR involves a delicate balance among several components of the CAR, including, but not limited to, the selection of appropriate structural domains (such as hinge or transmembrane domains), appropriate signaling or co-stimulatory domains (such as 4-1BB or CD3ζ), and appropriate antigen-binding domains. For example, preferably, the antigen-binding domain binds to antigens that are expressed on cancer cells and have relatively low (or absent) expression on non-cancer cells. Furthermore, the binding cannot be too strong or too weak so as not to result in no signaling or too much signaling.

[0052] Recent attempts to treat B-cell malignancies by targeting B-cell maturation antigens (BCMA, also known as CD269 or tumor necrosis factor receptor superfamily, member 17; TNFRSF17) through the use of BCMA-targeted therapeutic antibodies or chimeric antigen receptors (CARs) have been promising but limited in success. Indeed, many patients experience measurable therapeutic benefits not previously seen in these patient populations, but not all patients receiving these treatments experience complete remission and numerous relapses. Therefore, there remains a significant unmet need for improved treatments in these patient populations, including improvements in anti-BCMA antibodies and / or CARs (including CAR T therapy).

[0053] BCMA is a member of the tumor necrosis factor receptor superfamily (see, e.g., Thompson et al., J. Exp. Medicine, 192(1):129-135, 2000 and Mackay et al., Annu. Rev. Immunol, 21:231-264, 2003). BCMA binds to B-cell activator (BAFF) and proliferation-inducing ligand (APRIL) (see, e.g., Mackay et al., 2003 and Kalled et al., Immunological Reviews, 204:43-54, 2005). Among non-malignant cells, BCMA has been reported to be expressed mainly in a subset of plasma cells and mature B cells (see, for example, Laabi et al., EMBO J., 77(1):3897-3904, 1992; Laabi et al., Nucleic Acids Res., 22(7):1147-1154, 1994; Kalled et al., 2005; O'Connor et al., J.Exp.Medicine, 199(1):91-97, 2004; Ng et al., J.Immunol., 73(2):807-817, 2004). Mice lacking BCMA are healthy and have a normal number of B cells, but the survival of long-lived plasma cells is impaired (e.g., O'Connor et al., 2004; Xu et al., Mol. Cell. Biol, 21(12):4067-4074, 2001; Schiemann et al., Science, 293(5537):2 111-21 14, 2001). BCMA RNA has been universally detected in multiple myeloma cells and other lymphomas, and BCMA protein has been detected on the surface of plasma cells in multiple myeloma patients by multiple researchers (see, e.g., Novak et al., Blood, 103(2):689-694, 2004; Neri et al., Clinical Cancer Research, 73(19):5903-5909, 2007; Bellucci et al., Blood, 105(10):3945-3950, 2005; Moreaux et al., Blood, 703(8):3148-3157, 2004).

[0054] Accordingly, the improved compositions and methods of adoptive cell therapy disclosed herein provide genetically modified immunoeffector cells (e.g., CAR T cells) that target cells expressing BCMA, have a human-derived antigen-binding domain, and exhibit improved cytokine release and / or low antigen-independent signaling. In certain embodiments, CARs are provided comprising a human anti-BCMA antibody or antigen-binding fragment, a transmembrane domain, and one or more intracellular signaling domains. In further embodiments, the improved CAR T cells exhibit high IFNγ release in co-culture with low antigen-density cells.

[0055] In one embodiment, immune effector cells genetically modified to express the CAR intended herein are provided. T cells expressing the CAR are referred to herein as CAR T cells or CAR-modified T cells.

[0056] In various embodiments, the genetically modified immune effector cells intended herein are administered to patients with B-cell-related conditions, such as autoimmune diseases associated with B-cell or B-cell malignancies.

[0057] In another embodiment, an improved anti-BCMA antibody or a fragment thereof is provided.

[0058] The implementation of this invention employs conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology, unless otherwise indicated, which are within the scope of the art of the art, many of which are described below for illustrative purposes. Such techniques are fully described in the literature, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience;Glover, DNA Cloning:A Practical Approach, Volumes I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes (Academic Press, New York, 1992), Transcription and Translation (edited by B. Hames & S. Higgins, 1984), Perbal, A See also research articles in specialist journals such as Practical Guide to Molecular Cloning (1984), Harlow and Lane, Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Current Protocols in Immunology QEColigan, AMKruisbeek, DH Margulies, EM Shevach and W. Strober (eds.), 1991); Annual Review of Immunology; and Advances in Immunology.

[0059] B. Definition Before describing this disclosure in more detail, providing definitions of certain terms to be used herein may be helpful for understanding it.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those universally understood by those skilled in the art to which the present invention pertains. Similar or equivalent methods and materials may be used to implement or verify specific embodiments, but preferred compositions, methods, and materials are disclosed herein. For the purposes of this disclosure, the following terms are defined below:

[0061] The articles "a," "an," and "the" are used herein to refer to one or more grammatical objects (i.e., at least one, or one or more) of which the article is used. For example, "an element" means one or more elements.

[0062] The use of options (e.g., "or") should be understood to mean either one, both, or any combination of those options.

[0063] The terms "and / or" should be understood to mean either one of those options, or both of those options.

[0064] As used herein, the terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a base quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, the terms “about” or “approximately” refer to a range of quantities, levels, values, numbers, frequency, percentage, dimension, size, quantity, weight, or length of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% relative to a base quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0065] In one embodiment, for example, a range such as 1 to 5, approximately 1 to 5, or approximately 1 to approximately 5 refers to each numerical value included within that range. For example, in one non-limiting and merely illustrative embodiment, the range "1-5" is equivalent to expressions 1, 2, 3, 4, 5, or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0, or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0066] As used herein, the term “substantially” means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length. In one embodiment, “substantially the same” means an effect that is approximately the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length, e.g., a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that produces a physiological effect.

[0067] Throughout this specification, unless the context otherwise requires, the phrases “comprise,” “comprises,” and “comprise” imply that they include the specified process or element or group of processes or elements, but not that they imply that they exclude any other process or element or group of processes or elements. “Consists of” means that it includes and is limited to everything that precedes the phrase “consists of.” Thus, the phrase “consists of” indicates that the listed elements are necessary or required, and no other elements can exist. “Essentially consists of” means that it includes any elements listed after the phrase, and any other elements that do not interfere with or contribute to the activity or action of the listed elements as identified in this disclosure. Thus, the phrase “essentially consists of” indicates that the listed elements are essential or required, but no other elements substantially affect the activity or action of the listed elements.

[0068] Throughout this specification, any reference to “an embodiment,” “embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment,” or any combination thereof, means that a particular property, structure, or feature described in association with that embodiment is included in at least one embodiment. Thus, the occurrence of the aforementioned words in various parts of this specification does not necessarily all refer to the same embodiment. Furthermore, a particular property, structure, or feature may be combined in any suitable manner in one or more embodiments. Also, it is understood that a positive enumeration of a feature in an embodiment may serve as a basis for excluding that feature in a particular embodiment.

[0069] Additional definitions are provided throughout this disclosure.

[0070] C. Human anti-BCMA antibody In certain embodiments, an antibody or its antigen-binding fragment that binds to human BCMA is provided.

[0071] The term "antibody" refers to a binder which is a polypeptide containing at least a light chain or heavy chain immunoglobulin variable region or fragment thereof that specifically recognizes and binds to one or more antigen epitopes, such as peptides, lipids, polysaccharides, or nucleic acids containing antigenic determinants, including those recognized by immune cells.

[0072] The term “antibody” encompasses any naturally occurring, recombinant, modified, or engineered immunoglobulin-like structure or antigen-binding fragment or part thereof, or derivative thereof, as further described elsewhere herein. Therefore, the term refers to an immunoglobulin molecule that specifically binds to a target antigen, including, for example, chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. Intact antibodies generally contain at least two full-length heavy chains and two full-length light chains, but may in some cases contain fewer chains, such as antibodies naturally occurring in camelids that may contain only heavy chains. Antibodies can be derived from only a single source, or they can be “chimeric,” i.e., different parts of an antibody can be derived from two different antibodies. Antibodies or their antigen-binding portions may be produced in hybridomas by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies.

[0073] The terms “antigen-binding fragment” or “antigen-binding moiety” refer to one or more fragments of an antibody that possess the ability to specifically bind to an antigen (e.g., BCMA). Antigen-binding fragments include, but are not limited to, any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind to the antigen to form a complex. In some embodiments, the antigen-binding moiety of an antibody may be derived from the complete antibody molecule using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques, including, for example, manipulation and expression of DNA encoding antibody-variable and optionally constant domains. In preferred embodiments, the antigen-binding fragment is a single-stranded variable fragment (svFv).

[0074] A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of the antibody, where these domains are present in either orientation (e.g., VL-VH or VH-VL) within a single polypeptide chain. For example, in some embodiments, the scFv variable light chain is c-terminal to that of the variable heavy chain. In some embodiments, the scFv variable heavy chain is c-terminal to that of the variable light chain. Generally, the scFv polypeptide further contains a polypeptide linker between the VH and VL domains, thereby allowing the scFv to form a desired structure for antigen binding. For a review of scFv, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, (Springer-Verlag, New York, 1994), pp. 269–315.

[0075] "Isolated antibody or its antigen-binding fragment" refers to an antibody or its antigen-binding fragment identified, isolated, and / or recovered from its natural environment.

[0076] "Antigen (Ag)" broadly includes any molecule containing an antigenic determinant within a binding region to which an antibody or fragment specifically binds. In certain embodiments, "Antigen (Ag)" refers to a compound, composition, or substance that can stimulate antibody production or a T-cell response in an animal, and includes compositions (such as those containing cancer-specific proteins) that are injected into or absorbed by the animal. The antigen reacts with specific humoral or cellular immune products, including products induced by heterologous antigens such as the disclosed antigen. In certain embodiments, the target antigen is an epitope of a BCMA polypeptide (e.g., human BCMA polypeptide).

[0077] An antigen may be a single unit molecule (such as a protein monomer or fragment) or a complex composed of multiple components. An antigen may provide an epitope, e.g., a molecule or part of a molecule, or a complex of molecules or parts of molecules, which can be bound by a selective binder, such as an antigen-binding protein (e.g., an antibody). Thus, a selective binder can specifically bind to an antigen formed by two or more components in the complex. In some embodiments, an antigen can be used in animals to produce antibodies that can bind to that antigen. An antigen may have one or more epitopes that can interact with different antigen-binding proteins, e.g., antibodies.

[0078] An "epitope" or "antigenicity determinant" refers to the region of an antigen to which a binding substance binds. Epitopes can be formed from both continuous and discontinuous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed from continuous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly at least five, about nine, or about eight to ten amino acids in their unique spatial structure.

[0079] As used herein, the terms “specific binding affinity,” “specifically binding,” “specifically binding,” “specific binding,” or “specific target” describe the binding of an anti-BCMA antibody or its antigen-binding fragment (or a CAR containing it) to BCMA (e.g., human BCMA) at a binding affinity greater than background binding. The binding domain (or a CAR containing a binding domain or a fusion protein containing a binding domain) is, for example, about 10 5 M -1 The above affinity or K a If it binds to BCMA or associates with BCMA at a specific equilibrium association constant for a particular binding interaction with a unit of 1 / M, it "specifically binds" to BCMA. In certain embodiments, the binding domain (or fusion protein) is approximately 10 6M -1 、 10 7 M -1 、 10 8 M -1 、 10 9 M -1 、 10 10 M -1 、 10 11 M -1 、 10 12 M -1 、 or 10 13 M -1 binds to the target with the above Ka. A "high affinity" binding domain (or its single-chain fusion protein) has at least 10 7 M -1 、 at least 10 8 M -1 、 at least 10 9 M -1 、 at least 10 10 M -1 、 at least 10 11 M -1 、 at least 10 12 M -1 、 at least 10 13 M -1 or greater K a refers to a binding domain having.

[0080] Alternatively, the affinity is the equilibrium dissociation constant (K -5 M ~ 10 -13 M, or less) of a specific binding interaction at the M unit (e.g., 10 d) may be defined as. The affinity between the binding domain polypeptide and the CAR protein according to this disclosure can be readily determined using conventional techniques, such as competitive ELISA (enzyme-linked immunosorbent assay) or binding association, or by substitution assays using labeled ligands, or by using surface plasmon resonance instruments such as the Biacore T100 available from Biacore, Piscataway, NJ, or by optical biosensor technologies such as the EPIC system or EnSpire available from Corning and Perkin Elmer, respectively (see, for example, Scatchard et al. (1949) Ann. NYAcad. Sci. 51:660; and U.S. Patents 5,283,173; 5,468,614, or equivalent).

[0081] In one embodiment, the affinity of the specific binding is approximately twice as high as the background binding, approximately five times higher than the background binding, approximately ten times higher than the background binding, approximately twenty times higher than the background binding, approximately fifty times higher than the background binding, approximately one hundred times higher than the background binding, or approximately one thousand times higher than the background binding, or more.

[0082] In certain embodiments, the extracellular binding domain of the CAR contains an antibody or its antigen-binding fragment. In the context of CAR, “antibody” refers to a binder which is a polypeptide containing at least a light-chain or heavy-chain immunoglobulin variable region that specifically recognizes and binds to an antigen epitope, and includes, for example, peptides, lipids, polysaccharides, or nucleic acids containing antigenic determinants such as those recognized by immune cells.

[0083] As will be understood by those skilled in the art and as described elsewhere in this specification, a complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second, and third constant regions, while each light chain consists of a variable region and a constant region.

[0084] The light chain variable region and the heavy chain variable region include a “framework” region that is interrupted by three hypervariable regions, which is also called the “complementarity determination region” or “CDR”. CDRs can be defined or identified by conventional methods, such as the sequence by Kabat et al. (Wu, TT and Kabat, EA, J Exp Med. 132(2):211-50, (1970); Borden, P. and Kabat, EA, PNAS, 84:2440-2443 (1987); (Kabat et al., Sequences of Protein of Immunological Interest, USD Department of Health and Human Services, 1991, incorporated herein by reference), or by structure by Chothia et al. (Chothia, C. and Lesk, AM, J Mol. Biol., 196(4):901-917 (1987); Chothia, C. et al., Nature, 342:877-883 (1989)).

[0085] Other boundaries defining CDRs that overlap with the Kabat CDR are described in Padlan (1995) FASEB J.9:133-139 and MacCallum (1996) J.Mol.Biol.262(5):732-45. Further other CDR boundary definitions may not strictly adhere to one of the systems herein, but nevertheless overlap with the Kabat CDR, although they may be shortened or extended in light of predictions or experimental findings that certain residues or groups of residues, or even the entire CDR, do not significantly affect antigen binding. For example, the CDR of an antibody refers to the AbM hypervariable region representing a compromise between the Kabat CDR and the Chothia structural loop, and is modeled using Oxford Molecular's AbM antibody modeling software (Oxford Molecular This can be determined according to the AbM numbering scheme used by Group, Inc.

[0086] Furthermore, the CDR of the antibody can be determined according to the IMGT numbering system, as described in Lefranc MP, (1999) The Immunologist 7:132-136 and Lefranc MP et al., (1999) Nucleic Acids Res 27:209-212.

[0087] Further methods for CDR determination are disclosed in MacCallum RM et al., (1996) J Mol Biol 262:732-745. For example, Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domain,” in Antibody Engineering, Kontermann and D u See also bel, ed., section 31, pp. 422–439, Springer-Verlag, Berlin (2001). Proprietary and publicly available programs that can be used to determine a CDR based on any of the CDR definitions described herein, e.g., abysis (abysis.org / abysis / ) and IMGT / V-QUEST (imgt.org / IMGT_vquest), are known to those skilled in the art.

[0088] An exemplary example of a rule for predicting light chain CDRs is that CDRL1 begins at approximately 24 residues, preceded by Cys, followed by approximately 10-17 residues, then Trp (typically Trp-Tyr-Gln, but also Trp-Leu-Gln, Trp-Phe-Gln, Trp-Tyr-Leu). CDRL2 begins at approximately 16 residues after the end of CDRL1, generally preceded by Ile-Tyr, Val-Tyr, Ile-Lys, Ile-Phe, and consists of 7 residues. CDRL3 begins at approximately 33 residues after the end of CDRL2, preceded by Cys, followed by 7-11 residues, then Phe-Gly-XXX-Gly (SEQ ID NO: 108) (XXX is any amino acid).

[0089] Exemplary examples of rules for predicting heavy chain CDRs include: CDRH1 starts at approximately 26 residues, preceded by Cys-XXX-XXX-XXX (SEQ ID NO: 109), followed by 10-12 residues, then Trp (typically Trp-Val, but also Trp-Ile, Trp-Ala); CDRH2 starts at approximately 15 residues after the end of CDRH1, generally followed by Leu-Glu-Trp-Ile-Gly (SEQ ID NO: 110) or several residues. The lyation is preceded by 16-19 residues, followed by Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala, and the AbM definition ends 7 residues earlier; CDRH3 begins about 33 residues after the end of CDRH2, preceded by Cys-XXX-XXX (typically Cys-Ala-Arg), for 3-25 residues, followed by Trp-Gly-XXX-Gly (sequence number 111).

[0090] Accordingly, in certain embodiments, the Disclosure provides isolated antibodies, antigen-binding fragments thereof, that specifically bind to human BCMA proteins and include a heavy chain variable region comprising the CDRL1, CDRL2, and CDRL3 region amino acid sequences described in Variable Light Chain SEQ ID NOs. 7, 15, 23, 31, 39, or 47 and / or the CDRH1, CDRH2, and CDRH3 region amino acid sequences described in Variable Heavy Chain SEQ ID NOs. 8, 16, 24, 32, 40, or 48, wherein the CDR is defined according to the Kabat definition, the Chothia definition, a combination of the Kabat and Chothia definitions, the IMGT definition, the AbM definition, or the contact definition of the CDR. In some embodiments, the CDR is defined according to the Kabat definition. In some embodiments, the CDR is defined according to the Chothia definition. In some embodiments, the CDR is defined according to the AbM definition. In some embodiments, the CDR is defined according to the IMGT definition. In some embodiments, the CDR is defined according to the contact definition. In some embodiments, the CDR is defined by any combination of the above-described CDR definitions.

[0091] Examples of light chain CDRs suitable for antibodies or antigen-binding fragments intended herein include, but are not limited to, the CDR sequences described in SEQ ID NOs: 1-3, 9-11, 17-19, 25-27, 33-35, or 41-43. Examples of heavy chain CDRs suitable for antibodies or antigen-binding fragments intended herein include, but are not limited to, the CDR sequences described in SEQ ID NOs: 4-6, 12-14, 20-22, 28-30, 36-38, or 44-46.

[0092] References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including antibodies, Fv, scFv, dsFv, Fab, or other antibody fragments disclosed herein. References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, including antibodies, Fv, scFv, dsFv, Fab, or other antibody fragments disclosed herein.

[0093] In certain embodiments, the antigen-specific binding domain is an scFv that binds to a human BCMA polypeptide. Exemplary examples of variable heavy chains suitable for the antibodies or antigen-binding fragments envisioned herein include, but are not limited to, the amino acid sequences described in SEQ ID NOs: 8, 16, 24, 32, 40, and 48. Exemplary examples of variable light chains suitable for the antibodies or antigen-binding fragments envisioned herein include, but are not limited to, the amino acid sequences described in SEQ ID NOs: 7, 15, 23, 31, 39, and 47.

[0094] The BCMA-specific binding domains provided herein also comprise one, two, three, four, five, or six CDRs. Such CDRs may be human or non-human CDRs or modified non-human CDRs selected from the light chain CDRL1, CDRL2, and CDRL3 and the heavy chain CDRH1, CDRH2, and CDRH3. In certain embodiments, the BCMA-specific binding domain comprises (a) a light chain variable region comprising light chain CDRL1, light chain CDRL2, and light chain CDRL3, and (b) a heavy chain variable region comprising heavy chain CDRH1, heavy chain CDRH2, and heavy chain CDRH3. In preferred embodiments, the CDRs are human CDRs.

[0095] Examples of light chain CDRs suitable for antibodies or antigen-binding fragments intended herein include, but are not limited to, the CDR sequences described in SEQ ID NOs: 1-3, 9-11, 17-19, 25-27, 33-35, or 41-43. Examples of heavy chain CDRs suitable for antibodies or antigen-binding fragments intended herein include, but are not limited to, the CDR sequences described in SEQ ID NOs: 4-6, 12-14, 20-22, 28-30, 36-38, or 44-46.

[0096] In various embodiments, the antibody or its antigen-binding fragment contains one or more CDR sequences that are substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 compared to the corresponding CDR region. For example, the antibody or its antigen-binding fragment may contain one or more CDR sequences (e.g., SEQ ID NOs: 1-6, 9-14, 17-22, 25-30, 33-38, or 41-46) and each contains up to 1, 2, 3, 4, or 5 amino acid residue variations compared to any one of the corresponding CDR regions of SEQ ID NOs: 1-6, 9-14, 17-22, 25-30, 33-38, or 41-46.

[0097] As used herein, the terms “amino acid variation,” “amino acid change,” or “amino acid residue change” refer to a difference of one or more amino acids compared to a reference sequence, and include modifications, substitutions, insertions, and / or deletions of amino acids.

[0098] In one embodiment, the antibody or its antigen-binding fragment comprises at least three CDRs selected from the following, and optionally each CDR comprising up to three amino acid changes, e.g., one, two, or three amino acid changes: CDRL1: SEQ ID NO: 1; CDRL2: SEQ ID NO: 2; CDRL3: SEQ ID NO: 3; CDRH1: SEQ ID NO: 4; CDRH2: SEQ ID NO: 5; and CDRH3: SEQ ID NO: 6.

[0099] In one embodiment, the antibody or its antigen-binding fragment comprises at least three CDRs selected from the following, optionally comprising up to three amino acid changes for each CDR, e.g., one, two, or three amino acid changes: CDRL1: SEQ ID NO: 9; CDRL2: SEQ ID NO: 10; CDRL3: SEQ ID NO: 11; CDRH1: SEQ ID NO: 12; CDRH2: SEQ ID NO: 13; and CDRH3: SEQ ID NO: 14.

[0100] In one embodiment, the antibody or its antigen-binding fragment comprises at least three CDRs selected from the following, optionally comprising up to three amino acid changes for each CDR, e.g., one, two, or three amino acid changes: CDRL1: SEQ ID NO: 17; CDRL2: SEQ ID NO: 18; CDRL3: SEQ ID NO: 19; CDRH1: SEQ ID NO: 20; CDRH2: SEQ ID NO: 21; and CDRH3: SEQ ID NO: 22.

[0101] In one embodiment, the antibody or its antigen-binding fragment comprises at least three CDRs selected from the following, optionally comprising up to three amino acid changes for each CDR, e.g., one, two, or three amino acid changes: CDRL1: SEQ ID NO: 25; CDRL2: SEQ ID NO: 26; CDRL3: SEQ ID NO: 27; CDRH1: SEQ ID NO: 28; CDRH2: SEQ ID NO: 29; and CDRH3: SEQ ID NO: 30.

[0102] In one embodiment, the antibody or its antigen-binding fragment comprises at least three CDRs selected from the following, optionally comprising up to three amino acid changes for each CDR, e.g., one, two, or three amino acid changes: CDRL1: SEQ ID NO: 33; CDRL2: SEQ ID NO: 34; CDRL3: SEQ ID NO: 35; CDRH1: SEQ ID NO: 36; CDRH2: SEQ ID NO: 37; and CDRH3: SEQ ID NO: 38.

[0103] In one embodiment, the antibody or its antigen-binding fragment comprises at least three CDRs selected from the following, optionally comprising up to three amino acid changes for each CDR, e.g., one, two, or three amino acid changes: CDRL1: SEQ ID NO: 41; CDRL2: SEQ ID NO: 42; CDRL3: SEQ ID NO: 43; CDRH1: SEQ ID NO: 44; CDRH2: SEQ ID NO: 45; and CDRH3: SEQ ID NO: 46.

[0104] In some embodiments, the antibody or its antigen-binding fragment includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, the antibody or its antigen-binding fragment includes the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs: 4, 5, and 6, respectively. In certain embodiments, the antibody or its antigen-binding fragment includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs: 1, 2, and 3, respectively, as well as the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs: 4, 5, and 6.

[0105] In some embodiments, the antibody or its antigen-binding fragment includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs: 9, 10, and 11, respectively. In some embodiments, the antibody or its antigen-binding fragment includes the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs: 12, 13, and 14, respectively. In certain embodiments, the antibody or its antigen-binding fragment includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs: 9, 10, and 11, respectively, as well as the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs: 12, 13, and 14.

[0106] In some embodiments, the antibody or its antigen-binding fragment includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs. 17, 18, and 19, respectively. In some embodiments, the antibody or its antigen-binding fragment includes the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs. 20, 21, and 22, respectively. In certain embodiments, the antibody or its antigen-binding fragment includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs. 17, 18, and 19, respectively, as well as the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs. 20, 21, and 22.

[0107] In some embodiments, the antibody or its antigen-binding fragment includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs. 25, 26, and 27, respectively. In some embodiments, the antibody or its antigen-binding fragment includes the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs. 28, 29, and 30, respectively. In certain embodiments, the antibody or its antigen-binding fragment includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs. 25, 26, and 27, and the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs. 28, 29, and 30, respectively.

[0108] In some embodiments, the antibody or its antigen-binding fragment comprises the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs. 33, 34, and 35, respectively. In some embodiments, the antibody or its antigen-binding fragment comprises the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs. 36, 37, and 38, respectively. In certain embodiments, the antibody or its antigen-binding fragment comprises the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs. 33, 34, and 35, respectively, and the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs. 36, 37, and 38.

[0109] In some embodiments, the antibody or its antigen-binding fragment comprises the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs. 41, 42, and 43, respectively. In some embodiments, the antibody or its antigen-binding fragment comprises the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs. 44, 45, and 46, respectively. In certain embodiments, the antibody or its antigen-binding fragment comprises the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs. 41, 42, and 43, respectively, and the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs. 44, 45, and 46, respectively.

[0110] Aspects of the present invention relate to an antibody or its antigen-binding fragment that selectively binds to human BCMA, comprising a heavy chain variable region sequence and a light chain variable region sequence.

[0111] In various embodiments, the antibody or its antigen-binding fragment includes a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs. In one embodiment, the antibody or its antigen-binding fragment includes a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs. 8, 16, 24, 32, 40, or 48.

[0112] In one embodiment, the antibody or its antigen-binding fragment includes a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7, and / or a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.

[0113] In one embodiment, the antibody or its antigen-binding fragment includes a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, and / or a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16.

[0114] In one embodiment, the antibody or its antigen-binding fragment includes a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23, and / or a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24.

[0115] In one embodiment, the antibody or its antigen-binding fragment includes a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 31, and / or a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32.

[0116] In one embodiment, the antibody or its antigen-binding fragment includes a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 39, and / or a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 40.

[0117] In one embodiment, the antibody or its antigen-binding fragment includes a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 47, and / or a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 48.

[0118] In some embodiments, the light chain variable region and / or heavy chain variable region sequence remains unchanged in any of the CDR sequences provided herein. For example, in some embodiments, the degree of sequence variation (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) may occur within the heavy chain variable and / or light chain variable amino acid sequence, excluding any of the CDR sequences provided herein.

[0119] In some embodiments, the "identity percentage" of two amino acid sequences is determined using the algorithms described in Karlin and Altschul Proc.Natl.Acad.Sci.USA 87:2264-68, 1990, and Karlin and Altschul Proc.Natl.Acad.Sci.USA 90:5873-77, 1993. These algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) of Biol. 215:403-10, 1990. BLAST protein searches can be performed using the XBLAST program (score=50, word length=3) to obtain amino acid sequences homologous to the target protein molecule. When a gap exists between two sequences, Gapped BLAST can be used as described by Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using BLAST and Gapped BLAST programs, the default parameters for each program (such as XBLAST and NBLAST) can be used.

[0120] In various embodiments, the antibody or its antigen-binding fragment includes a light chain variable domain containing the amino acid sequence described in SEQ ID NO: 7 and / or a heavy chain variable domain containing the amino acid sequence described in SEQ ID NO: 8.

[0121] In various embodiments, the antibody or its antigen-binding fragment includes a light chain variable domain containing the amino acid sequence described in SEQ ID NO: 15 and / or a heavy chain variable domain containing the amino acid sequence described in SEQ ID NO: 16.

[0122] In various embodiments, the antibody or its antigen-binding fragment includes a light chain variable domain containing the amino acid sequence described in SEQ ID NO: 23 and / or a heavy chain variable domain containing the amino acid sequence described in SEQ ID NO: 24.

[0123] In various embodiments, the antibody or its antigen-binding fragment includes a light chain variable domain containing the amino acid sequence described in SEQ ID NO: 31 and / or a heavy chain variable domain containing the amino acid sequence described in SEQ ID NO: 32.

[0124] In various embodiments, the antibody or its antigen-binding fragment includes a light chain variable domain containing the amino acid sequence described in SEQ ID NO: 39 and / or a heavy chain variable domain containing the amino acid sequence described in SEQ ID NO: 40.

[0125] In various embodiments, the antibody or its antigen-binding fragment includes a light chain variable domain containing the amino acid sequence described in SEQ ID NO: 47 and / or a heavy chain variable domain containing the amino acid sequence described in SEQ ID NO: 48.

[0126] In any of the antibodies or antigen-binding fragments contemplated herein, one or more conserved mutations can be introduced into the CDR or framework sequence at positions where residues are unlikely to be involved in antibody-antigen interactions. In some embodiments, such conserved mutations may be introduced into the CDR or framework sequence at positions where residues are unlikely to be involved in interactions with BCMA, as determined based on the crystal structure. In some embodiments, likely interfaces (e.g., residues involved in antigen-antibody interactions) may be inferred from known structural information relating to other antigens that share structural similarities.

[0127] In various embodiments, the Disclosure provides an antibody or antigen-binding fragment that competes for binding to the antibody or antigen-binding fragment intended herein. In one embodiment, the Disclosure provides an antibody or antigen-binding fragment that binds to the same epitope as the antibody or antigen-binding fragment intended herein.

[0128] Aspects of this disclosure relate to antibodies that compete or cross-compete with any of the specific antibodies or their antigen-binding fragments provided herein, for example, antibodies having one or more CDR sequences (1, 2, 3, 4, 5, or 6 CDR sequences) as described above. In one embodiment, this disclosure provides antibodies and their antigen-binding fragments that compete or cross-compete with antibodies having light chain CDR sequences described in SEQ ID NOs: 1-3, 9-11, 17-19, 25-27, 33-35, or 41-43 and / or heavy chain CDR sequences described in SEQ ID NOs: 4-6, 12-14, 20-22, 28-30, 36-38, or 44-46. In one embodiment, this disclosure provides antibodies or their antigen-binding fragments that compete or cross-compete with antibodies having light chain variable region sequences including SEQ ID NOs: 7, 15, 23, 31, 39, or 47 and / or heavy chain variable region sequences including SEQ ID NOs: 8, 16, 24, 32, 40, or 48.

[0129] In one embodiment, the disclosure provides an antibody and its antigen-binding fragment that competes or cross-competes with an antibody having a light chain CDR sequence described in SEQ ID NOs: 1-3 and / or a heavy chain CDR sequence described in SEQ ID NOs: 4-6. In one embodiment, the disclosure provides an antibody and its antigen-binding fragment that competes or cross-competes with an antibody having a light chain variable region sequence including SEQ ID NO: 7 and / or a heavy chain variable region sequence including SEQ ID NO: 8.

[0130] In one embodiment, the disclosure provides an antibody and its antigen-binding fragment that competes or cross-competes with an antibody having a light chain CDR sequence described in SEQ ID NOs. 9-11 and / or a heavy chain CDR sequence described in SEQ ID NOs. 12-14. In one embodiment, the disclosure provides an antibody and its antigen-binding fragment that competes or cross-competes with an antibody having a light chain variable region sequence including SEQ ID NO. 15 and / or a heavy chain variable region sequence including SEQ ID NO. 16.

[0131] In one embodiment, the disclosure provides an antibody and its antigen-binding fragment that competes or cross-competes with an antibody having a light chain CDR sequence described in SEQ ID NOs. 17-19 and / or a heavy chain CDR sequence described in SEQ ID NOs. 20-22. In one embodiment, the disclosure provides an antibody and its antigen-binding fragment that competes or cross-competes with an antibody having a light chain variable region sequence including SEQ ID NOs. 23 and / or a heavy chain variable region sequence including SEQ ID NOs. 24.

[0132] In one embodiment, the disclosure provides an antibody that competes or cross-competes with an antibody or its antigen-binding fragment having a light chain variable region sequence including SEQ ID NOs. 25-27 and / or a heavy chain variable region sequence including SEQ ID NOs. 28-30. In one embodiment, the disclosure provides an antibody that competes or cross-competes with an antibody or its antigen-binding fragment having a light chain variable region sequence including SEQ ID NOs. 31 and / or a heavy chain variable region sequence including SEQ ID NOs. 32.

[0133] In one embodiment, the disclosure provides an antibody and its antigen-binding fragment that competes or cross-competes with an antibody having a light chain CDR sequence described in SEQ ID NOs.33-35 and / or a heavy chain CDR sequence described in SEQ ID NOs.36-38. In one embodiment, the disclosure provides an antibody and its antigen-binding fragment that competes or cross-competes with an antibody having a light chain variable region sequence including SEQ ID NOs.39 and / or a heavy chain variable region sequence including SEQ ID NOs.40.

[0134] In one embodiment, the disclosure provides an antibody and its antigen-binding fragment that competes or cross-competes with an antibody having a light chain CDR sequence described in SEQ ID NOs. 41-43 and / or a heavy chain CDR sequence described in SEQ ID NOs. 44-46. In one embodiment, the disclosure provides an antibody and its antigen-binding fragment that competes or cross-competes with an antibody having a light chain variable region sequence including SEQ ID NOs. 47 and / or a heavy chain variable region sequence including SEQ ID NOs. 48.

[0135] In some embodiments, an antibody or its antigen-binding fragment binds to or near the same epitope as any of the antibodies provided herein. In some embodiments, an antibody or its antigen-binding fragment binds near an epitope if it binds to within 15 or fewer amino acid residues of the epitope. In some embodiments, any of the antibodies or their antigen-binding fragments provided herein binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the epitope to which any of the antibodies provided herein bind.

[0136] In another embodiment, one of the antigens provided herein (e.g., human BCMA) is given 10 -8 Equilibrium dissociation constant K between antibodies and proteins less than M D Therefore, competing or cross-competing antibodies or antigen-binding fragments thereof are provided herein. In other embodiments, the antibody is 10 -11 M~10 -8 K in the range of M D Therefore, it competes with or cross-competes with BCMA. In some embodiments, BCMA-specific antibodies or antigen-binding fragments that compete with the antibodies or antigen-binding fragments intended herein are provided herein. In some embodiments, provided herein are BCMA-specific antibodies or antigen-binding fragments that bind to the same epitope as the antibodies or antigen-binding fragments intended herein.

[0137] The antibodies provided herein can be characterized using any suitable method. For example, one method is to identify the epitope to which the antigen binds, or "epitope mapping." There are many suitable methods for mapping and characterizing the location of epitopes on proteins, such as elucidating the crystal structure of antibody-antigen complexes, competitive assays, gene fragment expression assays, and synthetic peptide-based assays, as described in Section 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In an additional example, epitope mapping can be used to determine the sequence to which an antibody binds. An epitope may be a linear epitope, i.e., one that is contained in a single stretch of amino acids, or a three-dimensional epitope (primary structure linear sequence) formed by the three-dimensional interaction of amino acids that is not necessarily contained in a single stretch.

[0138] Peptides of various lengths (e.g., at least 4–19 amino acids) can be isolated or synthesized (e.g., recombinantly) and used in antibody binding assays. In another example, the epitope to which the antibody binds can be determined in a systematic screening by using duplicate peptides derived from the target antigen sequence and determining antibody binding. According to gene fragment expression assays, an open reading frame encoding the target antigen is fragmented randomly or by a specific gene structure, and the reactivity of the antigen expression fragment with the antibody being tested is determined. The gene fragment can be produced, for example, by PCR, and then transcribed and translated into a protein in vitro in the presence of radioactive amino acids. The binding of the antibody to the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis. Specific epitopes can also be identified by using a large library of random peptide sequences displayed on the surface of phage particles (phage library). Alternatively, a defined library of duplicate peptide fragments can be tested for binding to the test antibody in a simple binding assay. In additional examples, antigen-binding domain mutagenesis, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues necessary, sufficient, and / or required for epitope binding. For example, domain swapping experiments can be performed using variants of target antigens in which various fragments of BCMA are swapped with sequences from closely related but antigenically different proteins, such as another member of the BCMA protein family. By evaluating antibody binding to mutant BCMA, the importance of specific antigen fragments for antibody binding can be assessed.

[0139] Alternatively, a competitive assay can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as other antibodies. Competitive assays are well known to those skilled in the art.

[0140] Furthermore, the interaction between any antibody provided herein and one or more residues in BCMA can be determined by routine techniques. For example, the crystal structure can be determined, and the distance between a residue in BCMA and one or more residues in the antibody (or antigen-binding fragment) can be determined accordingly. Based on such distances, it can be determined whether a particular residue in BCMA interacts with one or more residues in the antibody. In addition, the preferential binding of candidate antibodies can be determined by applying appropriate methods such as competitive assays and targeted mutagenesis assays.

[0141] In some embodiments, the antibody or antigen-binding fragment of the Disclosure that selectively binds to BCMA comprises one or more complementarity-determining regions (CDRs) as intended herein. In some embodiments, the Disclosure provides a nucleic acid molecule encoding an antibody or antigen-binding fragment that selectively binds to BCMA as intended herein. In one embodiment, the nucleic acid molecule encodes one or more of the CDR sequences as intended herein.

[0142] D. Chimeric antigen receptor In various embodiments, improved genetically engineered receptors are provided that redirect the cytotoxicity of immune effector cells toward BCMA-expressing cells (e.g., B cells). These genetically engineered receptors are referred to herein as chimeric antigen receptors (CARs). CARs are molecules that combine antibody-based specificity against a desired antigen (such as BCMA) with a T-cell receptor-activating intracellular domain to produce chimeric proteins that exhibit specific anti-BCMA cellular immune activity. As used herein, the term "chimeric" refers to a molecule composed of different protein or DNA portions of different origins.

[0143] The CARs contemplated herein include an extracellular domain that binds to BCMA (also called a binding domain or antigen-specific binding domain), a transmembrane domain, and an intracellular signaling domain. When the anti-BCMA antigen-binding domain of a CAR binds to BCMA on the surface of a target cell, clustering of the CAR occurs, resulting in an activation stimulus to the CAR-containing cells. A key characteristic of CARs is their ability to redirect the specificity of immune effector cells, which induces the production of molecules that can mediate proliferation, cytokine production, phagocytosis, or cell death of target antigen-expressing cells in a major histocompatibility complex (MHC)-independent manner, thereby eliciting the cell-specific targeting ability of monoclonal antibodies, soluble ligands, or cell-specific coreceptors.

[0144] In various embodiments, the CAR comprises an extracellular binding domain including an anti-BCMA-specific binding domain, a transmembrane domain, one or more intracellular costimulatory signaling domains, and a primary signaling domain.

[0145] In certain embodiments, the CAR comprises an extracellular binding domain containing an anti-BCMA antibody or its antigen-binding fragment, one or more hinge domains or spacer domains, and a transmembrane domain, the transmembrane domain comprising one or more intracellular costimulatory signaling domains and a primary signaling domain.

[0146] 1. Joint domain In certain embodiments, the CAR as envisioned herein comprises an extracellular binding domain containing an anti-BCMA antibody or its antigen-binding fragment that specifically binds to a human BCMA polypeptide expressed on B cells. In some embodiments, the antibody or its antigen-binding fragment specifically binds to one or more epitopes of the human BCMA polypeptide. In certain embodiments, the anti-BCMA antibody or antigen-binding fragment is a human antibody or antigen-binding fragment. In various embodiments, the CAR comprises the anti-BCMA antibody or antigen-binding fragment as envisioned herein.

[0147] As used herein, the terms “binding domain,” “extracellular domain,” “extracellular binding domain,” “antigen-specific binding domain,” and “extracellular antigen-specific binding domain” are interchangeable and provide a CAR having the ability to specifically bind to a target antigen of interest, such as BCMA. The binding domain may be derived from a natural, synthetic, semi-synthetic, or recombinant source.

[0148] In certain embodiments, the CAR contemplated herein comprises an antigen-specific binding domain which is scFv. In various embodiments, the scFv domain is present in a single polypeptide chain and in either orientation (e.g., VL-VH or VH-VL). For example, in some embodiments, the scFv variable light chain is c-terminal to that of the variable heavy chain. In other embodiments, the scFv variable heavy chain is c-terminal to that of the variable light chain. See, for example, Figure 1. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain, thereby allowing the scFv to form a desired structure for antigen binding.

[0149] Examples of variable heavy (VH) chains suitable for constructing the BCMA CARs intended herein include, but are not limited to, the amino acid sequences described in SEQ ID NOs: 8, 16, 24, 32, 40, and 48. Examples of variable light (VL) chains suitable for constructing the BCMA CARs intended herein include, but are not limited to, the amino acid sequences described in SEQ ID NOs: 7, 15, 23, 31, 39, and 47.

[0150] The BCMA-specific binding domains provided herein also comprise one, two, three, four, five, or six CDRs. Such CDRs may be non-human CDRs or modified non-human CDRs selected from the light chain CDRL1, CDRL2, and CDRL3 and the heavy chain CDRH1, CDRH2, and CDRH3. In certain embodiments, the BCMA-specific binding domain comprises (a) a light chain variable region comprising light chain CDRL1, light chain CDRL2, and light chain CDRL3, and (b) a heavy chain variable region comprising heavy chain CDRH1, heavy chain CDRH2, and heavy chain CDRH3.

[0151] Accordingly, in certain embodiments, the Disclosure provides a CAR extracellular binding domain which binds to a human BCMA protein and comprises a heavy chain variable region including the CDRL1, CDRL2, and CDRL3 region amino acid sequences described in Variable Light Chain SEQ ID NOs. 7, 15, 23, 31, 39, or 47, and / or the CDRH1, CDRH2, and CDRH3 region amino acid sequences described in Variable Heavy Chain SEQ ID NOs. 8, 16, 24, 32, 40, or 48. In certain embodiments, each CDR is defined according to the Kabat definition, the Chothia definition, a combination of the Kabat and Chothia definitions, the IMGT definition, the AbM definition, or the contact definition of the CDR. In some embodiments, the CDR is defined according to the Kabat definition. In some embodiments, the CDR is defined according to the Chothia definition. In some embodiments, the CDR is defined according to the AbM definition. In some embodiments, the CDR is defined according to the IMGT definition. In some embodiments, the CDR is defined according to the contact definition. In some embodiments, the CDR is defined by any combination of the above-described CDR definitions.

[0152] Examples of light chain CDRs suitable for constructing the humanized BCMA CARs intended herein include, but are not limited to, the CDR sequences described in SEQ ID NOs: 1-3, 9-11, 17-19, 25-27, 33-35, or 41-43. Examples of heavy chain CDRs suitable for constructing the humanized BCMA CARs intended herein include, but are not limited to, the CDR sequences described in SEQ ID NOs: 4-6, 12-14, 20-22, 28-30, 36-38, or 44-46.

[0153] In various embodiments, the BCMA-specific binding domain includes one or more CDR sequences that are substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 compared to the corresponding CDR region. For example, the BCMA-specific binding domain includes one or more CDR sequences (e.g., SEQ ID NOs: 1-6, 9-14, 17-22, 25-30, 33-38, or 41-46) that have a variation of 1, 2, 3, 4, or 5 amino acid residues compared to the corresponding CDR region, in any one of SEQ ID NOs: 1-6, 9-14, 17-22, 25-30, 33-38, or 41-46.

[0154] As used herein, the terms “amino acid variation,” “amino acid change,” or “amino acid residue change” include amino acid substitutions and / or deletions.

[0155] In one embodiment, the BCMA-specific binding domain comprises at least three CDRs selected from the following, optionally comprising up to three amino acid changes, for example, one, two, or three amino acid changes for each CDR: CDRL1: SEQ ID NO: 1; CDRL2: SEQ ID NO: 2; CDRL3: SEQ ID NO: 3; CDRH1: SEQ ID NO: 4; CDRH2: SEQ ID NO: 5; and CDRH3: SEQ ID NO: 6.

[0156] In one embodiment, the BCMA-specific binding domain comprises at least three CDRs selected from the following, optionally comprising up to three amino acid changes, for example, one, two, or three amino acid changes for each CDR: CDRL1: SEQ ID NO: 9; CDRL2: SEQ ID NO: 10; CDRL3: SEQ ID NO: 11; CDRH1: SEQ ID NO: 12; CDRH2: SEQ ID NO: 13; and CDRH3: SEQ ID NO: 14.

[0157] In one embodiment, the BCMA-specific binding domain comprises at least three CDRs selected from the following, and optionally comprising up to three amino acid changes, for example, one, two, or three amino acid changes for each CDR: CDRL1: SEQ ID NO: 17; CDRL2: SEQ ID NO: 18; CDRL3: SEQ ID NO: 19; CDRH1: SEQ ID NO: 20; CDRH2: SEQ ID NO: 21; and CDRH3: SEQ ID NO: 22.

[0158] In one embodiment, the BCMA-specific binding domain comprises at least three CDRs selected from the following, optionally comprising up to three amino acid changes, for example, one, two, or three amino acid changes for each CDR: CDRL1: SEQ ID NO: 25; CDRL2: SEQ ID NO: 26; CDRL3: SEQ ID NO: 27; CDRH1: SEQ ID NO: 28; CDRH2: SEQ ID NO: 29; and CDRH3: SEQ ID NO: 30.

[0159] In one embodiment, the BCMA-specific binding domain comprises at least three CDRs selected from the following, and optionally comprising up to three amino acid changes, for example, one, two, or three amino acid changes for each CDR: CDRL1: SEQ ID NO: 33; CDRL2: SEQ ID NO: 34; CDRL3: SEQ ID NO: 35; CDRH1: SEQ ID NO: 36; CDRH2: SEQ ID NO: 37; and CDRH3: SEQ ID NO: 38.

[0160] In one embodiment, the BCMA-specific binding domain comprises at least three CDRs selected from the following, and optionally comprising up to three amino acid changes, for example, one, two, or three amino acid changes for each CDR: CDRL1: SEQ ID NO: 41; CDRL2: SEQ ID NO: 42; CDRL3: SEQ ID NO: 43; CDRH1: SEQ ID NO: 44; CDRH2: SEQ ID NO: 45; and CDRH3: SEQ ID NO: 46.

[0161] In some embodiments, the BCMA-specific binding domain includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, the BCMA-specific binding domain includes the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs: 4, 5, and 6, respectively. In certain embodiments, the BCMA-specific binding domain includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs: 1, 2, and 3, respectively, and the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs: 4, 5, and 6, respectively.

[0162] In some embodiments, the BCMA-specific binding domain includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs: 9, 10, and 11, respectively. In some embodiments, the BCMA-specific binding domain includes the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs: 12, 13, and 14, respectively. In certain embodiments, the BCMA-specific binding domain includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs: 9, 10, and 11, respectively, and the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs: 12, 13, and 14, respectively.

[0163] In some embodiments, the BCMA-specific binding domain includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs. 17, 18, and 19, respectively. In some embodiments, the BCMA-specific binding domain includes the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs. 20, 21, and 22, respectively. In certain embodiments, the BCMA-specific binding domain includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs. 17, 18, and 19, respectively, and the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs. 20, 21, and 22, respectively.

[0164] In some embodiments, the BCMA-specific binding domain includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs. 25, 26, and 27, respectively. In some embodiments, the BCMA-specific binding domain includes the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs. 28, 29, and 30, respectively. In certain embodiments, the BCMA-specific binding domain includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs. 25, 26, and 27, respectively, and the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs. 28, 29, and 30, respectively.

[0165] In some embodiments, the BCMA-specific binding domain includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs. 33, 34, and 35, respectively. In some embodiments, the BCMA-specific binding domain includes the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs. 36, 37, and 38, respectively. In certain embodiments, the BCMA-specific binding domain includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs. 33, 34, and 35, respectively, and the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs. 36, 37, and 38, respectively.

[0166] In some embodiments, the BCMA-specific binding domain includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs. 41, 42, and 43, respectively. In some embodiments, the BCMA-specific binding domain includes the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs. 44, 45, and 46, respectively. In certain embodiments, the BCMA-specific binding domain includes the light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs. 41, 42, and 43, respectively, and the heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs. 44, 45, and 46, respectively.

[0167] Aspects of this disclosure relate to a CAR comprising an extracellular binding domain (BCMA-specific binding domain) that selectively binds to human BCMA, which includes heavy chain variable region sequences and light chain variable region sequences.

[0168] In various embodiments, the BCMA-specific binding domain includes a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs. In one embodiment, the antibody or its antigen-binding fragment includes a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs. 8, 16, 24, 32, 40, or 48.

[0169] In one embodiment, the BCMA-specific binding domain includes a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7, and / or a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.

[0170] In one embodiment, the BCMA-specific binding domain includes a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, and / or a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16.

[0171] In one embodiment, the BCMA-specific binding domain includes a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23, and / or a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24.

[0172] In one embodiment, the BCMA-specific binding domain includes a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 31, and / or a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32.

[0173] In one embodiment, the BCMA-specific binding domain includes a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 39, and / or a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 40.

[0174] In one embodiment, the BCMA-specific binding domain includes a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 47, and / or a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 48.

[0175] In some embodiments, the light chain variable region and / or heavy chain variable region sequence remains unchanged in any of the CDR sequences provided herein. For example, in some embodiments, the degree of sequence variation (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) may occur within the heavy chain variable and / or light chain variable amino acid sequence, excluding any of the CDR sequences provided herein.

[0176] In various embodiments, the BCMA-specific binding domain is a light chain variable domain containing the amino acid sequence described in SEQ ID NO: 7 and / or a heavy chain variable domain containing the amino acid sequence described in SEQ ID NO: 8.

[0177] In various embodiments, the BCMA-specific binding domain includes a light chain variable domain containing the amino acid sequence described in SEQ ID NO: 15 and / or a heavy chain variable domain containing the amino acid sequence described in SEQ ID NO: 16.

[0178] In various embodiments, the BCMA-specific binding domain includes a light chain variable domain containing the amino acid sequence described in SEQ ID NO: 23 and / or a heavy chain variable domain containing the amino acid sequence described in SEQ ID NO: 24.

[0179] In various embodiments, the BCMA-specific binding domain includes a light chain variable domain containing the amino acid sequence described in SEQ ID NO: 31 and / or a heavy chain variable domain containing the amino acid sequence described in SEQ ID NO: 32.

[0180] In various embodiments, the BCMA-specific binding domain includes a light chain variable domain containing the amino acid sequence described in SEQ ID NO: 39 and / or a heavy chain variable domain containing the amino acid sequence described in SEQ ID NO: 40.

[0181] In various embodiments, the BCMA-specific binding domain includes a light chain variable domain containing the amino acid sequence described in SEQ ID NO: 47 and / or a heavy chain variable domain containing the amino acid sequence described in SEQ ID NO: 48.

[0182] In any of the BCMA-specific binding domains contemplated herein, one or more conserved mutations can be introduced into the CDR or framework sequence at a position where the residue is unlikely to be involved in antibody-antigen interaction. In some embodiments, such conserved mutations may be introduced into the CDR or framework sequence at a position where the residue is unlikely to be involved in interaction with BCMA, as determined based on the crystal structure. In some embodiments, likely interfaces (e.g., residues involved in antigen-antibody interaction) may be inferred from known structural information relating to other antigens that share structural similarities.

[0183] 2. Linker In certain embodiments, the CARs contemplated herein may be linker residues between various domains, for example, for proper spacing and conformation of the molecule. In certain embodiments, the linker is a sequence that links variable regions. The "sequence that links variable regions" is V H and V L The linker is an amino acid sequence that connects the two lower binding domains, providing an interactive and adaptable spacer function, and the resulting polypeptide retains the same specific binding affinity to the target molecule as an antibody containing the same light chain variable region and heavy chain variable region. The CARs contemplated herein may comprise one, two, three, four, or five or more linkers. In certain embodiments, the linker length is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any length of intervening amino acids. In some embodiments, the linker has an amino acid 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, or more amino acids.

[0184] An example of a linker is glycine polymer (G). n , Glycine-serine polymer (G 1-5 S 1-5 ) n Examples include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art (wherein n is an integer of at least 1, 2, 3, 4, or 5). Glycine and glycine-serine polymers are relatively amorphous and can therefore function as neutral tethers between domains of fusion proteins such as CARs contemplated herein. Glycine accesses significantly more phi-psi spaces than alanine and is far less restrictive than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). 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 consequently, the linker may include one or more parts that confer less flexible structures to provide a flexible linker as well as a desired CAR structure.

[0185] Other exemplary linker sequences include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 71); TGEKP (SEQ ID NO: 72) (see, for example, Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 73) (Pomerantz et al. 1995, see above); (GGGGS) n, where = 1, 2, 3, 4 or 5 (SEQ ID NO: 74) (Kim et al., PNAS 93, 1156-1160 (1996); EGKSSGSGSGSESKVD (SEQ ID NO: 75) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 76) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 77); LRQRDGERP (SEQ ID NO: 78); LRQKDGGGSERP (SEQ ID NO: 79); LRQKd(GGGS)2ERP (SEQ ID NO: 80). Alternatively, computer programs that can model both the DNA binding site and the peptide itself (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS Flexible linkers can be rationally designed by 91:11099-11103 (1994) or by phage display. In one embodiment, the linker contains the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 81) (Cooper et al., Blood, 101(4):1637-1644 (2003)).

[0186] 3. Spacer Domain In certain embodiments, the CAR binding domain is followed by one or more “spacer domains,” which refer to regions that move the antigen-binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999; 6:412-419). The spacer domains may be derived from natural, synthetic, semi-synthetic, or recombinant sources. In some embodiments, the spacer domain is an immunoglobulin moiety containing one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may also contain amino acid sequences of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0187] In one embodiment, the spacer domain includes the CH2 and CH3 domains of IgG1, IgG2, or IgG4, or an appropriate combination thereof.

[0188] 4. Hinged Domain The binding domain of a CAR is generally followed by one or more "hinge domains," which position the antigen-binding domain away from the effector cell surface. The hinge domains play a role in enabling proper cell / cell contact, antigen binding, and activation. CARs typically contain one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domains may originate from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domains may contain amino acid sequences from naturally occurring immunoglobulin hinge regions or modified immunoglobulin hinge regions.

[0189] Exemplary hinge domains suitable for use in CARs as envisioned herein include hinge domains derived from the extracellular regions of type 1 membrane proteins, including but not limited to CD8α, CD4, CD28, and CD7, which may be wild-type hinge domains derived from these molecules or may be modified. In one embodiment, the hinge is a PD-1 hinge or a CD152 hinge. In another embodiment, the hinge domain includes naturally occurring immunoglobin hinge domains, e.g., IgG1, IgG2, IgG3, or IgG4 hinges or appropriate combinations thereof. In yet another embodiment, the hinge domain includes IgG1 hinge / CH2 / CH3, IgG1 hinge / CH3 / hinge / M1, IgG4 hinge / CH2 / CH3, or IgG4 hinge / CH2.

[0190] In various embodiments, the CARs contemplated herein include a modified hinge region. Where used herein, the terms “modified hinge region,” “modified hinge region,” and “modified hinge domain” are used interchangeably and refer to (a) 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), and (b) a portion of a naturally occurring hinge region having at least 10 amino acids (e.g., at least 12, 1 (c) a length of 3, 14 or 15 amino acids, with up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or (c) a portion of a naturally occurring hinge region including the core hinge region (which is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long).

[0191] In various embodiments, the modified hinge region includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity with respect to suitable hinge domains / regions intended herein and / or known in the art. In some embodiments, the modified hinge region including the hinge sequence intended herein has four or fewer, three or fewer, or two or fewer amino acid substitutions and / or deletions.

[0192] In certain embodiments, one or more cysteine ​​residues in a naturally occurring hinge region / domain may be replaced by one or more other amino acid residues to generate a modified hinge domain. In some embodiments, the modified hinge domain includes one or more cysteine ​​residues replaced with serine or alanine. In another embodiment, the modified hinge domain includes one or more cysteine ​​residues replaced with serine. In yet another embodiment, the modified hinge domain includes one or more cysteine ​​residues replaced with alanine.

[0193] In certain embodiments, the modified hinge region includes the substitution of a proline residue with another amino acid residue (e.g., a serine residue).

[0194] 5. Transmembrane (TM) domain The "transmembrane domain" is the portion of the CAR that fuses the extracellular binding portion with the intracellular signaling domain, fixing the CAR to the plasma membrane of an immunoeffector cell. The TM domain may originate from any of the following sources: native, synthetic, semi-synthetic, or recombinant. The TM domain may originate from the alpha or beta chain of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1 (i.e., including at least one transmembrane region). In certain embodiments, the TM domain is synthetic and mainly contains hydrophobic residues such as leucine and valine.

[0195] In one embodiment, the CAR comprises a TM domain derived from PD1, CD152, CD28, or CD8α. In another embodiment, the CAR comprises a TM domain derived from PD1, CD152, CD28, or CD8α and a short oligolinker or polypeptide linker, which preferably has a length between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids linking the TM domain to the intracellular signaling or co-stimulatory domain of the CAR. Glycine-serine-based linkers provide particularly suitable linkers.

[0196] 6. Intracellular signal transduction domains In certain embodiments, the CARs contemplated herein include an intracellular signaling domain. The “intracellular signaling domain” refers to a portion of the CAR that is involved in transmitting the message of an effective BCMA CAR that binds to a human BCMA polypeptide into the interior of an immune effector cell to induce effector cell function, including, for example, activation, cytokine production, proliferation and cytotoxic activity, release of cytotoxic factors to CAR-binding target cells, or other cellular responses induced by antigens that bind to the extracellular CAR domain.

[0197] The term "effector function" refers to the specialized function of immune effector cells. The effector function of a T cell may be, for example, cytolytic activity or help activity, including cytokine secretion. Therefore, the term "intracellular signaling domain" refers to the portion of a protein that transduces effector function signals and directs the cell to perform specialized functions. While the entire intracellular signaling domain may usually be employed, it is often not necessary to use the entire domain. Such cleaved portions of an intracellular signaling domain can be used in place of the entire domain, to the extent that they transduce effector function signals. The term "intracellular signaling domain" means that it includes any cleaved portion of an intracellular signaling domain sufficient to transduce effector function signals.

[0198] It is known that signals generated by TCRs alone are insufficient for complete T cell activation, and that secondary, or co-stimulatory, signals are also required. Therefore, it can be said that T cell activation is mediated by two distinct classes of intracellular signaling domains: a primary signaling domain that initiates antigen-dependent primary activation by a TCR (e.g., the TCR / CD3 complex), and a co-stimulatory signaling domain that acts in an antigen-dependent manner to provide secondary, or co-stimulatory, signals. In a preferred embodiment, the CAR contemplated herein comprises an intracellular signaling domain including one or more "co-stimulatory signaling domains" and "primary signaling domains."

[0199] Primary signaling domains regulate the primary activation of the TCR complex either through stimulation or inhibition. Stimulative primary signaling domains may include signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0200] Exemplary examples of ITAMs containing primary signaling domains particularly used in the present invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In a particularly preferred embodiment, the CAR comprises the CD3ζ primary signaling domain and one or more co-stimulatory signaling domains. The intracellular primary signaling domain and the co-stimulatory signaling domains may be tandemly linked to the carboxyl terminus of the transmembrane domain in any order.

[0201] The CARs contemplated herein include one or more costimulatory signaling domains for enhancing the efficacy and proliferation of T cells expressing CAR receptors. As used herein, the terms “costimulatory signaling domain” or “costimulatory domain” refer to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that, upon binding to an antigen, provides a secondary signal required for the efficient activation and function of T lymphocytes. Exemplary examples of such co-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70.

[0202] In one embodiment, the CAR includes one or more co-stimulatory signaling domains selected from the group consisting of CD28, CD137, CD134, and CD3ζ primary signaling domains.

[0203] In another embodiment, the CAR includes CD28 and CD137 co-stimulatory signaling domains and a CD3ζ primary signaling domain.

[0204] In yet another embodiment, the CAR includes CD28 and CD134 co-stimulatory signaling domains and a CD3ζ primary signaling domain.

[0205] In one embodiment, the CAR includes CD137 and CD134 co-stimulatory signaling domains and a CD3ζ primary signaling domain.

[0206] Exemplary Embodiment of E.CAR In one embodiment, the CAR comprises an antibody or an antigen-specific binding fragment that binds to an antigen, a hinge region, a transmembrane domain, one or more intracellular costimulatory signaling domains from a costimulatory molecule, and a primary signaling domain. In a particular embodiment, the CAR contemplated herein comprises an anti-BCMA antibody or an antigen-binding fragment that specifically binds to a BCMA polypeptide expressed on B cells.

[0207] In one embodiment, CAR comprises: anti-BCMA scFv that binds to BCMA polypeptide; a spacer or hinge domain; a transmembrane domain derived from a polypeptide selected from the group consisting of: α, β, or zeta chains of T cell receptors, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1; and one or more intracellular costimulatory signaling domains from costimulatory molecules selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CA It includes primary signaling domains from RD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (IQOS), DAP10, LAT, NKD2C, SLP76, TRIM, TNFR2, and ZAP70; as well as primary signaling domains from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0208] In one embodiment, the CAR is an anti-BCMA scFv that binds to a BCMA polypeptide; a spacer domain or hinge domain selected from the group consisting of: CD8α hinge, CD4 hinge, CD28 hinge, CD7 hinge, PD-1 hinge, CD152 hinge, IgG1 hinge, IgG2 hinge, IgG3 hinge, IgG4 hinge, IgG1 hinge / CH2 / CH3, IgG1 hinge / CH3 / hinge / M1, IgG4 hinge / CH2 / CH3, or IgG4 hinge / CH2; Transmembrane domains derived from polypeptides selected from the following group: α, β, or zeta chains of T cell receptors, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1; co-scinae selected from the following group. One or more intracellular co-stimulatory signaling domains from stimulator molecules: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD It includes primary signaling domains from 223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (IQOS), DAP10, LAT, NKD2C, SLP76, TRIM, TNFR2, and ZAP70; as well as primary signaling domains from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0209] In one embodiment, the CAR is a transmembrane domain derived from a polypeptide selected from the group consisting of anti-BCMA scFv that binds to a BCMA polypeptide; and hinge domains selected from the group consisting of: CD8α hinge, CD4 hinge, CD28 hinge, CD7 hinge, PD-1 hinge, CD152 hinge, IgG1 hinge, IgG2 hinge, IgG3 hinge, IgG4 hinge, IgG1 hinge / CH2 / CH3, IgG1 hinge / CH3 / hinge / M1, IgG4 hinge / CH2 / CH3, or IgG4 hinge / CH2; and transmembrane domains selected from the group consisting of: anti-BCMA scFv that binds to a BCMA polypeptide; and hinge domains selected from the group consisting of: CD8α hinge, CD4 hinge, CD28 hinge, CD28 hinge, CD7 hinge, PD-1 hinge, CD152 hinge, IgG1 hinge, IgG2 hinge, IgG3 hinge, IgG4 hinge, IgG4 hinge, IgG1 hinge / CH2 / CH3, or IgG4 hinge / CH2. n: The α, β, or zeta chains of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1;TM domains are linked to the intracellular signaling domain of CAR, preferably with a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. A short oligolinker or polypeptide linker and one or more intracellular co-stimulatory signaling domains from a co-stimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAM) It includes primary signaling domains from F1), CD152(CTLA4), CD223(LAG3), CD270(HVEM), CD273(PD-L2), CD274(PD-L1), CD278(IQOS), DAP10, LAT, NKD2C, SLP76, TRIM, TNFR2, and ZAP70; as well as primary signaling domains from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0210] In certain embodiments, the CAR comprises an anti-BCMA scFv that binds to a BCMA polypeptide, a spacer domain containing one or more fragments of IgG2 and / or IgG4 hinge / CH2 / CH3 polypeptides, a CD28 transmembrane domain, a CD137 intracellular costimulatory signaling domain, and a CD3ζ primary signaling domain.

[0211] In certain embodiments, the CAR comprises a hinge domain containing an anti-BCMA scFv that binds to a BCMA polypeptide, an IgG1 hinge / CH2 / CH3 polypeptide, and a CD8α polypeptide; a CD8α transmembrane domain containing a polypeptide linker of about 3 to about 10 amino acids; a CD137 intracellular costimulatory signaling domain; and a CD3ζ primary signaling domain.

[0212] In certain embodiments, the CAR comprises a hinge domain containing an anti-BCMA scFv, CD8α polypeptide that binds to a BCMA polypeptide; a CD8α transmembrane domain containing a polypeptide linker of about 3 to about 10 amino acids; a CD134 intracellular costimulatory signaling domain; and a CD3ζ primary signaling domain.

[0213] In certain embodiments, the CAR comprises a hinge domain containing an anti-BCMA scFv that binds to a BCMA polypeptide, a CD8α polypeptide, a CD8α transmembrane domain containing a polypeptide linker of about 3 to about 10 amino acids, a CD28 intracellular costimulatory signaling domain, and a CD3ζ primary signaling domain.

[0214] Furthermore, the CAR designs envisioned herein enable improved proliferation, long-term persistence, and acceptable cytotoxicity in CAR-expressing T cells compared to unmodified T cells or T cells modified to express other CARs.

[0215] In various embodiments, the improved compositions and methods of adoptive cell therapy disclosed herein provide genetically modified immune effector cells (e.g., CAR T cells) that target cells expressing BCMA, have human-derived antigen-binding domains, exhibit improved cytokine release, and low antigen-independent signaling.

[0216] In certain embodiments, improved CAR T cells exhibit high IFNγ release in co-culture with BCMA-expressing cells. In some embodiments, improved CAR T cells exhibit similar or higher IFNγ release in co-culture with BCMA-expressing cells compared to the same CAR T cells, except that the CAR contains an extracellular domain containing mouse-derived anti-BCMA scFv. In some embodiments, the co-cultured BCMA-expressing cells are Daudi cells and / or HT1080.BCMA cells.

[0217] In certain embodiments, improved CAR T cells exhibit high IFNγ release in co-culture with low antigen density (low BCMA expression) cells. Cells or cell lines are characterized as having low BCMA expression if they have at least 5 times (e.g., at least 10 times, at least 5 times, at least 15 times, or at least 20 times) less surface BCMA expression than Daudi, HT1080.BCMA, and / or RPMI-8226 cells. In some embodiments, cells are cultured under the same or similar culture conditions. In some embodiments, low BCMA-expressing cells have at least 5 times less surface BCMA expression compared to Daudi, HT1080.BCMA, and / or RPMI-8226 cells. In some embodiments, low BCMA-expressing cells have at least 10 times less surface BCMA expression compared to HT1080.BCMA cells. In some embodiments, low BCMA-expressing cells have at least 10 times less surface BCMA expression compared to RPMI-8226 cells. Assays for measuring protein surface expression are known to those skilled in the art (e.g., FACS analysis).

[0218] In some embodiments, improved CAR T cells exhibit higher IFNγ release in co-culture with cells of lower antigen density (low BCMA expression) compared to the same CAR T cells, except that the CAR contains an extracellular domain containing mouse-derived anti-BCMA scFv. In some embodiments, the low BCMA-expressing cells are RL cells and / or Toledo cells.

[0219] In some embodiments, improved CAR T cells exhibit lower antigen-independent signaling compared to the same CAR T cells, except that the CAR contains an extracellular domain containing mouse-derived anti-BCMA scFv.

[0220] In some embodiments, improved CAR T cells exhibit lower antigen-independent signaling compared to the same CAR T cells, except that the CAR contains an extracellular domain containing mouse-derived anti-BCMA scFv.

[0221] F. polypeptide This disclosure intends, in part, to describe CAR polypeptides and their fragments, cells and compositions containing them, and vectors for expressing polypeptides. In preferred embodiments, polypeptides containing one or more CARs described in SEQ ID NOs. 50, 52, 54, 56, 58, 60, 62, 64, 66, and 68 are provided.

[0222] The terms “polypeptide,” “polypeptide fragment,” “peptide,” and “protein” are used interchangeably in their conventional sense, i.e., as amino acid sequences, unless otherwise specified. Polypeptides are not limited to a specific length; for example, a polypeptide may contain a full-length protein sequence or a fragment of a full-length protein, and a polypeptide may include post-translational modifications of the polypeptide, such as glycosylation, acetylation, and phosphorylation, as well as other natural and non-natural modifications known in the art. In various embodiments, the CAR polypeptides contemplated herein include a signal (or leader) sequence at the N-terminus of a protein, which directs cotranslational or posttranslational transmission of the protein. Exemplary examples of suitable signal sequences useful in the CARs disclosed herein include, but are not limited to, the IgG1 heavy chain signal sequence and the CD8α signal sequence. Polypeptides may be prepared using any of a variety of well-known recombinant and / or synthetic techniques. The polypeptides contemplated herein primarily encompass sequences having deletions, additions, and / or substitutions from one or more amino acids of the CARs disclosed herein.

[0223] Where used herein, “isolated peptide” or “isolated polypeptide,” etc., refers to the in vitro isolation and / or purification of a peptide or polypeptide molecule from the cellular environment and from association with other components of the cell; that is, not substantially related to the substance in vivo. Similarly, “isolated cell” refers to a cell obtained from an in vivo tissue or organ that is substantially free of extracellular matrix.

[0224] Polypeptides include "polypeptide variants." Polypeptide variants may differ from naturally occurring polypeptides by one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be synthetically produced, for example, by modifying one or more of the polypeptide sequences described above. For example, in certain embodiments, it may be desirable to improve the binding affinity and / or other biological properties of the CAR by introducing one or more substitutions, deletions, additions, and / or insertions into the binding domain, hinge, TM domain, co-stimulatory signaling domain, or primary signaling domain of the CAR polypeptide. Preferably, the polypeptides intended herein include polypeptides having at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% amino acid identity with respect to them.

[0225] Polypeptides include "polypeptide fragments." A polypeptide fragment refers to a monomeric or polymeric polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion or substitution of a naturally occurring or recombinantly produced polypeptide. In certain embodiments, a polypeptide fragment may contain an amino acid chain of at least 5 to about 500 amino acids in length. In certain embodiments, the fragment is at least 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acid lengths. Particularly useful polypeptide fragments include a functional domain containing an antigen-binding domain or an antibody fragment. In the case of mouse anti-BCMA antibodies, useful fragments include, but are not limited to, the following: CDR regions, CDR3 regions of the heavy or light chain, heavy or light chain variable regions, antibody chains containing two CDRs, or parts of variable regions, etc.

[0226] Polypeptides may also be fused in-frame or conjugated to a linker or other sequence for ease of synthesis, purification, or identification of polypeptides (e.g., poly-His), or to enhance binding of polypeptides to a solid support.

[0227] As described above, polypeptides may be modified in various ways, including amino acid substitution, deletion, cleavage, and insertion. Methods for such operations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be produced by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification are known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al. (1987, Methods in Enzymol, 154:367-382), U.S. Patent No. 4,873,192, Watson, JD et al. (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and the references cited therein. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0228] In certain embodiments, the variant includes a conservative substitution. A “conservative substitution” is a substitution in which an amino acid is replaced by another amino acid having similar properties, and a person skilled in the art of peptide chemistry would predict that such a substitution does not substantially alter the secondary structure and hydrophobic / hydrophilic properties of the polypeptide. Modifications may be made in the structures of the polynucleotides and polypeptides of this disclosure to obtain functional molecules encoding variant or derivative polypeptides that still have the desired properties. If it is desired to modify the amino acid sequence of a polypeptide to create an equivalent or improved variant polypeptide as intended herein, a person skilled in the art may, for example, modify one or more codons in the coding DNA sequence according to Table 1. [Table 1]

[0229] Guidelines for determining which amino acid residues can be substituted, inserted, or deleted without loss of biological activity can be found using computer programs well known in the art, such as DNASTARTM software. Amino acid changes in protein variants disclosed herein are preferably conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of one of the amino acid families associated in its side chain. Natural amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. Appropriate conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art recognize that a single amino acid substitution in a non-essential region of a polypeptide generally does not significantly alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.Co., p.224). Exemplary conservative substitutions are described in U.S. Provisional Patent Application No. 61 / 241,647, the disclosure of which is incorporated herein by reference.

[0230] When making such modifications, the hydrophobicity and hydrophilicity indices of amino acids can be taken into consideration. The importance of the hydropathic amino acid index in conferring interactive biological functions to proteins is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). Each amino acid has been assigned a hydrophobicity and hydrophilicity index based on its hydrophobic and charge properties (Kyte and Doolittle, 1982). These values ​​are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0231] It is known in the art that certain amino acids can be substituted with other amino acids having similar hydrophobicity or hydrophilicity indices or scores, resulting in proteins with similar biological activity, i.e., proteins that are still biologically and functionally equivalent. When making such modifications, substitutions of amino acids with hydrophobicity indices within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more preferred. It is also understood in the art that substitutions of similar amino acids can be effectively carried out based on hydrophilicity.

[0232] As detailed in U.S. Patent No. 4,554,101, the following hydrophilic values ​​are assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that amino acids can be substituted with other amino acids having similar hydrophilic values, and that biologically equivalent, and especially immunologically equivalent, proteins can still be obtained. In such changes, substitution of amino acids with a hydrophilicity value of ±2 or less is preferred, amino acids within ±1 are particularly preferred, and amino acids within ±0.5 are even more preferred.

[0233] As described above, amino acid substitutions may be based on the relative similarities of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, and size.

[0234] Polypeptide variants further include glycosylated forms, aggregated conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (e.g., pegylated molecules). Covalent variants can be prepared by linking functionality to groups present in amino acid chains or in N-terminal or C-terminal residues, as is known in the art. Variants also include allelic variants, species variants, and mutant proteins. Cleavage or deletion of regions that do not affect the functional activity of a protein are also variants.

[0235] In one embodiment, when the expression of two or more polypeptides is desired, the polynucleotide sequences encoding them can be separated by an IRES sequence as discussed elsewhere herein. In another embodiment, two or more polypeptides can be expressed as a fusion protein comprising one or more self-cleaving polypeptide sequences.

[0236] The polypeptides of the present disclosure include fusion polypeptides. In a preferred embodiment, a fusion polypeptide and a polynucleotide encoding the fusion polypeptide, such as a CAR, are provided. Fusion polypeptides and fusion proteins refer to polypeptides having at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more polypeptide segments. Fusion polypeptides are typically linked from the C-terminus to the N-terminus, although they may be linked from C-terminus to C-terminus, from N-terminus to N-terminus, or from N-terminus to C-terminus. The polypeptides of the fusion protein may be in any order or in a specified order. The fusion polypeptide or fusion protein can also include conservatively modified variants, polymorphic variants, alleles, variants, subsequences, and interspecies homologs as long as the desired transcriptional activity of the fusion polypeptide is maintained. The fusion polypeptide may be made by chemical synthesis methods or by chemical bonding between two parts, or may be generally prepared using other standard methods. The linked DNA sequences comprising the fusion polypeptide are operably linked to appropriate transcriptional or translational control elements as discussed elsewhere herein.

[0237] In one embodiment, the fusion partner includes a sequence that aids in expressing the protein (expression enhancer) at a higher yield than a native recombinant protein. Other fusion partners can be selected to increase the solubility of the protein, or to enable the protein to be targeted to a desired intracellular compartment, or to facilitate the transport of the fusion protein across the cell membrane.

[0238] The fusion polypeptide may further include a polypeptide cleavage signal between each of the polypeptide domains contemplated herein. The polypeptide moieties can be incorporated into any linker peptide sequence. Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004, Traffic, 5(8);616-26).

[0239] Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Examples of protease cleavage sites include, but are not limited to, potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, byovirus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (Waikavirus) 3C-like protease, PYVF (Parsnip yellow fleck virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase cleavage sites. Due to its high cleavage stringency, the TEV (tobacco etch virus) protease cleavage site is preferred in one embodiment, for example, EXXYXQ(G / S) (SEQ ID NO: 82), for example, ENLYFQG (SEQ ID NO: 83) and ENLYFQS (SEQ ID NO: 84), where X represents any amino acid (cleavage by TEV occurs between Q and G or Q and S).

[0240] In certain embodiments, the polypeptide cleavage signal is a viral autocleavage peptide or a ribosome skipping sequence.

[0241] Exemplary examples of ribosome skipping sequences include, but are not limited to, the following: 2A or 2A-like sites, sequences, or domains (Donnelly et al., 2001 J. Gen. Virol. 82:1027-1041). In certain embodiments, the viral 2A peptide is an aphthous virus 2A peptide, a potivirus 2A peptide, or a cardiovirus 2A peptide.

[0242] In one embodiment, the virus 2A peptide is selected from the group consisting of foot-and-mouth disease virus (FMDV) 2A peptide, equine rhinitis A virus (ERAV) 2A peptide, zosea signal virus (TaV) 2A peptide, porcine tescovirus-1 (PTV-1) 2A peptide, tyrovirus 2A peptide, and encephalomyocarditis virus 2A peptide.

[0243] Table 2 shows examples of area 2A. [Table 2]

[0244] In a preferred embodiment, the polypeptides intended herein include CAR polypeptides.

[0245] G. Polynucleotide In preferred embodiments, polynucleotides encoding one or more CAR polypeptides, such as SEQ ID NOs: 49, 51, 53, 55, 57, 59, 61, 63, 65, and 67, are provided. In some embodiments, the polynucleotide encodes an amino acid sequence described in any of SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, and 68.

[0246] In other embodiments, polynucleotides, antibodies, or fragments thereof encoding anti-BMCA CARs are provided. In some embodiments, the polynucleotides encode anti-BMCA CARs, antibodies, or fragments thereof comprising variable light chain CDRL1, CDRL2, and CDRL3 sequences described in SEQ ID NOs: 1-3, 9-11, 17-19, 25-27, 33-35, or 41-43, and / or variable heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs: 4-6, 12-14, 20-22, 28-30, 36-38, or 44-46.

[0247] In some embodiments, the polynucleotide contains a variable light chain comprising an amino acid sequence described in any of SEQ ID NOs: 7, 15, 23, 31, 39, or 47, and is an anti-BMCA The polynucleotide encodes an anti-BMCA CAR, antibody, or fragment thereof. In some embodiments, the polynucleotide encodes an anti-BMCA CAR, antibody, or fragment thereof comprising a variable light chain containing an amino acid sequence described in any of SEQ ID NOs: 8, 16, 24, 32, 40, or 48. In some embodiments, the polynucleotide encodes an anti-BMCA CAR, antibody, or fragment thereof comprising a variable light chain containing an amino acid sequence described in any of SEQ ID NOs: 7, 15, 23, 31, 39, or 47, and a variable heavy chain containing an amino acid sequence described in any of SEQ ID NOs: 8, 16, 24, 32, 40, or 48.

[0248] As used herein, the terms “polynucleotide” or “nucleic acid” refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides may be single-stranded or double-stranded, and may be recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. A polynucleotide refers to a multimer of nucleotides having a length of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 nucleotides, and includes ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide, as well as all intermediate lengths. In this context, “intermediate length” will be readily understood to mean any length between the cited values, such as 6, 7, 8, 9, 101, 102, 103, 151, 152, 153, 201, 202, 203, etc. In certain embodiments, the polynucleotide or variant has at least, or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the reference sequence.

[0249] As used herein, “isolated polynucleotide” refers to a polynucleotide purified from a sequence adjacent to it in its naturally occurring state, for example, a DNA fragment removed from a sequence normally adjacent to the fragment. In certain embodiments, “isolated polynucleotide” also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not exist in nature but are artificially produced. In certain embodiments, the isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, or a polynucleotide obtained from or derived from a recombinant source.

[0250] In various embodiments, the polynucleotide contains mRNA encoding the polypeptide intended herein. In some embodiments, the mRNA contains a cap, one or more nucleotides, and a poly(A) tail.

[0251] In certain embodiments, polynucleotides may be codon-optimized. As used herein, the term “codon optimization” refers to the substitution of codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability, and / or activity of the polypeptide. Factors influencing codon optimization include, but are not limited to, one or more of the following: (i) variations in codon bias between two or more organisms or genes or synthetically constructed bias tables; (ii) variations in the degree of codon bias within an organism, gene, or gene set; (iii) systematic variations in codons, including context; (iv) variations in codons associated with their decoded tRNA; (v) variations in codons associated with the GC% of the entire triad or any one position in the triad; (vi) variations in similarity to a reference sequence, such as a natural sequence; (vii) variations in codon frequency cutoffs; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence underlying the design of the codon substitution set; (x) synthetic variations in the codon set for each amino acid; and / or (xi) isolated removal of incorrect translation start sites.

[0252] As used herein, terms such as “polynucleotide variant” and “variant” refer to a polynucleotide that exhibits a corresponding sequence identity with a reference polynucleotide sequence, or a polynucleotide that hybridizes with a reference sequence under stringent conditions as defined herein. These terms include polynucleotides in which one or more nucleotides are added, deleted, or substituted with different nucleotides compared to the reference polynucleotide. In this regard, it is understood in the art that a modification, including mutation, addition, deletion, and substitution, is made to a reference polynucleotide, and the modified polynucleotide may retain the biological function or biological activity of the reference polynucleotide.

[0253] Polynucleotide variants include polynucleotide fragments encoding biologically active polypeptide fragments or variants. As used herein, the term “polynucleotide fragment” means at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 22, 23, 24, 25, 26, 27, 22, 23, 24, 25, 26, 27, 22, 23, 24, 25, 26, 27, 22, 23, 24, 25, 26, 27, 22, 23, 24, 25, 26, 27, 22, 23, 24, 25, 26, 27, 22, 23, 24, 25, 26, 27, 22, 23, 24, 25, 26, 27, 22, 23, 26 This refers to polynucleotide fragments with nucleotide lengths of 8, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, and 1700 or more. A polynucleotide fragment refers to a polynucleotide encoding a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion, or a naturally occurring or recombinant polypeptide with one or more amino acid substitutions.

[0254] When used herein, the term "sequence identity," or for example, "sequences that are 50% identical to ~," refers to the degree to which sequences are identical nucleotide-wise or amino acid-wise on a comparison window. Thus, the "percentage of sequence identity" may be calculated by comparing two optimal alignment sequences on a comparison window, determining the number of positions in both sequences where identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) are present, calculating the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. The materials include nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity with respect to any of the reference sequences contemplated herein, wherein the polypeptide variant maintains the biological activity of at least one of the reference polypeptides.

[0255] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity.” A “reference sequence” is a sequence containing nucleotides and amino acid residues, with a length of at least 12 monomer units, often 15 to 18 monomer units, and often at least 25 monomer units. Each of the two polynucleotides may contain (1) sequences similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence) and (2) sequences that diverge between the two polynucleotides. Sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides in a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to at least six consecutive positions, usually about 50 to about 100, more commonly about 100 to about 150 conceptual segments, and the sequences are compared to the reference sequence in the same number of consecutive positions after the two sequences have been optimally aligned. The comparison window may contain approximately 20% or less of additions or deletions (i.e., gaps) compared to the reference sequence (which contains no additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning the comparison window can be achieved by computerized execution of algorithms (GAP, BESTFIT, FASTA, and TFASTA) in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, or by inspection and best alignment (i.e., yielding the highest homology across the comparison window) generated by one of various selected methods. See, for example, the BLAST family of programs disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389.A detailed discussion of array analysis can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994 - 1998, Unit 19.3 in Chapter 15.

[0256] Terms used to describe the directionality of polynucleotides include 5' (usually the end of the polynucleotide with a free phosphate group) and 3' (usually the end of the polynucleotide with a free hydroxyl (OH) group). A polynucleotide sequence can be annotated in the 5' to 3' orientation or the 3' to 5' orientation. For DNA and mRNA, the 5' to 3' strand is designated the "sense" strand, "plus" strand, or "coding" strand because its sequence is identical to the sequence of the pre - messenger (pre - mRNA) [except that uracil (U) in RNA replaces thymine (T) in DNA]. For DNA and mRNA, the complementary 3' to 5' strand, which is the strand transcribed by RNA polymerase, is designated the "template", "antisense", "minus", or "non - coding" strand. As used herein, the term "reverse orientation" refers to a 5' - 3' sequence written in the 3' - 5' orientation, or a 3' - 5' sequence written in the 5' - 3' direction.

[0257] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., sequences of nucleotides) that are related by base - pairing rules. For example, the complementary strand of the DNA sequence 5'AGTCATG3' is 3'TCAGTAC5'. The latter sequence is often written as the reverse complement, 5' C A T G A C T 3', with the 5' end on the left and the 3' end on the right. A sequence that is equal to its reverse complement is said to be a palindromic sequence. Complementarity can be "partial", where only some of the nucleic acid bases match according to the base - pairing rules. Or, "complete" or "total" complementarity can exist between nucleic acids.

[0258] Furthermore, as a result of the degeneracy of the genetic code, it will be understood by those skilled in the art that there are many nucleotide sequences that encode fragments of polypeptides or variants thereof, as intended herein. Some of these polynucleotides carry minimal homology to the nucleotide sequences of any native gene. Nevertheless, polynucleotides that are altered by differences in codon usage frequency are intended, and in certain embodiments, for example, polynucleotides optimized for human and / or primate codon selection are intended. Furthermore, alleles of genes containing the polynucleotide sequences provided herein may also be used. Alleles are endogenous genes that are modified as a result of one or more mutations, such as nucleotide deletions, additions, and / or substitutions.

[0259] As used herein, the terms “nucleic acid cassette” or “expression cassette” refer to a gene sequence in a vector capable of expressing RNA and subsequently a polypeptide. In one embodiment, the nucleic acid cassette contains the gene of interest, for example, the polynucleotide of interest. In another embodiment, the nucleic acid cassette contains one or more expression regulatory sequences, for example, a promoter, an enhancer, a poly(A) sequence, and the gene of interest, such as the polynucleotide of interest. A vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleic acid cassettes. The nucleic acid cassettes are locologically and sequentially oriented within the vector. This allows the nucleic acids in the cassette to be transcribed into RNA, translated into proteins or polypeptides as needed, undergo appropriate post-translational modifications required for activity in transformed cells, and be targeted to appropriate intracellular compartments, thereby being moved to compartments suitable for biological activity or secreted into extracellular compartments. The cassettes preferably have 3' and 5' ends adapted for immediate insertion into the vector, for example, each end having a restriction endonuclease site. In a preferred embodiment, the nucleic acid cassette encodes a CAR. The cassette can be removed and inserted as a single unit into a plasmid or viral vector.

[0260] The polynucleotide comprises the polynucleotide of interest. As used herein, the term “polynucleotide of interest” refers to a polynucleotide encoding a polypeptide, polypeptide variant, or fusion polypeptide. The vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polynucleotides of interest. In certain embodiments, the polynucleotide of interest encodes a polypeptide that provides a therapeutic effect in the treatment or prevention of a disease or injury. The polynucleotide of interest, and the polypeptide encoded therefrom, comprises both the wild-type polypeptide and the polynucleotides encoding the functional variant and fragment. In certain embodiments, the functional variant has at least 80%, at least 90%, at least 95%, or at least 99% identity with respect to the corresponding wild-type reference polynucleotide or polypeptide sequence. In certain embodiments, the functional variant or fragment has at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the biological activity of the corresponding wild-type polypeptide.

[0261] The polynucleotides envisioned herein, regardless of the length of the coding sequence itself, may be combined with other DNA sequences disclosed elsewhere herein or known in the art, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kossack sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRESs), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, etc., and as a result, their overall lengths may vary considerably. Therefore, polynucleotide fragments of substantially any length may be used in particular embodiments, and the full length is preferably limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0262] Polynucleotides may be prepared, manipulated, and / or expressed using any of the various established techniques known and available in the art. To express a desired polypeptide, the nucleotide sequence encoding the polypeptide may be inserted into a suitable vector.

[0263] Examples of vectors, but not limited to, include plasmids, self-replicating sequences, and transposition factors, such as piggyBac, Sleeping Beauty, Mos1, Tc1 / mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince, and their derivatives.

[0264] Additional examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phages or M13 phages, and animal viruses.

[0265] Examples of viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40).

[0266] Examples of expression vectors, but not limited to, include the pClneo vector for expression in mammalian cells (Promega), and pLenti4 / V5-DEST®, pLenti6 / V5-DEST®, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequences of polypeptides disclosed herein may be ligated into such expression vectors for polypeptide expression in mammalian cells.

[0267] In one embodiment, the vector encoding the CAR intended herein comprises the polynucleotide sequences described in SEQ ID NOs: 49, 51, 53, 55, 57, 59, 61, 63, 65, and 67.

[0268] In certain embodiments, the vector is an episomal vector, or a vector maintained outside of chromosomes. As used herein, the term “episomal” means a vector that can replicate without being integrated into the host's chromosomal DNA and is not gradually reduced by host cell division, and therefore also means that the vector replicates outside of chromosomes or episomally.

[0269] The "regulatory elements" and "regulatory sequences" present in an expression vector are the untranslated regions of the vector, including the replication origin, selection cassette, promoter, enhancer, translation initiation signal (Shine Dalgarno sequence or Kozak sequence), introns, polyadenylated sequences, and 5' and 3' untranslated regions, which interact with host cellular proteins to perform transcription and translation. These factors may have altered strength and specificity. Depending on the vector system and host used, any number of appropriate transcription and translation factors, including ubiquitous and inducible promoters, may be used.

[0270] In certain embodiments, vectors include, but are not limited to, expression vectors and viral vectors, and include exogenous, endogenous, or heterologous regulatory sequences such as promoters and / or enhancers. “Endogenous” regulatory sequences are sequences that are naturally linked to a given gene in the genome. “Exogenous” regulatory sequences are those that are positioned alongside a gene by means of genetic engineering (i.e., molecular biological techniques) so that the transcription of that gene is directed by the linked enhancer / promoter. “Hexlogous” regulatory sequences are exogenous sequences that originate from a different species than the cell being genetically engineered.

[0271] As used herein, the term “promoter” refers to a recognition site of a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes the polynucleotide operably ligated to the promoter. In certain embodiments, a promoter operating in mammalian cells contains an AT-rich region located approximately 25–30 bases upstream from the transcription initiation site, and / or a CNCAAT region which is another sequence located 70–80 bases upstream from the transcription initiation site, where N can be any nucleotide.

[0272] The term "enhancer" refers to a DNA segment containing a sequence that can provide transcriptional enhancement, and in some cases can function independently of the orientation of other regulatory sequences. Enhancers can function in cooperation with or additively with promoters and / or other enhancer elements. The term "promoter / enhancer" refers to a DNA segment containing a sequence that can provide both promoter and enhancer functions.

[0273] The term “operably linked” means a juxtaposition of the described components in a relationship that allows them to function in the manner intended. In one embodiment, the term refers to a functional linkage between a nucleic acid expression regulatory sequence (such as a promoter and / or enhancer) and a second polynucleotide sequence, e.g., the polynucleotide of interest, where the expression regulatory sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0274] As used herein, the term “structural regulatory sequence” refers to a promoter, enhancer, or promoter / enhancer that enables continuous or sequential transcription of a manipulably linked sequence. A structural regulatory sequence may be a “ubiquitous” promoter, enhancer, or promoter / enhancer that enables expression in a variety of cell and tissue types, or it may be a “cell-specific,” “cell type-specific,” “cell line-specific,” or “tissue-specific” promoter, enhancer, or promoter / enhancer that enables expression in limited cell and tissue types, respectively.

[0275] Examples of ubiquitous expression regulatory sequences suitable for use in specific embodiments include, but are not limited to, the cytomegalovirus (CMV) pre-early promoter, viral Simianvirus 40 (SV40) (e.g., early or late), Moroni mouse leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, vaccinia virus-derived H5, P7.5 and P11 promoters, elongation factor 1 alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde triphosphate dehydrogenase (GAPDH), eukaryotic cell translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), and heat shock protein 90kDa. Examples include beta, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (β-KIN), human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer, negative regulatory region deletion type, and dl587rev primer binding site substitution (MND) U3 promoter (Haas et al. Journal of Virology. 2003; 77(17): 9439-9450).

[0276] In one embodiment, the vector contains an MNDU3 promoter.

[0277] In one embodiment, the vector contains the EF1a promoter, which includes the first intron of the human EF1a gene.

[0278] In one embodiment, the vector contains an EF1a promoter lacking the first intron of the human EF1a gene.

[0279] In certain embodiments, it may be desirable to express polynucleotides containing CARs derived from a T cell-specific promoter.

[0280] As used herein, “conditional expression” may refer to any type of conditional expression, including but not limited to inducible expression, repressive expression, and expression in cells or tissues having a particular physiological, biological, or disease state. This definition is not intended to exclude cell-type-specific or tissue-specific expression. Some embodiments provide conditional expression of a polynucleotide of interest, where expression is controlled, for example, by exposing cells, tissues, or organisms to a treatment or condition in which the polynucleotide is expressed, or to a treatment or condition in which the expression of the polynucleotide encoded by the polynucleotide of interest is increased or decreased.

[0281] Examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, e.g., promoters of genes encoding glucocorticoid receptors or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionein promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible with interferon), and the "GeneSwitch" mifepristone regulatory system (Sirin et al.). Examples include al., 2003, Gene, 323:67), cumate-inducible gene switches (WO2002 / 088346), and tetracycline-dependent regulatory systems.

[0282] Conditional expression can also be achieved by using site-specific DNA recombinases. According to certain embodiments, the vector contains at least one (typically two) sites for recombination mediated by the site-specific recombinase. As used herein, the terms “recombinase” or “site-specific recombinase” include excisive or integrated proteins, enzymes, cofactors or related proteins involved in a recombination reaction that includes one or more recombination sites (e.g., 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 50, etc.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing recombinant protein sequences or fragments thereof), fragments and variants thereof. Exemplary examples of recombinases suitable for use in specific embodiments include, but are not limited to, Cre, Int, IHF, Xis, Flop, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolverase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0283] The vector may contain one or more recombination sites for any of a wide range of site-specific recombinases. It should be understood that the target sites of site-specific recombinases are added to any(yes) sites required for integration of the vector, e.g., a retroviral vector or a lentiviral vector. As used herein, the terms “recombinant sequence,” “recombinant site,” or “site-specific recombinant site” refer to the specific nucleic acid sequence that a recombinase recognizes and binds to.

[0284] For example, one recombination site of Cre recombinase is loxP, a 34-base pair sequence containing two 13-base pair inverse repeats (which remain as recombinase binding sites) adjacent to an 8-base pair core sequence (Sauer, B., Current Opinion in See Figure 1 in Biotechnology 5:521-527 (1994). Other exemplary loxP sites include, but are not limited to, lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), lox71 (Albert et al., 1995), and lox66 (Albert et al., 1995).

[0285] Preferred recognition sites for FLP recombinase include, but are not limited to, FRT (McLeod et al., 1996), F1, F2, F3 (Schlake and Bode, 1994), F4, F5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), and FRT(RE) (Senecoff et al., 1988).

[0286] Other examples of recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme λ integrase, e.g., phi-c31. The φC31 SSR mediates recombination only between the heterotype sites attB (length 34 bp) and attP (length 39 bp) (Groth et al., 2000). attB and attP are named after the attachment sites of phage integrases on bacterial and phage genomes, respectively, but both contain incomplete inverted repeats that are likely linked by the φC31 homodimer (Groth et al., 2000). The product sites, attL and attR, are further effectively inactive to φC31-mediated recombination (Belteki et al., 2003), making the reaction irreversible. To catalyze insertion, it has been found that attB-carrying DNA is more readily inserted into genomic attP sites than attP sites are inserted into genomic attB sites (Thyagarajan et al., 2001; Belteki et al., 2003). Therefore, a typical strategy is to position the attP-carrying "docking site" at a defined locus through homologous recombination, and then bind it to the attB-carrying incoming sequence for insertion.

[0287] As used herein, "internal ribosome entry site" or "IRES" refers to a factor that facilitates the direct entry of internal ribosomes into the start codon of a cistron (protein-coding region), such as ATG, thereby producing cap-independent gene translation. For example, Jackson et al., 1990. Trends Biochem See Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. In certain embodiments, the vector comprises one or more target polynucleotides encoding one or more polypeptides. In certain embodiments, to achieve efficient translation of each of the plurality of polypeptides, the polynucleotide sequence may be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides. In one embodiment, the IRES used for the polynucleotides expected herein is an EMCV IRES.

[0288] As used herein, the term “Kozak sequence” refers to a short nucleotide sequence that greatly promotes the initial binding of mRNA to the ribosomal small subunit and increases translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO: 107), where R is purine (A or G) (Kozak, 1986 Cell. 44(2):283-92 and Kozak, 1987 Nucleic Acids Res. 15(20):8125-48). In certain embodiments, the vector comprises a polynucleotide having a consensus Kozak sequence encoding a desired polypeptide, e.g., CAR.

[0289] Factors that induce efficient termination and polyadenylation of heterologous nucleic acid transcripts increase the expression of heterologous genes. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, the vector contains a polyadenylation sequence 3' of the polynucleotide encoding the polypeptide to be expressed. As used herein, the terms “polyA site” or “polyA sequence” refer to a DNA sequence that is directed by RNA polymerase II to both terminate and polyadenylate a developing RNA transcript. Polyadenylation sequences can enhance mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thus contributing to improved translation efficiency. Cleavage and polyadenylation are directed by poly(A) sequences in RNA. The core poly(A) sequence of mammalian premRNA has two recognition elements adjacent to the cleavage polyadenylation site. Typically, a nearly immutable AAUAAA hexamer is located 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the initial transcript occurs between these two elements, with up to 250 adenosines added to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is the best poly(A) sequence (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is the poly(A) sequence of SV40, the bovine growth hormone poly(A) sequence (BGHpA), the rabbit β-globin poly(A) sequence (rβgpA), their variants, or another suitable heterologous or endogenous poly(A) sequence known in the art.

[0290] In some embodiments, polynucleotides, or cells containing polynucleotides, utilize suicide genes, including inducible suicide genes, to reduce the risk of direct toxicity and / or uncontrolled amplification. In certain embodiments, the suicide genes are not immunogenic to the polynucleotide-containing host or cells. Certain examples of suicide genes that may be used are caspase-9, caspase-8, or cytosine deaminase. Caspase-9 may be activated using a specific chemical inducer of dimerization (CID).

[0291] H. Vector In certain embodiments, one or more polynucleotides encoding a CAR are introduced into cells (e.g., immune effector cells) by a non-viral or viral vector. In some embodiments, a polycistronic polynucleotide encoding a CAR is introduced into cells by a non-viral or viral vector.

[0292] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is typically inserted into, for example, the vector nucleic acid molecule. The vector may contain sequences directed toward self-replication within the cell, or it may contain sequences sufficient to enable integration into host cell DNA. In certain embodiments, a non-viral vector is used to deliver one or more polynucleotides intended herein to T cells.

[0293] Examples of nonviral vectors include, but are not limited to, mRNA, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0294] Exemplary methods for nonviral delivery of polynucleotides or vectors intended in certain embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, bioristics, virosomes, liposomes, immunoliposomes, nanoparticles, polycations or lipids; nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran-mediated transcription, gene guns, and heat shock.

[0295] Examples of polynucleotide delivery systems suitable for use in specific embodiments intended in particular embodiments include, but are not limited to, systems provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides are described in the literature, e.g., Liu et al. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal See of drug delivery. 2011:1-12. Antibody-targeted delivery, bacterial-inducible delivery, and non-biological nanocell-based delivery are also anticipated in certain embodiments.

[0296] In various embodiments, the polynucleotide is an mRNA introduced into a cell to transiently express a desired polypeptide.

[0297] As used herein, “transient” refers to the expression of a non-integrated transgene over a period of several hours, days, or weeks, the expression period being shorter than the expression period of a polynucleotide if it is integrated into the intracellular genome or contained within a stable plasmid replicon.

[0298] In certain embodiments, the mRNA encoding the polypeptide is mRNA transcribed in vitro. As used herein, “RNA transcribed in vitro” refers to RNA synthesized in vitro, preferably mRNA. Generally, RNA transcribed in vitro is produced from an in vitro transcription vector, which contains a template used to produce in vitro transcribed RNA.

[0299] In certain embodiments, mRNA may further comprise a 5' cap or a modified 5' cap, and / or a poly(A) sequence. As used herein, the 5' cap (RNA cap, RNA 7-methylguanosine cap, or RNA m) may further comprise a 5' cap or a modified 5' cap. 7G A cap (also called a 5' cap) is a modified guanine nucleotide added to the “pre” or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap contains a terminal group that is ligated to the first transcription nucleotide, recognized by ribosomes, and protected from RNase. The capping portion may be modified to regulate mRNA function, such as translation stability or efficiency. In certain embodiments, the mRNA contains a poly(A) sequence of about 50 to about 5000 adenines. In one embodiment, the mRNA contains a poly(A) sequence of about 100 to about 1000 bases, about 200 to about 500 bases, or about 300 to about 400 bases. In one embodiment, the mRNA contains a poly(A) sequence of about 65 bases, about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, or about 1000 bases or more. Poly(A) sequences can be chemically or enzymatically modified to regulate mRNA function, such as localization, stability, or translation efficiency.

[0300] Viral vectors containing polynucleotides as envisioned in certain embodiments can be delivered in vivo by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or topical application to individual patients, typically as described below. Alternatively, the vector may be delivered in vitro to cells such as those explanted from individual patients (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirate, tissue biopsy, etc.) or hematopoietic stem cells from a universal donor, and then re-implanted into the patient.

[0301] In one embodiment, a viral vector containing a polynucleotide encoding a CAR is administered directly to an organism for in vivo cell transduction. Alternatively, naked DNA may be administered. Administration is by any route commonly used to introduce molecules to final contact with blood or tissue cells, including but not limited to injection, infusion, topical application, and electroporation. While suitable methods for administering such nucleic acids are available and known to those skilled in the art, a particular composition may be administered using multiple routes, and a particular route can often provide a more immediate and effective response than another route.

[0302] Examples of viral vector systems suitable for use in the specific embodiments intended herein include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0303] In various embodiments, one or more polynucleotides encoding a CAR are introduced into immune effector cells, such as T cells, by transducing cells with recombinant adeno-associated virus (rAAV) containing one or more polynucleotides.

[0304] AAV is a small (approximately 26 nm) replication-defective virus, primarily covered by an episomal non-enveloping membrane. AAV can infect both dividing and non-dividing cells and integrate its genome into the host cell's genome. Recombinant AAV (rAAV) typically consists at least of a transgene and its regulatory sequence, as well as 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequence is approximately 145 bp long. In certain embodiments, rAAV contains ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0305] In some embodiments, chimeric rAAVs are used. The ITR sequence is isolated from one AAV serotype, and the capsid sequence is isolated from another AAV serotype. For example, an rAAV containing an ITR sequence from AAV2 and a capsid sequence from AAV6 is called AAV2 / AAV6. In certain embodiments, an rAAV vector may contain an ITR from AAV2 and a capsid protein derived from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In preferred embodiments, the rAAV contains an ITR sequence from AAV2 and a capsid sequence from AAV6. In preferred embodiments, the rAAV contains an ITR sequence from AAV2 and a capsid sequence from AAV2.

[0306] In some embodiments, the manipulation and selection method may be performed on AAV capsids to increase the probability that they will be transduced into target cells.

[0307] The construction, manufacture, and purification of rAAV vectors are disclosed, for example, in U.S. Patents 9,169,494, 9,169,492, 9,012,224, 8,889,641, 8,809,058, and 8,784,799, each of which is incorporated herein by reference in whole.

[0308] In various embodiments, one or more polynucleotides encoding CARs are introduced into immunoeffector cells, and the cells are transduced with a retrovirus, such as a lentivirus, thereby containing one or more polynucleotides.

[0309] As used herein, the term “retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then co-integrates its genomic DNA into the host genome. Exemplary retroviruses suitable for use in particular embodiments include, but are not limited to, Moloney mouse leukemia virus (M-MuLV), Moloney mouse sarcoma virus (MoMSV), Harvey mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, friend mouse leukemia virus, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV), as well as lentiviruses.

[0310] As used herein, the term “lentivirus” refers to a group (or genus) of compound retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (including human immunodeficiency virus, HIV1 and HIV2), bisnamaedi virus (VMV) viruses, Caprin arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and Simian immunodeficiency virus (SIV). In one embodiment, an HIV-based vector scaffold (i.e., an HIV cis-acting sequence factor) is preferred.

[0311] In various embodiments, the lentiviral vectors intended herein include one or more LTRs and one or more or all of the following accessory factors: cPPT / FLAP, Psi(Ψ) package signal, export factor. The elements, poly(A) sequences, and optionally, as otherwise discussed herein, may include WPRE or HPRE, insulator factors, selection markers, and suicide genes.

[0312] In certain embodiments, the lentiviral vectors contemplated herein may be integrating, non-integrating, or lentivirus lacking integration ability. As used herein, the terms “integrating-deficient lentivirus” or “IDLV” refer to lentiviruses having an integrase that lacks the ability to integrate the viral genome into the genome of a host cell. An embedded-incompetent viral vector is described in patent application WO2006 / 010834, which is incorporated herein by reference in its entirety.

[0313] Exemplary mutations in the HIV-1 pol gene suitable for reducing integrase activity include, but are not limited to, H12N, H12C, H16C, H16V, and S81. R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116 N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E 157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K1 86T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 Examples include L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A, and K264H.

[0314] In one embodiment, the HIV-1 integrase-deficient pol gene includes D64V, D116I, D116A, E152G, or E152A mutations, D64V, D116I, and E152G mutations, or D64V, D116A, and E152A mutations.

[0315] In one embodiment, the HIV-1 integrase-deficient pol gene contains the D64V mutation.

[0316] The term "long terminal repeat (LTR)" refers to a domain of base pairs located at the end of retroviral DNA. In its original sequence, an LTR is a direct repeat and contains the U3, R, and U5 regions.

[0317] As used herein, the terms “FLAP factor” or “cPPT / FLAP” refer to nucleic acids whose sequences include the central polypurine tract and central termination sequences (cPPT and CTS) of retroviruses, such as HIV-1 and HIV-2. Suitable FLAP factors are described in U.S. Patent No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173. In another embodiment, the lentiviral vector contains a FLAP element having one or more mutations in the cPPT and / or CTS elements. In yet another embodiment, the lentiviral vector contains either the cPPT or CTS element. In yet another embodiment, the lentiviral vector does not contain either the cPPT or CTS element.

[0318] As used herein, the terms “package signal” or “package sequence” refer to the psi[Ψ] sequence located within the retroviral genome that is required to insert viral RNA into the viral capsid or viral particle. See, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4, pp. 2101–2109.

[0319] The term "export factor" refers to a cis-acting post-transcriptional regulator that controls the transport of RNA transcripts from the cell nucleus to the cytoplasm. Examples of RNA export factors, though not limited to them, include rev-reactive factors (RREs) of human immunodeficiency virus (HIV) (see, e.g., Cullen et al., 1991. J. Virol. 65:1053; and Cullen et al., 1991. Cell 58:423) and post-transcriptional regulators (HPREs) of hepatitis B virus.

[0320] In certain embodiments, the expression of heterologous sequences in viral vectors is increased by incorporating post-transcriptional regulators, efficient polyadenylation sites, and optionally transcription termination signals into the vector. Various post-transcriptional regulatory elements can increase the expression of heterologous nucleic acids in proteins, such as the woodchuck hepatitis virus post-transcriptional regulator (WPRE; Zufferey et al., 1999 J. Virol., 73:2886); the post-transcriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and similar elements (Liu et al., 1995 Genes Dev., 9:1766).

[0321] Lentiviral vectors preferably include several safety enhancements as a result of modifications to the LTR. A “self-inactivating” (SIN) vector refers to a vector that lacks replication ability, for example, the right (3')LTR enhancer-promoter region, known as the U3 region, which is modified (e.g., by deletion or substitution) to inhibit viral transcription beyond the first round of viral replication. Additional safety enhancements are provided by replacing the U3 region of the 5'LTR with a heterologous promoter that induces transcription of the viral genome during viral particle production. Examples of heterologous promoters that may be used include, for example, promoters for Simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., pre-early), Moloney's mouse leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase).

[0322] As used herein, the terms “pseudotype” or “pseudotype” refer to a virus having a viral envelope protein that has been replaced with the envelope protein of another virus having preferred properties. For example, the HIV envelope protein (encoded in the env gene) is typically CD4 + While the virus is targeted to presenting cells, HIV can infect a wide range of cells by pseudotyping it with the G protein (VSV-G) envelope protein of varicella stomatitis virus.

[0323] In one embodiment, the lentiviral vector is produced by a known method. See, for example, Kutner et al., BMC Biotechnol.2009;9:10.doi:10.1186 / 1472-6750-9-10; Kutner et al. Nat. Protoc.2009;4(4):495-505.doi:10.1038 / nprot.2009.22.

[0324] According to certain embodiments contemplated herein, the skeletal sequences of almost all viral vectors are derived from lentiviruses, such as HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used, or that a number of combined substitutions and modifications of certain lentiviral sequences can be adapted without impairing the ability of the transfer vector to perform the functions described herein. Furthermore, various lentiviral vectors are known in the art; see Naldini et al. (1996a, 1996b, and 1998); Zufferey et al. (1997), Dull et al., 1998, U.S. Patent No. 6,013,516, and No. 5,994,136, many of which can be adapted for the production of viral vectors or transfer plasmids contemplated herein.

[0325] In various embodiments, one or more polynucleotides encoding a CAR are introduced into immunoeffector cells by transducing the cells with an adenovirus containing one or more polynucleotides.

[0326] Adenovirus-based vectors enable extremely high transduction efficiency in many cell types and do not require cell division. High titers and high levels of expression can be obtained using such vectors. These vectors can be produced in large quantities using relatively simple systems. Most adenovirus vectors are engineered so that the transgene replaces the Ad E1a, E1b, and / or E3 genes, and then the replication-deficient vector is grown in human 293 cells that supply the trans-deleted gene function. Ad vectors can transduce in vivo into multiple types of tissues, including non-dividing cells and differentiated cells, such as those found in the liver, kidneys, and muscles. Conventional Ad vectors have high transport capacity.

[0327] The current production and amplification of adenovirus vectors, which lack replication ability, sometimes utilizes a unique helper cell line called 293. This cell line is derived from human embryonic kidney cells and Ad5 These cells are transformed with DNA fragments and structurally express the E1 protein (Graham et al., 1977). Since the E3 region is unnecessary in the adenovirus genome (Jones & Shenk, 1978), current adenovirus vectors utilize 293 cells to deliver foreign DNA to either the E1, D3, or both regions (Graham & Prevec, 1991). Adenovirus vectors are used for eukaryotic cell gene expression (Levrero et al., 1991; Gomez-Foix et al.). It has been used in vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Experiments involving the administration of recombinant adenovirus to various tissues include tracheal infusion (Rosenfeld et al., 1992). Examples of Ad vector use include intramuscular injection (Ragot et al., 1993), peripheral intravenous injection (Herz & Gerard, 1993), and stereotactic intracerebral injection (Le Gal La Salle et al., 1993). Examples of Ad vector use in clinical trials include polynucleotide therapy for antitumor immunity using intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).

[0328] In various embodiments, one or more polynucleotides encoding a CAR are introduced into immune effector cells by transducing cells with herpes simplex virus, e.g., HSV-1, HSV-2, and comprise one or more polynucleotides. In some embodiments, one or more polynucleotides encoding a polycistronic message encoding a CAR are introduced into immune effector cells by transducing cells with herpes simplex virus, e.g., HSV-1, HSV-2, and comprise one or more polynucleotides.

[0329] A mature HSV virion consists of an icosahedral capsid covered by an envelope containing a viral genome consisting of a 152kb linear double-stranded DNA molecule. In one embodiment, an HSV-based viral vector is deficient in one or more essential or non-essential HSV genes. In one embodiment, an HSV-based viral vector is replication-deficient. Most replication-deficient HSV vectors contain deletions to remove one or more early, early, or late HSV genes to prevent replication. For example, an HSV vector may be deficient in an early gene selected from the group consisting of ICP4, ICP22, ICP27, ICP47, and combinations thereof. The advantages of HSV vectors are their ability to enter a latent period that can result in prolonged DNA expression, and their large viral DNA genome that can accommodate up to 25kb of exogenous DNA. HSV-based vectors are described, for example, in U.S. Patents 5,837,532, 5,846,782 and 5,804,413, and in international patent applications WO 91 / 02788, WO 96 / 04394, WO 98 / 15637 and WO 99 / 06583, which are incorporated herein by reference in their entirety.

[0330] I. Genetically modified cells In various embodiments, cells are used to treat B-cell-associated conditions, having been genetically modified to express the CARs intended herein. As used herein, the terms “genetically engineered” or “genetically modified” refer to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms “genetically modified cell,” “modified cell,” and “redirected cell” are interchangeable. As used herein, the term “gene therapy” refers to the introduction of extra genetic material in the form of DNA or RNA into the total genetic material of a cell to restore, modify, or alter gene expression, or to the introduction of extra genetic material in the form of DNA or RNA for the purpose of expressing a therapeutic polypeptide, such as a CAR.

[0331] In certain embodiments, the CARs contemplated herein are introduced and expressed in immune effector cells to redirect their specificity to a target antigen of interest, such as a BCMA polypeptide. “Immune effector cells” are any cells of the immune system having one or more effector functions (e.g., cytotoxic cytotoxic activity, cytokine secretion, induction of ADCC and / or CDC). Exemplary immune effector cells contemplated herein are T lymphocytes, including but not limited to cytotoxic T cells (CTLs; CD8+ T cells), TILs, and helper T cells (HTLs; CD4+ T cells). In certain embodiments, the cells include αβ T cells. In certain embodiments, the cells include γδ T cells. In one embodiment, the immune effector cells include natural killer (NK) cells. In one embodiment, the immune effector cells include natural killer T (NKT) cells.

[0332] Immune effector cells may be self or non-self (e.g., allogeneic, syngeneic, or heterogeneous). As used herein, “self” means cells derived from the same subject. As used herein, “allogeneic” means cells of the same species but genetically different from the cells being compared. As used herein, “synogeneic” means cells of a different subject that are genetically identical to the cells being compared. As used herein, “heterogeneous” means cells of a different species from the cells being compared. In a preferred embodiment, the cells are self.

[0333] Exemplary immune effector cells used with CARs as intended in certain embodiments include T lymphocytes. The terms “T cell” or “T lymphocyte” are recognized in the art and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, quiescent T lymphocytes, or activated T lymphocytes. T cells may be T helper (Th) cells, e.g., T helper 1 (Th1) or T helper 2 (Th2) cells. T cells may be helper T cells (HTL; CD4+ T cells), CD4+ T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), CD4+CD8+ T cells, CD4-CD8-T cells, or any other subset of T cells. Other exemplary populations of T cells suitable for use in certain embodiments include naive T cells (TN), T memory stem cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), and effector T cells (TEFF).

[0334] As will be understood by those skilled in the art, other cells may also be used herein as CAR-containing immune effector cells. In particular, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include effector cell precursors, such progenitor cells may be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in certain embodiments, immune effector cells include immune effector cell precursors such as hematopoietic stem cells (HSCs) contained within a CD34+ population of cells derived from umbilical cord blood, bone marrow, or recruited peripheral blood, which may differentiate into mature immune effector cells upon administration to a subject, or may be induced in vitro to differentiate into mature immune effector cells.

[0335] As used herein, the term “CD34+ cell” refers to a cell that expresses the CD34 protein on its cell surface. As used herein, “CD34” refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion molecule and is involved in the entry of T cells into lymph nodes. The CD34+ cell population contains hematopoietic stem cells (HSCs), which differentiate when administered to a patient and become the basis for all hematopoietic cells, including T cells, NK cells, NKT cells, neutrophils, and monocyte / macrophage lineages.

[0336] Methods for producing immunoeffector cells expressing the CARs intended herein are provided in specific embodiments. In one embodiment, the method comprises transfecting or transfecting immunoeffector cells isolated from an organism to express one or more CARs intended herein. In one embodiment, immunoeffector cells are isolated from an organism and genetically modified without further in vitro manipulation. Such cells may then be directly re-administered to the organism. In a further embodiment, immunoeffector cells are initially activated and stimulated to proliferate in vitro before being genetically modified to express the CARs. In this regard, immunoeffector cells may be cultured before and / or after being genetically modified (i.e., transfected or transfected to express the CARs intended herein).

[0337] In certain embodiments, the cell source is obtained from a subject prior to the in vitro manipulation or genetic modification of the immunoeffector cells intended herein. In certain embodiments, the modified immunoeffector cells contain T cells.

[0338] In certain embodiments, PBMCs may be directly genetically modified using the methods contemplated herein to express CARs. In certain embodiments, after isolation of PBMCs, T lymphocytes may be further isolated, and in certain embodiments, both cytotoxic T lymphocytes and helper T lymphocytes may be stored in naive, memory, and effector T cell subpopulations either before or after genetic modification and / or proliferation.

[0339] For example, immune effector cells such as T cells may be genetically modified after isolation using known methods, or immune effector cells may be activated and expanded in vitro (or differentiated in the case of progenitor cells) and then genetically modified. In certain embodiments, immune effector cells such as T cells are genetically modified with the chimeric antigen receptor intended herein (e.g., transduced with a nucleic acid encoding a CAR or a viral vector containing a polycistronic message encoding a CAR), and then activated and proliferated in vitro. In various embodiments, T cells can be activated and proliferated before or after genetic modification to express a CAR, for example, U.S. Patents 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, and 7,144,575. The genes may be activated and expanded before or after gene modification using the methods described in Patent Nos. 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication 20060121005.

[0340] In one embodiment, CD34+ cells are transduced with nucleic acid constructs anticipated herein. In a particular embodiment, the transduced CD34+ cells are administered to a subject, generally the subject from which the cells were initially isolated, and then differentiated in vivo into mature immunoeffector cells. In another embodiment, CD34+ cells may be stimulated in vitro with one or more of the following cytokines: Flt-3 ligand (FLT3), stem cell factor (SCF), megakaryocyte growth and differentiation factor (TPO), IL-3, and IL-6, before or after genetic modification with the CARs intended herein, according to the methods described above (Asheuer et al., 2004; 2004).

[0341] In certain embodiments, populations of modified immune effector cells for the treatment of cancer include CARs as envisioned herein. For example, populations of modified immune effector cells are prepared from peripheral blood mononuclear cells (PBMCs) obtained from a patient (autologous donor) diagnosed with a B-cell malignancy as described herein. PBMCs form a heterogeneous population of T lymphocytes, which may be CD4+, CD8+, or CD4+ and CD8+.

[0342] PBMCs may also include other cytotoxic lymphocytes such as NK cells or NKT cells. An expression vector carrying the coding sequence of the CAR intended in a particular embodiment is introduced into a population of human donor T cells, NK cells, or NKT cells. In a particular embodiment, successfully transduced T cells carrying the expression vector can be isolated as CD3-positive T cells using flow cytometry and then further proliferated to increase the number of these CAR proteins expressing the T cells, in addition to cell activation using anti-CD3 antibodies and / or anti-CD28 antibodies and IL-2 or any other method known in the art as described elsewhere herein. A standard procedure is used for cryopreservation of T cells expressing CAR protein T cells for storage and / or preparation for use in human subjects. In one embodiment, in vitro transduction, culture, and / or proliferation of T cells are carried out in the absence of non-human animal-derived products such as fetal calf serum and fetal bovine serum. Since a heterogeneous population of PBMCs is genetically modified, the resulting transdextrins are a heterogeneous population of modified cells, including CAR-targeting BCMAs as intended herein.

[0343] In further embodiments, for example, a mixture of one, two, three, four, five, or more different expression vectors can be used to genetically modify a donor population of immune effector cells, each vector encoding a different chimeric antigen receptor protein as intended herein. The resulting modified immune effector cells form a mixed population of modified cells.

[0344] Genetically modified cells, including T cells, can be produced using various methods known in the art; see, for example, International Publication No. 2016 / 094304, which is incorporated herein by reference in its entirety.

[0345] J. Compositions and Formulations In certain embodiments, formulations of pharmaceutically acceptable carrier solutions are well known to those skilled in the art and include, for example, enteral and parenteral, intravascular, intravenous, intrauterine, intraosseous, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulations, as well as the development of suitable dosing and therapeutic regimens for using the particular compositions contemplated herein in various therapeutic regimens. Those skilled in the art will understand that the particular embodiments anticipated herein may include, for example, other formulations, such as those described in Remington: The Science and Practice of Pharmacy, volume I and volume II, 22nd Edition, Edited by Loyd V. Allen Jr., Philadelphia, PA: Pharmaceutical Press; 2012, which are, for example, well known in the pharmaceutical field and are incorporated herein in their entirety by reference.

[0346] The compositions envisioned herein may include one or more anti-BCMA antibodies or fragments thereof, CAR polypeptides, polynucleotides, vectors containing the same, or genetically modified immunoeffector cells, etc. Examples of compositions, though not limited to pharmaceutical compositions, include pharmaceutical compositions. In a preferred embodiment, the composition includes one or more cells modified to express CAR.

[0347] "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 modalities. It should also be understood that, where desired, the composition may also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharmaceutically active agents. There are virtually no limitations on other components that may be contained in the composition, however, any additional agents should not adversely affect the composition's ability to deliver the intended therapy. In preferred embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, or excipient, and one or more cells modified to express the CAR contemplated herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, or excipient, and an anti-BCMA antibody or a fragment thereof.

[0348] In this specification, the term “pharmaceutically acceptable” is used to mean compounds, substances, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio, within the bounds of appropriate medical judgment.

[0349] As used herein, “pharmaceutically acceptable carriers, diluents, or excipients” includes, but is not limited to, adjuvants, carriers, excipients, lubricants, sweeteners, diluents, preservatives, dyes / colorants, flavorings, surfactants, humectants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, surfactants, or emulsifiers that are approved by the U.S. Food and Drug Administration as acceptable for use in human or animal husbandry. 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; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, phosphate buffer, and any other suitable substances used in pharmaceutical formulations.

[0350] In certain embodiments, the composition comprises CAR-expressing immunoeffector cells in the amounts intended herein. In other embodiments, the composition comprises anti-BCMA antibody or a fragment thereof in the amounts intended herein. As used herein, the term “amount” means an “effective amount” or “effective quantity” of genetically modified therapeutic cells, such as T cells, to achieve a beneficial or desired preventive or therapeutic outcome, including clinical results.

[0351] The "prophylactic effective dose" refers to the amount of genetically modified therapeutic cells effective in achieving the desired prophylactic outcome. Typically, though not always, the prophylactic effective dose is less than the therapeutic effective dose because prophylactic doses are used before or in early-stage patients with the disease.

[0352] The “therapeutic dose” of genetically modified therapeutic cells may vary depending on factors such as the individual’s condition, age, sex, and weight, as well as the ability of stem cells and progenitor cells to elicit the desired response in the individual. The therapeutic dose is also the amount at which the therapeutically beneficial effects outweigh any toxic or adverse effects of the virus or transduced therapeutic cells. The term “therapeutic dose” includes the amount effective in “treating” a subject (e.g., a patient). Where a therapeutic dose is indicated, the exact amount of the composition administered may be determined by a physician, taking into account individual differences in the patient’s (subject’s) age, weight, tumor size, degree of infection or metastasis, and condition.

[0353] Generally, the pharmaceutical compositions containing T cells intended herein are 10 2 ~101 0 Cells / kg body weight, preferably 10 5 ~10 6 It can be stated that the dosage may be given in units of cells / kg body weight, including all integer values ​​within that range. The number of cells depends on the intended end use of the composition and the type of cells contained in the composition. With respect to the uses presented herein, the amount of cells is generally less than or equal to liters, and may be less than or equal to 500 mL, and even less than or equal to 250 mL or 100 mL. Thus, the desired cell density is often 10 6 It is greater than cells / ml, and generally 10 7 Cells / ml or more, generally 10 8 The number of cells / ml or more is clinically relevant. The number of clinically relevant immune cells may be divided into multiple infusions, and the cumulative total is 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 1012 The number of cells is greater than one. In some embodiments, specifically all injected cells are redirected to a particular target antigen, so 10 6 / kilogram (10 per patient) 6 ~10 11 A small number of cells within the range of ) may be administered. The composition may be administered multiple times in doses within these ranges. The cells may be allogeneic, syngeneic, heterogeneic, or autologous to the patient receiving the therapy. If desired, the therapy may also include the administration of mitogens (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) intended herein to enhance the induction of an immune response.

[0354] In general, compositions comprising activated and proliferated cells as intended herein may be used for the treatment and prevention of diseases occurring in immunocompromised individuals. In certain embodiments, compositions comprising immunoeffector cells modified to express the CARs intended herein are used for the treatment of cancer (e.g., B-cell malignancies). Modified immunoeffector cells may be administered alone, or as part of a pharmaceutical composition with carriers, diluents, and excipients, and / or as part of a pharmaceutical composition with other components such as IL-2 or other cytokines or other cell populations. In certain embodiments, the pharmaceutical composition comprises a quantity of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0355] A pharmaceutical composition comprising a population of immunoeffector cells (e.g., T cells) or an antibody, or a fragment thereof, modified to express CARs, may include a buffer, such as neutral buffered saline or phosphate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, or mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The composition is preferably formulated for parenteral administration, such as intravascular (intravenous or intra-arterial), intraperitoneal, or intramuscular administration.

[0356] Liquid pharmaceutical compositions, whether in solution, suspension, or other similar forms, may contain one or more of the following: water for injection, physiological saline, preferably physiological saline solution, Ringer's solution, isotonic saline, fixing oils such as synthetic mono or diglycerides that can serve as solvents or suspension media, sterile diluents such as polyethylene glycol, glycerin, propylene glycol, or other solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium sulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering agents such as acetates, citrates, or phosphates; and agents for adjusting isotonicity such as sodium chloride or dextrose. Parenteral preparations may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Pharmaceutical compositions for injection are preferably sterile.

[0357] In one embodiment, the immunoeffector cell (e.g., T cell) composition intended herein is formulated in a pharmaceutically acceptable cell culture medium. Such a composition is suitable for administration to human subjects. In certain embodiments, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0358] Serum-free media offer several advantages over serum-containing media, including simpler and more obvious composition, reduced levels of contaminants, elimination of the potential for them to be a source of infectious entities, and lower costs. In various embodiments, serum-free media may be free of animal matter and optionally protein-free. Optionally, the media may contain biopharmaceutically acceptable recombinant proteins. A “free of animal matter” medium refers to a medium whose components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in a free of animal matter medium, and their nutrients are obtained from synthetic, plant, or microbial sources. A “protein-free” medium, in contrast, is defined as substantially protein-free.

[0359] Examples of serum-free media used in specific compositions include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0360] In one preferred embodiment, a composition comprising immunoeffector cells as anticipated herein is formulated in a solution containing PlasmaLyte A.

[0361] In another preferred embodiment, the composition comprising the immunoeffector cells anticipated herein is formulated in a solution comprising a cryopreservation medium. For example, a cryopreservation medium containing a cryopreservative may be used to maintain high cell activity after thawing. Examples of cryopreservation media used in a particular composition include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0362] In a more preferred embodiment, the composition comprising the immunoeffector cells anticipated herein is formulated in a solution containing PlasmaLyte A in a 50:50 ratio to CryoStor CS10.

[0363] In certain embodiments, the composition comprises an effective amount of immunoeffector cells modified to express CARs, 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. The composition may also be administered in combination with antibiotics. Such therapeutic agents may be accepted in the art as standard treatments for specific disease conditions contemplated herein, such as certain cancers. Examples of therapeutic agents anticipated in certain embodiments include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapy, therapeutic antibodies, or other active adjunct agents.

[0364] In certain embodiments, a composition comprising immunoeffector cells modified to express CARs may be administered in combination with any number of chemotherapeutic agents.

[0365] Various other therapeutic agents may be used in combination with the compositions intended herein. In one embodiment, a composition comprising immune effector cells, CARs, is administered together with an anti-inflammatory agent.

[0366] In one embodiment, a composition comprising CAR-modified immune effector cells is administered together with a therapeutic antibody. Exemplary examples of therapeutic antibodies suitable for use in combination with CAR-modified T cells as intended in a particular embodiment include, but are not limited to, atezolizumab, avelumab, bavituximab, bevacizumab (Avastin), vibatuzumab, blinatumomab, semiprimab, conatumumab, crizotinib, daratumumab, duligotumab, dacetuzumab, dalotuzumab, and du Examples include ruvalumab, elotuzumab (HuLuc63), gemtuzumab, ibritumomab, indatuximab, inotuzumab, ipilimumab, rorbotuzumab, lucatumumab, milatuzumab, moxetumomab, nivolumab, okalatuzumab, ofatumumab, pembrolizumab, rituximab, siltuximab, teprotumumab, and ubrituximab.

[0367] K. Treatment method The genetically modified immune effector cells expressing CARs as intended herein provide an improved method of adoptive immunotherapy for use in the prevention, treatment, and improvement of immunomodulatory conditions and B-cell-related conditions, including but not limited to hematological malignancies.

[0368] In various embodiments, the genetically modified immunoeffector cells intended herein provide an improved method of adoptive immunotherapy used to increase cytotoxicity in cancer cells in a subject or to reduce the number of cancer cells in a subject.

[0369] In certain embodiments, the specificity of primary immune effector cells is redirected to cells expressing a specific antigen, such as cancer cells, by genetically modifying the primary immune effector cells with the CAR, as intended herein. In various embodiments, a viral vector is used to genetically modify immune effector cells with a specific polynucleotide encoding the CAR. In certain embodiments, the CAR comprises an anti-BCMA antigen-binding domain that binds to a BCMA polypeptide, a hinge domain, a transmembrane (TM) domain, a short oligolinker or polypeptide linker that ligates the TM domain to the intracellular signaling domain of the CAR, one or more intracellular costimulatory signaling domains, and a primary signaling domain.

[0370] In one embodiment, a type of cell therapy is provided in which T cells are genetically modified to express a CAR targeting a BCMA expressed on cancer cells, and the T cells are injected into a recipient who needs it. The injected cells can kill disease-causing cells in the recipient. Unlike antibody therapy, T cell therapy can replicate in vivo, resulting in long-lasting effects that can lead to sustained cancer treatment.

[0371] In one embodiment, CAR-expressing T cells can undergo robust in vivo T cell proliferation and persist for a long period. In another embodiment, CAR-expressing T cells evolve into specific memory T cells or stem cell memory T cells that can be reactivated to inhibit any further tumor formation or proliferation.

[0372] In certain embodiments, compositions comprising immune effector cells expressing the CARs intended herein are used to treat conditions associated with specific antigen-expressing cancer cells or cancer stem cells.

[0373] Examples of conditions that can be treated, prevented or improved using CAR-containing immune effector cells as intended herein include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas disease, Graves' disease, Wegener's granulomatosis, polyarteritis nodosa, Sjögren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis, Goodpasture disease, Kawasaki disease, and rapidly progressive glomerulonephritis.

[0374] Modified immune effector cells can also be applied to plasma cell disorders such as heavy chain diseases, primary or immune cell-associated amyloidosis, and monoclonal gammopathy of unknown importance (MGUS).

[0375] In this specification, "B-cell malignancy" refers to a type of cancer that develops in B cells (a type of immune system cell), as will be discussed later.

[0376] In certain embodiments, a composition comprising T cells expressing the CAR intended herein is used for the treatment of osteosarcoma or Ewing's sarcoma.

[0377] In certain embodiments, compositions comprising T cells expressing the CARs intended herein are used to treat liquid cancer or hematological cancer.

[0378] In certain embodiments, humoral cancer or hematological cancer is selected from the group consisting of leukemia, lymphoma, and multiple myeloma.

[0379] In certain embodiments, liquid or hematological cancers are selected from the group consisting of: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), polycythemia vera, Hodgkin lymphoma, nodular lymphocytosis-predominant Hodgkin lymphoma, Burkitt lymphoma, small lymphoma Lymphoma globulinum (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Sézary syndrome, precursor T-cell lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, sclerosing myeloma, solitary plasmacytoma of bone, extramedullary plasmacytoma.

[0380] In certain embodiments, humoral cancer or hematological cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML).

[0381] In a preferred embodiment, the liquid or hematological cancer is multiple myeloma (MM).

[0382] In a preferred embodiment, the liquid or hematological cancer is relapsed / refractory multiple myeloma (MM).

[0383] In certain embodiments, a method is provided comprising administering a therapeutically effective amount of immunoeffector cells expressing the CARs contemplated herein, or a composition comprising them, to a patient in need, either alone or in combination with one or more therapeutic agents. In certain embodiments, the cells are used to treat patients at risk of developing cancer or a cancer cell-related condition. Thus, in certain embodiments, a method for the treatment, prevention, or improvement of at least one symptom of cancer or a condition related to abnormal B cell activity (e.g., B cell malignancy) is provided, comprising administering a therapeutically effective amount of modified T cells expressing the CARs contemplated herein to a subject in need.

[0384] Where used herein, the terms “individual” and “subject” are often used interchangeably and refer to any animal exhibiting symptoms of a disease, injury, or illness that can be treated with gene therapy vectors, cell-based therapeutics, and methods as otherwise anticipated herein. In preferred embodiments, a subject includes any animal exhibiting symptoms of a cancer-related disease, injury, or condition that can be treated using gene therapy vectors, cell-based therapeutics, and methods as otherwise anticipated herein. Suitable subjects (e.g., patients) include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, domestic animals, or pets (e.g., cats or dogs). Non-human primates, preferably human patients, are also included. Typical subjects include human patients who have cancer (e.g., B-cell malignancies), have been diagnosed with cancer (e.g., B-cell malignancies), or are at risk of having cancer (e.g., B-cell malignancies).

[0385] As used herein, the term “patient” refers to a subject diagnosed with a particular disease, injury or condition that can be treated with gene therapy vectors, cell lineage therapeutics, and other methods disclosed elsewhere herein.

[0386] As used herein, “treatment” or “treating” includes any beneficial or desirable effect on the symptoms or pathology of a disease or condition, and may even include the smallest reduction of one or more measurable markers of the disease or condition being treated. Treatment may optionally include either a reduction of the disease or condition, or a delay in the progression of the disease or condition. “Treatment” does not necessarily mean the complete elimination or cure of the disease or condition, or its associated symptoms.

[0387] As used herein, “prevent,” and similar terms such as “preventable,” “preventing,” etc., refer to an approach aimed at preventing, inhibiting, or reducing the likelihood of a disease or condition occurring or recurring. Furthermore, it also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, “prevention” and similar terms also include a reduction in the intensity, action, symptoms, and / or burden of a disease or condition before the onset or recurrence of the disease or symptoms.

[0388] As used herein, “improvement of at least one symptom of ~” means a reduction in one or more symptoms of the disease or condition being treated. In certain embodiments, the disease or condition being treated is cancer, and the one or more symptoms improved in this case include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, lymph node dilation, abdominal distension or pain (due to distended abdominal organs), bone or joint pain, fractures, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination (due to impaired renal function).

[0389] "Enhance," "promote," "increase," or "magnify" generally refers to the ability of a composition contemplated herein, e.g., recombinant T cells expressing CAR, to produce, induce, or cause a greater physiological response (i.e., downstream effect) compared to the response induced by either the vehicle or the regulatory molecule / composition. Measurable physiological responses include, in particular, increased T cell dilation, activation, persistence, and / or cancer cell-killing ability, as is evident from the understanding of the art and the description herein. The amount "increased" or "enhanced" is typically a "statistically significant" amount and may include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more (e.g., 500 times, 1000 times) (including all integers and decimals in between and greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response induced by the vehicle or control composition.

[0390] "Decrease," "lower," "reduce," "decrease," or "weaken" generally refers to the ability of a composition expected herein to produce, induce, or generate a smaller physiological response (i.e., a downstream effect) compared to the response by the vehicle or control molecule / composition. The amount "decreased" or "reduced" is typically a "statistically significant" amount and may include a decrease of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more (e.g., 500 times, 1000 times) (including all integers and decimals in between and greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by the vehicle, control composition, or in a particular cell line.

[0391] "Maintain," "preserve," "maintain," "unchanged," "substantial change," or "substantial decrease" generally means that the composition expected herein produces, induces, or causes a similar or equivalent physiological response (i.e., downstream effect) in cells compared to the reaction produced by the vehicle, the control molecule / composition, or the reaction in a particular cell line. An equivalent reaction is one that is substantially indistinguishable from, or measurably indistinguishable from, the reference reaction.

[0392] In one embodiment, a method for treating a B-cell-associated condition or cancer in a subject requiring such treatment involves administering an effective dose, for example, a therapeutically effective dose of a composition comprising genetically modified immune effector cells as intended herein. The dose and frequency of administration are determined by factors such as the patient's condition, the type and severity of the patient's disease, but the appropriate dose may be determined by clinical trials.

[0393] In one embodiment, the amount of CAR-expressing immune effector cells, such as T cells, in the composition administered to the subject is 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 × 10⁶ 10 It is a cell.

[0394] In certain embodiments, about 1×10 7 T cells to about 1×10 9 T cells, about 2×10 7 T cells to about 0.9×10 9 T cells, about 3×10 7 T cells to about 0.8×10 9 T cells, about 4×10 7 T cells to about 0.7×10 9 T cells, about 5×10 7 T cells to about 0.6×10 9 T cells, or about 5×10 7 T cells to about​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​6 T cells / kg body weight ~ approx. 0.9×10 8 T cells / kg body weight, approximately 3 x 10 6 T cells / kg body weight ~ approx. 0.8×10 8 T cells / kg body weight, approximately 4 x 10 6 T cells / kg body weight ~ approx. 0.7×10 8 T cells / kg body weight, approximately 5 x 10 6 T cells / kg body weight ~ approx. 0.6×10 8 T cells / kg of body weight, or approximately 5 × 10⁻⁶ 6 T cells / kg body weight ~ approx. 0.5×10 8 The target population is administered T cells per kg of body weight.

[0397] Those skilled in the art will recognize that multiple administrations of the compositions intended herein may be required to achieve the desired therapeutic effect. For example, the compositions may be administered one, two, three, one, two, three, four, five, six months, one, two, five, ten years, or more times over periods of one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year, two years, five years, ten years, or more.

[0398] In one embodiment, it may be desirable to administer activated immune effector cells to a subject, then collect blood from the subject (or perform apheresis therapy) to activate immune effector cells derived from it, and then reinject these activated and expanded immune effector cells into the patient. This process may be performed multiple times at intervals of several weeks. In a particular embodiment, immune effector cells may be activated from a blood draw of 10cc to 400cc. In a particular embodiment, immune effector cells are activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, 100cc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more. While not bound by theory, using a multi-blood-draw / multiple-reinjection protocol may be helpful in selecting a particular group of immune effector cells.

[0399] The compositions anticipated herein may be administered by any convenient method, including aerosol inhalation, injection, ingestion, transfusion, transplantation, or implantation. In preferred embodiments, the compositions are administered parenterally. As used herein, the terms “parenteral administration” and “administered parenterally” refer to modes of administration other than enteral and topical administration, and typically include, but are not limited to, injection, intravascular, intravenous, intramuscular, intraarterial, subarachnoid, intra-articular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions. In one embodiment, the compositions anticipated herein are administered to a target by direct injection into a tumor, lymph node, or site of infection.

[0400] In one embodiment, an effective amount of a composition that enhances the cellular immune response to a target B cell-associated state is administered to a subject in need. The immune response may include a cellular immune response mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses that can kill infected cells. A humoral immune response, mediated primarily by helper T cells that can activate B cells and thus resulting in antibody production, may also be induced. Various techniques may be used to analyze the types of immune responses induced by the composition, such as in Current Protocols in Immunology, edited by John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, and Warren Strober (2001), John Wiley & Sons, NY, NY.

[0401] In one embodiment, a method is provided for treating a subject diagnosed with a B cell-associated condition or cancer, comprising removing immune effector cells from the subject diagnosed with cancer or a BCMA-expressing B cell-associated condition, and genetically modifying the immune effector cells with a vector containing nucleic acids encoding CARs as intended herein, thereby creating a population of modified immune effector cells, and administering the population of modified immune effector cells to the subject. In a preferred embodiment, the immune effector cells include T cells.

[0402] In certain embodiments, a method is provided for stimulating immune effector cells that mediate an immune modulator response against a target cell population in a subject, comprising the step of administering to a population of immune effector cells expressing a nucleic acid construct encoding a CAR molecule.

[0403] Methods for administering cell compositions intended in particular embodiments include any method effective for resulting in the reintroduction of genetically modified immune effector cells ex vivo, either by directly expressing CAR in a subject or by reintroducing genetically modified precursors of immune effector cells that differentiate into mature immune effector cells expressing CAR upon introduction into a subject. One method includes transducing peripheral blood T cells in vitro using nucleic acid constructs anticipated herein and returning the transduced cells to a subject. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11]

[0404] All publications, patent applications, and registered patents cited herein are incorporated by reference in such a manner as if each individual publication, patent application, or registered patent were specifically and individually indicated as being incorporated by reference.

[0405] While the embodiments described above are described in detail with reference to the figures and examples for clarity and understanding, it will be readily apparent to those skilled in the art that certain changes and modifications can be made in light of the teachings intended herein without departing from the spirit or scope of the appended claims. The following embodiments are provided for illustrative purposes only and are not limiting. Those skilled in the art will readily recognize a variety of noncritical parameters that can be changed or modified to produce essentially similar results. [Examples]

[0406] Example 1 Construction of a human anti-BCMA CAR Lentiviral vectors containing constructs with human anti-BCMA CAR were designed, constructed, and validated. Constructs containing an MNDU3 promoter operably linked to an anti-BCMA CAR, including a CD8α signaling sequence, human anti-BCMA scFv, CD8α hinge and transmembrane domains, a CD137 costimulatory domain, and a CD3ζ primary signaling domain, were cloned into lentiviral vectors. Anti-BCMA scFv was designed and evaluated in both VH / VL and VL / VH orientations using a polyglycine-serine linker. Exemplary anti-BCMA CAR polypeptide sequences are described in SEQ ID NOs. {ut} 50, 52, 54, 56, 58, 60, 62, 64, 66, and 68, and exemplary anti-BCMA CAR polynucleotide sequences are described in SEQ ID NOs. {ut} 49, 51, 53, 55, 57, 59, 61, 63, 65, and 67 {ut}.

[0407] Example 2 Evaluation of human anti-BCMA CAR T cells Chimeric antigen receptors (CARs) specific to BCMA and possessing human scFvs (e.g., SEQ ID NOs. 50, 52, 54, 56, 58, 60, 62, 64, 66, and 68) were compared with known anti-BCMA CARs ("comparators") possessing mouse-derived scFvs to evaluate CAR expression and biological activity on BCMA-expressing cells. Anti-BCMA CAR T cells were generated using a 7-day process with G-REX® flasks. Briefly, peripheral blood mononuclear cells (PBMCs) were cultured in a medium containing IL-2 (CellGenix, GmbH) and antibodies specific to CD3 and CD28 (Miltenyi Biotec, Inc.). Lentivirus encoding anti-BCMA CARs was added 1 day after the start of culture. On day 4, CAR T cells were transferred from 24-well plates to 24-well G-REX flasks, where the cells were maintained until harvesting on day 7. CAR T cells were investigated for the integration of lentiviral vectors into their genomic DNA. The number of vector copies per cell ranged from 1 to 4 copies of the transgene in transduced cells. As shown in Figure 2A, all transduced cells exhibited vector copy numbers equivalent to or greater than those of comparator anti-BMCA CAR T cells.

[0408] CAR T cells were also analyzed for cell surface CAR expression using flow cytometry. CAR T cells were stained with recombinant, phycoerythrin (PE)-labeled, BCMA extracellular domain-FC fusion protein (Creative BioMart, Inc.). Surface CAR expression mediated by positive Fc-BCMA binding was detected at various levels for all transduction conditions, except for CAR3, which did not express CAR. These reagents confirm that T cells specifically express anti-BCMA CARs. As shown in Figure 2B, T cells transduced with CAR1, CAR4, or CAR5 have CAR expression levels equivalent to or higher than those of comparator anti-BCMA CAR T cells.

[0409] Furthermore, the biological activity of CAR T cells was evaluated for interferon-gamma production, either alone or in co-culture with tumor cell lines. Specifically, antigen-independent IFNγ production by CAR T cells in isolation or co-culture with BCMA-nonexpressing RD cell lines or HT1080 cells was assessed. As shown in Figures 3A and 3B, all CARs except CAR4 produced minimal IFNγ independently of the antigen. Similar results were observed for CAR7-10 in Figures 3D and 3E. Interferon-gamma production was also measured after co-culture of CAR T cells with Burkitt lymphoma cells (Daudi cells) or BCMA-expressing HT.1080.BCMA cells. As shown in Figures 3C and 3F, CAR1, CAR4, CAR5, CAR9, and CAR10 produced IFNγ equivalent to or greater than that of comparator anti-BCMA CARs. Similarly, when CAR1 or CAR5 were evaluated for IFNγ production either alone or in co-culture with antigen-negative (HT1080) or antigen-positive (HT1080) tumor cell lines, CAR1 and CAR5 produced minimal antigen-dependent IFNγ (as shown in Figure 3I, and Figures 3G and 3H), while similarly producing comparable or greater amounts of IFNγ in an antigen-dependent manner.

[0410] Furthermore, when CAR1, CAR5, CAR9, and CAR10 were evaluated for IL2 cytokine production in co-culture with low-antigen (Jeko1) or high-antigen (RPMI8336) tumor cell lines, CAR1 and CAR5 produced more IL2 than comparator CARs, as shown in Figures 3J and 3K.

[0411] Example 3 Evaluation of human anti-BCMA CAR T cell activation in low- and high-antigen-expressing tumor cells. In another experiment, anti-BCMA CAR T cells were produced using a system directly scalable to large-scale clinical manufacturing processes. Briefly, peripheral blood mononuclear cells (PBMCs) were cultured in a medium containing IL-2 (CellGenix, GmbH) and antibodies specific to CD3 and CD28 (Miltenyi Biotec, Inc.). Lentivirus encoding anti-BCMA CAR was added at a specified multiplicity of infection (MOI) one day after the start of culture. CAR T cells were maintained in the logarithmic phase by culturing for a total of 10 days with the addition of fresh medium containing IL-2. Surface CAR expression of anti-BCMA CAR T cells was analyzed by flow cytometry analysis of bound BCMA-Fc antigen, and normalized numbers of CAR-positive CAR T cells were added to cultures alone or with tumor cells of varying antigen densities. The B-cell lymphoma cell lines RL and Toledo exhibit lower BCMA responsiveness compared to the Burket lymphoma cell line (Daudi) and the manipulated cell line HT1080.BCMA.

[0412] As shown in Figure 4A, both CAR1 and CAR5 cells produce minimal IFNγ independently of the antigen and less than comparator anti-BCMA CAR T cells. However, IFNγ production from co-culture with low-BCMA-density cell lines suggests that IFNγ production of CAR1 and CAR5 is higher than that of comparators (Figure 4B). Furthermore, IFNγ production from co-culture with high-BCMA-density cell lines suggests that IFNγ production of CAR1 and CAR5 is similar to that of comparators (Figure 4C). These data suggest that human CAR1 and CAR5 T cells are 1) potentially more potent than comparator CARs against low-antigen-density cells, 2) at least as potent against high-antigen-density cells compared to comparator CARs, and 3) exhibit low antigen-independent IFNγ release.

[0413] Example 4 Evaluation of human anti-BCMA CAR T cell toxicity against antigen-expressing tumor cells To evaluate the ability of anti-BCMA CAR T cells to kill BCMA-expressing cancer cells, anti-BCMA CAR T cells were initially generated using a system directly scalable to the large-scale clinical manufacturing process described in Example 3. After 10 days of culture, the same number of anti-BCMA-expressing CAR T cells were cultured at various effector-to-target cell ratios (E:T) using HT.1080 cell lines expressing nuclear red fluorescent protein (HT1080-nucRed) or a derivative strain engineered to express BCMA (HT.1080-nucRed.BCMA).

[0414] As shown in Figure 5, varying degrees of antigen-dependent cell damage were observed over time in co-cultures of CAR1, CAR5, CAR9, and CAR10 cells with the HT.1080-nucRed.BCMA cell line.

[0415] Example 5 BCMA antigen density in endogenous and modified cell lines In another experiment, BCMA receptor density was assessed in various cell lines containing both endogenously expressed BCMA (e.g., RL, Toledo, Daudi, and RPMI-8226) and cells engineered to express BCMA (e.g., HT.1080.BCMA). BCMA density was assessed using anti-BCMA antibody clone 19F2 (Biolegend) with Quantum® Simply Cellular® Assay (Bangs Laboratories, Inc.). As shown in Figure 6, the modified cell line HT.1080BCMA and the multiple myeloma cell line RPMI-8226 expressed the highest levels of BCMA, with an average of 20,000 receptors. Lymphoma lines Daudi, Toledo, and RL expressed 10 to 50 times less BCMA antigen than HT.1080.BCMA or RPMI-8226.

[0416] Example 6 Evaluation of human anti-BCMA CAR T cell antigen-dependent proliferation To evaluate the ability of anti-BCMA CAR T cells to proliferate in the presence of BCMA-expressing cancer cells, anti-BCMA CAR T cells were initially generated using a system similar to that described in Example 2. After 10 days of culture, the same number of anti-BCMA-expressing CAR T cells were cultured from 5E3 cells of each cell type using either an HT.1080 cell line expressing nuclear red fluorescent protein (HT1080-nucRed) or a derivative cell line engineered to express BCMA (HT.1080-nucRed.BCMA) under different conditions.

[0417] As shown in Figure 7A, no proliferation was observed in the HT.1080-nucRed cell line on day 6 after the addition of CAR T cells, ranging from minimal to no proliferation at all. However, proliferation was observed in co-culture with BCMA expressing the HT.1080-nucRed.BCMA cell line (Figure 7B).

[0418] In general, the terms used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed herein and herein, but rather as encompassing all possible embodiments, along with the full scope of the equivalents for which such claims are granted. Therefore, the claims are not limited by this disclosure. In certain embodiments, for example, the following are provided: (Item 1) It is a chimeric antigen receptor (CAR), a) An anti-BCMA (B cell maturation antigen) antibody or its antigen-binding fragment that binds to one or more epitopes of a human BCMA polypeptide, including the variable light chain CDRL1, CDRL2, and CDRL3 regions in the variable light chain amino acid sequence described in SEQ ID NOs. 7, 15, 23, 31, 39, or 47, and the variable heavy chain CDRH1, CDRH2, and CDRH3 regions in the variable heavy chain amino acid sequence described in SEQ ID NOs. 8, 16, 24, 32, 40, or 48, comprising an extracellular domain, b) Transmembrane domain, c) One or more intracellular costimulatory signaling domains, and d) A chimeric antigen receptor (CAR) containing a primary signaling domain. (Item 2) The CAR described in item 1, wherein the variable light chain amino acid sequence is described in SEQ ID NO: 7 and / or the variable heavy chain amino acid sequence is described in SEQ ID NO: 8. (Item 3) The CAR described in item 1, wherein the variable light chain amino acid sequence is described in SEQ ID NO: 15 and / or the variable heavy chain amino acid sequence is described in SEQ ID NO: 16. (Item 4) The CAR described in item 1, wherein the variable light chain amino acid sequence is described in SEQ ID NO: 23 and / or the variable heavy chain amino acid sequence is described in SEQ ID NO: 24. (Item 5) The CAR described in item 1, wherein the variable light chain amino acid sequence is described in SEQ ID NO: 31 and / or the variable heavy chain amino acid sequence is described in SEQ ID NO: 32. (Item 6) The CAR described in item 1, wherein the variable light chain amino acid sequence is described in SEQ ID NO: 39 and / or the variable heavy chain amino acid sequence is described in SEQ ID NO: 40. (Item 7) The variable light chain amino acid sequence is described in Sequence ID No. 47, and / or the variable heavy chain The CAR described in item 1, whose mino acid sequence is described in sequence number 48. (Item 8) It is a chimeric antigen receptor (CAR), a) An extracellular domain comprising an anti-BCMA (B cell maturation antigen) antibody or its antigen-binding fragment that binds to one or more epitopes of a human BCMA polypeptide containing variable light chain CDRL1, CDRL2, and CDRL3 sequences as described in SEQ ID NOs: 1-3, 9-11, 17-19, 25-27, 33-35, or 41-43, and variable heavy chain CDRH1, CDRH2, and CDRH3 sequences as described in SEQ ID NOs: 4-6, 12-14, 20-22, 28-30, 36-38, or 44-46, b) Transmembrane domain, c) One or more intracellular costimulatory signaling domains, and d) A chimeric antigen receptor (CAR) containing a primary signaling domain. (Item 9) The CAR described in any one of items 1 to 8, wherein the anti-BCMA antibody or antigen-binding fragment is selected from the group consisting of camel Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody ("scFv"), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide-stabilized Fv protein ("dsFv"), and single-domain antibody (sdAb, nanobody). (Item 10) The CAR according to any one of items 1 to 9, wherein the anti-BCMA antibody or antigen-binding fragment is scFv. (Item 11) The CAR according to any one of items 1 to 10, comprising the anti-BCMA antibody or its antigen-binding fragment, one or more light chain CDRs described in any one of SEQ ID NOs: 1 to 3 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs: 4 to 6. (Item 12) The CAR according to any one of items 1 to 10, comprising one or more light chain CDRs described in any one of SEQ ID NOs: 9 to 11 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs: 12 to 14. (Item 13) The CAR according to any one of items 1 to 10, comprising one or more light chain CDRs described in any one of SEQ ID NOs: 17 to 19 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs: 20 to 22. (Item 14) The CAR according to any one of items 1 to 10, comprising one or more light chain CDRs described in any one of SEQ ID NOs. 25 to 27 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs. 28 to 30. (Item 15) The CAR according to any one of items 1 to 10, wherein the anti-BCMA antibody or its antigen-binding fragment comprises one or more light chain CDRs described in any one of SEQ ID NOs. 33 to 35 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs. 36 to 38. (Item 16) The CAR according to any one of items 1 to 10, comprising one or more light chain CDRs described in any one of SEQ ID NOs: 41 to 43 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs: 44 to 46. (Item 17) The anti-BCMA antibody or its antigen-binding fragment has an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the variable light chain amino acid sequence described in any one of SEQ ID NOs: 7, 15, 23, 31, 39, or 47. A CAR according to any one of items 1 to 16, comprising a variable light chain containing and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in any one of SEQ ID NOs. 8, 16, 24, 32, 40, or 48. (Item 18) The CAR according to any one of items 1 to 16, wherein the anti-BCMA antibody or its antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 7, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 8. (Item 19) The CAR according to any one of items 1 to 16, wherein the anti-BCMA antibody or its antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 15, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 16. (Item 20) The CAR according to any one of items 1 to 16, wherein the anti-BCMA antibody or its antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 23, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 24. (Item 21) The CAR according to any one of items 1 to 16, wherein the anti-BCMA antibody or its antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 31, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 32. (Item 22) The CAR according to any one of items 1 to 16, wherein the anti-BCMA antibody or its antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 39, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 40. (Item 23) The CAR according to any one of items 1 to 16, wherein the anti-BCMA antibody or its antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 31, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 48. (Item 24) The anti-BCMA antibody or its antigen-binding fragment comprises a variable light chain sequence described in any one of SEQ ID NOs: 7, 15, 23, 31, 39, or 47 and / or a variable heavy chain sequence described in any one of SEQ ID NOs: 8, 16, 24, 32, 40, or 48. A CAR as described in any one of items 1 through 16. (Item 25) The CAR according to any one of items 1 to 16, comprising the anti-BCMA antibody or its antigen-binding fragment, wherein the CAR comprises the variable light chain sequence described in SEQ ID NO: 7 and / or the variable heavy chain sequence described in SEQ ID NO: 8. (Item 26) The CAR according to any one of items 1 to 16, comprising the anti-BCMA antibody or its antigen-binding fragment, wherein the CAR comprises the variable light chain sequence described in SEQ ID NO: 15 and / or the variable heavy chain sequence described in SEQ ID NO: 16. (Item 27) The CAR according to any one of items 1 to 16, comprising the anti-BCMA antibody or its antigen-binding fragment, wherein the CAR comprises the variable light chain sequence described in SEQ ID NO: 23 and / or the variable heavy chain sequence described in SEQ ID NO: 24. (Item 28) The CAR according to any one of items 1 to 16, wherein the anti-BCMA antibody or its antigen-binding fragment comprises the variable light chain sequence described in SEQ ID NO: 31 and / or the variable heavy chain sequence described in SEQ ID NO: 32. (Item 29) The CAR according to any one of items 1 to 16, comprising the anti-BCMA antibody or its antigen-binding fragment, wherein the CAR comprises the variable light chain sequence described in SEQ ID NO: 39 and / or the variable heavy chain sequence described in SEQ ID NO: 40. (Item 30) The CAR according to any one of items 1 to 16, comprising the anti-BCMA antibody or its antigen-binding fragment, wherein the CAR comprises the variable light chain sequence described in SEQ ID NO: 47 and / or the variable heavy chain sequence described in SEQ ID NO: 48. (Item 31) The antibody or antigen-binding fragment according to any one of items 1 to 30, wherein the antibody or antigen-binding fragment is scFv, and the variable light chain is located c-terminus relative to that of the variable heavy chain. (Item 32) The antibody or antigen-binding fragment according to any one of items 1 to 30, wherein the antibody or antigen-binding fragment is scFv, and the variable heavy chain is located c-terminus with respect to that of the variable light chain. (Item 33) The CAR according to any one of items 1 to 32, wherein the transmembrane domain is isolated from a polypeptide selected from the group consisting of the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. (Item 34) The CAR according to any one of items 1 to 33, wherein the transmembrane domain is isolated from a polypeptide selected from the group consisting of CD8α, CD4, CD45, PD1, and CD152. (Item 35) The CAR described in any one of items 1 to 34, wherein the transmembrane domain is isolated from CD8α. (Item 36) The CAR described in any one of items 1 to 34, wherein the transmembrane domain is isolated from PD1. (Item 37) The CAR described in any one of items 1 to 34, wherein the transmembrane domain is isolated from CD152. (Item 38) The CAR described in any one of items 1 to 37, wherein one or more of the aforementioned co-stimulatory signaling domains are isolated from a co-stimulatory molecule selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. (Item 39) The CAR according to any one of items 1 to 38, wherein one or more co-stimulatory signaling domains are isolated from a co-stimulatory molecule selected from the group consisting of CD28, CD134, and CD137. (Item 40) A CAR as described in any one of items 1 to 39, wherein one or more of the aforementioned co-stimulus signaling domains are isolated from CD28. (Item 41) The CAR described in any one of items 1 to 39, wherein one or more of the aforementioned co-stimulatory signaling domains are isolated from CD134. (Item 42) The CAR described in any one of items 1 to 39, wherein one or more of the aforementioned co-stimulatory signaling domains are isolated from CD137. (Item 43) The CAR according to any one of items 1 to 42, wherein the primary signaling domain is isolated from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. (Item 44) The CAR described in any one of items 1 to 43, wherein the primary signaling domain is isolated from CD3ζ. (Item 45) A CAR as described in any one of items 1 to 44, further comprising a hinge region polypeptide. (Item 46) The CAR according to item 45, wherein the hinge region polypeptide includes the hinge region of CD8α. (Item 47) The CAR according to item 45, wherein the hinge region polypeptide includes the hinge region of PD1. (Item 48) The CAR according to item 45, wherein the hinge region polypeptide includes the hinge region of CD152. (Item 49) A CAR as described in any one of items 1 to 48, further comprising a signal peptide. (Item 50) A CAR as described in any one of items 1 through 49, further including a spacer area. (Item 51) The CAR according to item 50, wherein the spacer region polypeptide includes the CH2 and CH3 regions of IgG1, IgG2, IgG4, or IgD. (Item 52) Any of the sequence numbers 50, 52, 54, 56, 58, 60, 62, 64, 66, and 68 A CAR described in any one of items 1 to 51, containing the amino acid sequence described in one of the items. (Item 53) A CAR described in any one of items 1 to 52, including the amino acid sequence described in SEQ ID NO: 50. (Item 54) A CAR described in any one of items 1 to 52, including the amino acid sequence described in SEQ ID NO: 52. (Item 55) A CAR described in any one of items 1 to 52, including the amino acid sequence described in SEQ ID NO: 54. (Item 56) A CAR described in any one of items 1 to 52, including the amino acid sequence described in SEQ ID NO: 56. (Item 57) A CAR described in any one of items 1 to 52, including the amino acid sequence described in SEQ ID NO: 58. (Item 58) A CAR described in any one of items 1 to 52, including the amino acid sequence described in SEQ ID NO: 60. (Item 59) A CAR described in any one of items 1 to 52, including the amino acid sequence described in SEQ ID NO: 62. (Item 60) A CAR described in any one of items 1 to 52, including the amino acid sequence described in SEQ ID NO: 64. (Item 61) A CAR described in any one of items 1 to 52, including the amino acid sequence described in SEQ ID NO: 66. (Item 62) A CAR described in any one of items 1 to 52, including the amino acid sequence described in SEQ ID NO: 68. (Item 63) A polypeptide comprising the amino acid sequence of a CAR described in any one of items 1 through 62. (Item 64) A polynucleotide encoding a CAR or polypeptide as described in any one of items 1 through 63. (Item 65) The polynucleotide according to item 64, wherein the polynucleotide comprises a polynucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of the polynucleotide sequences described in SEQ ID NOs. 49, 51, 53, 55, 57, 59, 61, 63, 65, and 67. (Item 66) The polynucleotide according to item 64, wherein the polynucleotide comprises a polynucleotide sequence described in any one of SEQ ID NOs: 49, 51, 53, 55, 57, 59, 61, 63, 65, and 67. (Item 67) A vector containing a polynucleotide as described in any one of items 64-66. (Item 68) The vector described in item 67 is an expression vector. (Item 69) The vector described in item 67 or 68, wherein the vector is an episomal vector. (Item 70) The vector described in any one of items 67 to 69, wherein the vector is a viral vector. (Item 71) The vector described in any one of items 67 to 70 is a retroviral vector. (Item 72) The vector described in any one of items 67 to 71, wherein the vector is a lentiviral vector. (Item 73) The vector described in item 72, wherein the lentiviral vector is selected from the group essentially consisting of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), bisnamaedi virus (VMV) virus, Caprin arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and Simian immunodeficiency virus (SIV). (Item 74) A vector according to any one of items 70-73, comprising a left (5') retroviral LTR, a Psi(Ψ) packaging signal, a central polypurine tube / DNA flap (cPPT / FLAP), a retroviral export element, a promoter operably conjugated to a polynucleotide as described in item 45, and a right (3') retroviral LTR. (Item 75) A vector as described in any one of items 70-74, further comprising heterologous polyadenylated sequences. (Item 76) A vector according to any one of items 70 to 75, further comprising a hepatitis B virus post-transcriptional modifier (HPRE) or a woodchuck post-transcriptional modifier (WPRE). (Item 77) The vector according to any one of items 70 to 76, wherein the promoter of the 5'-LTR is replaced with a heterologous promoter. (Item 78) The vector described in item 77, wherein the heterogeneous promoter is a cytomegalovirus (CMV) promoter, a rassarcoma virus (RSV) promoter, or a Simianvirus 40 (SV40) promoter. (Item 79) A vector according to any one of items 74-78, wherein the 5'LTR or 3'LTR is a lentiviral LTR. (Item 80) The vector described in any one of items 74-79, wherein the aforementioned 3'LTR includes one or more modifications. (Item 81) The vector according to any one of items 74-80, wherein the 3'LTR contains one or more deletions. (Item 82) The vector according to any one of items 74 to 81, wherein the 3'LTR is a self-inactivated (SIN)LTR. (Item 83) The polyadenylated sequence is a bovine growth hormone polyadenylated sequence or a signal rabbit β-globin polyadenylated sequence, as described in any one of items 75 to 82. Tar. (Item 84) A vector according to any one of items 67-83, comprising a polynucleotide according to any one of items 64-66, which contains an optimized Kozak sequence. (Item 85) The vector according to any one of items 74 to 84, wherein the promoter operably linked to the polynucleotide described in item 64 is selected from the group consisting of the cytomegalovirus immediate early gene promoter (CMV), elongation factor 1 alpha promoter (EF1-α), phosphoglycerate kinase-1 promoter (PGK), ubiquitin-C promoter (UBQ-C), cytomegalovirus enhancer / chicken beta-actin promoter (CAG), polyoma enhancer / herpes simplex thymidine kinase promoter (MC1), beta-actin promoter (β-ACT), Simian virus 40 promoter (SV40), and myeloproliferative sarcoma virus enhancer, wherein the (MND)U3 promoter has a deleted negative control region and a substituted dl587rev primer binding site. (Item 86) A cell expressing any one of the CARs or polypeptides described in item 1 to 63. (Item 87) A cell containing a polynucleotide as described in any one of items 64-66 or a vector as described in any one of items 67-85. (Item 88) The cells described in item 86 or 87, wherein the aforementioned cells are genetically modified host cells. (Item 89) The cells described above are hematopoietic cells, as described in any one of items 86 to 88. (Item 90) The cells described in any one of items 86 to 89, wherein the aforementioned cells are hematopoietic stem cells or progenitor cells. (Item 91) The cells described in any one of items 86 to 90, wherein the aforementioned cells are CD34+ hematopoietic stem cells or progenitor cells. (Item 92) The aforementioned cells are immune effector cells, as described in any one of items 86 to 89. (Item 93) The cell is a T cell, as described in any one of items 86-89 and 92. (Item 94) The aforementioned cells, CD3+ CD4 + and / or CD8 + A cell, as described in any one of items 86-89, 92, and 93. (Item 95) The cells described in any one of items 86-89 and 92-94, wherein the cells are cytotoxic T lymphocytes (CTLs), tumor-infiltrating lymphocytes (TILs), or helper T cells. (Item 96) The T cell described above is an αβ-T cell, as described in any one of items 93 to 95. (Item 97) The T cell is a γδ-T cell, as described in any one of items 93 to 95. (Item 98) The cell described in any one of items 86-89 and 92, wherein the host cell is a natural killer (NK) cell. (Item 99) The aforementioned natural killer cells are natural killer T (NKT) cells, see item 98. The cells described. (Item 100) The cells described in any one of items 86-89 and 92, wherein the aforementioned cells are macrophages. (Item 101) The immunoeffector cells according to any one of items 92 to 100, wherein the immunoeffector cells are transduced with a vector according to any one of items 67 to 85, activated and stimulated in the presence of a PI3K pathway inhibitor, thereby maintaining the proliferation of the transduced immunoeffector cells compared to the proliferation of transduced immunoeffector cells activated and stimulated in the absence of the PI3K pathway inhibitor. (Item 102) The immune effector cells described in item 101, wherein the activated and stimulated immune effector cells, in the presence of the inhibitor of the PI3K pathway, show increased expression of i) one or more markers selected from the group consisting of CD62L, CD127, CD197, and CD38, or ii) all of the markers CD62L, CD127, CD197, and CD38, compared to immune effector cells activated and stimulated in the absence of the inhibitor of the PI3K pathway. (Item 103) The immune effector cells described in item 101, wherein, in the presence of the inhibitor of the PI3K pathway, the activated and stimulated immune effector cells increased the expression of i) one or more markers selected from the group consisting of CD62L, CD127, CD27, and CD8, or ii) all of the markers CD62L, CD127, CD27, and CD8, compared to activated and stimulated immune effector cells in the absence of the inhibitor of the PI3K pathway. (Item 104) The immunoeffector cells described in any one of items 101 to 103, wherein the PI3K inhibitor is ZSTK474. (Item 105) The cells or their offspring described in any one of items 86 to 104, wherein the cells or their offspring exhibit high IFNγ release in co-culture with BCMA-expressing cells. (Item 106) The cells or their offspring described in any one of items 86-105, wherein the cells or their offspring exhibit similar or higher IFNγ release in co-culture with BCMA-expressing cells compared to the same cells, except that the CAR contains an extracellular domain comprising mouse-derived anti-BCMA scFv. (Item 107) The co-cultured BCMA-expressing cells are Daudi cells, HT1080.BCMA cells, and / or RPMI-8226 cells, as described in item 105 or item 106, or their offspring. (Item 108) The cells described in any one of items 86 to 107, wherein the cells or their offspring exhibit high IFNγ release in co-culture with low BCMA-expressing cells. (Item 109) The low-BCMA-expressing cells described in item 108 have at least 5-fold less surface BCMA expression compared to Daudi cells, HT1080.BCMA cells, and / or RPMI-8226 cells. (Item 110) The cells described in item 108 or 109, wherein the low-BCMA-expressing cells have at least 10 times less surface BCMA expression compared to HT1080.BCMA cells. (Item 111) The low-BCMA-expressing cells are those cells according to any one of items 108 to 110, wherein the low-BCMA-expressing cells have at least 10 times less surface BCMA expression compared to RPMI-8226 cells. (Item 112) The cells described in any one of items 108-111, wherein the low-BCMA-expressing cells are RL cells and / or Toledo cells. (Item 113) The cells described in any one of items 108 to 112, wherein the CAR T cells exhibit higher IFNγ release in co-culture with low antigen-density cells compared to the same CAR T cells, except that the CAR contains an extracellular domain comprising mouse-derived anti-BCMA scFv. (Item 114) The cells described above are those that exhibit low antigen-independent signaling, as described in any one of items 86 to 113. (Item 115) The cells described in any one of items 86-114, wherein the cells exhibit low antigen-independent signaling compared to the same CAR T cells, except that the CAR contains an extracellular domain comprising mouse-derived anti-BCMA scFv. (Item 116) A composition comprising any one of items 86 to 115, and a physiologically acceptable excipient. (Item 117) A method for generating immune effector cells comprising a CAR or polypeptide as described in any one of items 1 to 63, comprising introducing a vector as described in any one of items 67 to 85 into the immune effector cells. (Item 118) The method according to item 117, further comprising stimulating the immune effector cells and inducing the proliferation of the cells by contacting them with an antibody that binds to CD3 and an antibody that binds to CD28, thereby generating a population of immune effector cells. (Item 119) The method according to item 118, wherein the immune effector cells are stimulated and proliferated before the introduction of the vector. (Item 120) The method according to item 118 or item 119, wherein the immune effector cells include T lymphocytes. (Item 121) The method according to item 118 or item 119, wherein the immune effector cells include NK cells. (Item 122) The method according to any one of items 117 to 121, wherein, in the presence of the inhibitor of the PI3K pathway, the immune effector cells are activated and stimulated to increase the expression of i) one or more markers selected from the group consisting of CD62L, CD127, CD197, and CD38, or ii) all of the markers CD62L, CD127, CD197, and CD38, compared to immune effector cells activated and stimulated in the absence of the inhibitor of the PI3K pathway. (Item 123) In the presence of the PI3K pathway inhibitor, the immune effector cells are activated and stimulated to: i) one or more markers selected from the group consisting of CD62L, CD127, CD27, and CD8, or ii) all of the markers CD62L, CD127, CD27, and CD8, compared to the immune effector cells activated and stimulated in the absence of the PI3K pathway inhibitor. The method described in any one of items 117-121, wherein the expression is increased compared to effector cells. (Item 124) The method according to item 122 or item 123, wherein the PI3K inhibitor is ZSTK474. (Item 125) A method for treating a B-cell-associated condition in a subject requiring the use of the composition described in item 116, comprising administering a therapeutically effective amount of the composition described in item 116 to the subject. (Item 126) The method according to item 125, wherein the B-cell-associated condition is cancer. (Item 127) The method according to item 126, wherein the cancer is a solid tumor. (Item 128) The method according to item 126, wherein the cancer is a humoral carcinoma. (Item 129) The method according to item 126, wherein the cancer is a hematological malignancy. (Item 130) The method according to item 125, wherein the B-cell-associated condition is multiple myeloma (MM), non-Hodgkin lymphoma (NHL), B-cell proliferation of uncertain malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, immunomodulatory disorder, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas disease, Graves' disease, Wegener's granulomatosis, polyarteritis nodosa, Sjögren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis, Goodpasture disease, Kawasaki disease, autoimmune hemolytic anemia, and rapidly progressing glomerulonephritis, heavy chain disease, primary or immune cell-associated amyloidosis, or monoclonal ganmopathies of unknown importance. (Item 131) The method according to any one of items 125 to 130, wherein the B-cell associated condition is a B-cell malignancy. (Item 132) The method according to item 131, wherein the B-cell malignancy is multiple myeloma (MM) or non-Hodgkin lymphoma (NHL). (Item 133) The method according to item 132, wherein the MM is selected from the group consisting of overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerosing myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma. (Item 134) The method according to item 132, wherein the NHL is selected from the group consisting of Burkitt lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and mantle cell lymphoma. (Item 135) The method according to item 125, wherein the B cell-associated condition is a plasma cell malignancy. (Item 136) The method according to item 125, wherein the B cell-associated condition is an autoimmune disease. (Item 137) The method according to item 136, wherein the autoimmune disease is systemic lupus erythematosus. (Item 138) The method according to item 125, wherein the B cell-associated condition is rheumatoid arthritis. (Item 139) The aforementioned B cell-associated condition is idiopathic thrombocytopenic purpura, or myasthenia gravis, or autologous purpura. The method described in item 125 for immunological hemolytic anemia. (Item 140) A method for improving one or more symptoms associated with cancer expressing BCMA in a subject, comprising administering to the subject an amount sufficient to improve at least one symptom associated with cancer cells expressing BCMA, the composition described in item 116. (Item 141) The method according to item 140, wherein one or more of the symptoms to be improved are selected from the group consisting of weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, abdominal distension or pain, bone or joint pain, fractures, unplanned weight loss, loss of appetite, night sweats, persistent mild fever, and decreased urination. (Item 142) A method for reducing the number of cells expressing BCMA in a subject, comprising administering to the subject an amount of the composition described in item 116 sufficient to reduce the number of cells expressing BCMA compared to the number of cells expressing BCMA before administration. (Item 143) An antibody or antigen-binding fragment thereof that binds to one or more epitopes of a human BCMA polypeptide, comprising the variable light chain CDRL1, CDRL2, and CDRL3 regions within the variable light chain amino acid sequence described in SEQ ID NOs: 7, 15, 23, 31, 39, or 47, and the variable heavy chain CDRH1, CDRH2, and CDRH3 regions within the variable heavy chain amino acid sequence described in SEQ ID NOs: 8, 16, 24, 32, 40, or 48. (Item 144) An antibody or antigen-binding fragment thereof that binds to one or more epitopes of a human BCMA polypeptide, comprising variable light chain CDRL1, CDRL2, and CDRL3 sequences described in any one of SEQ ID NOs: 1-3, 9-11, 17-19, 25-27, 33-35, or 41-43, and variable heavy chain CDRH1, CDRH2, and CDRH3 sequences described in SEQ ID NOs: 4-6, 12-14, 20-22, 28-30, 36-38, or 44-46. (Item 145) The antibody or antigen-binding fragment described in item 143 or item 144, wherein the antibody or antigen-binding fragment is selected from the group consisting of camel Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody ("scFv"), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide-stabilized Fv protein ("dsFv"), and single-domain antibody (sdAb, nanobody). (Item 146) The antibody or antigen-binding fragment described in item 145, wherein the antibody or antigen-binding fragment is scFv. (Item 147) The antibody or antigen-binding fragment according to any one of items 143 to 146, comprising one or more light chain CDRs described in any one of SEQ ID NOs: 1 to 3 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs: 4 to 6. (Item 148) The antibody or antigen-binding fragment according to any one of items 143 to 146, comprising one or more light chain CDRs described in any one of SEQ ID NOs: 9 to 11 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs: 12 to 14. (Item 149) The antibody or its antigen-binding fragment comprises one or more light chain CDRs described in any one of SEQ ID NOs. 17-19 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs. 20-22, as described in any one of items 143-146. Antigen-binding fragment. (Item 150) The antibody or antigen-binding fragment according to any one of items 143 to 146, comprising one or more light chain CDRs described in any one of SEQ ID NOs. 25 to 27 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs. 28 to 30. (Item 151) The antibody or antigen-binding fragment according to any one of items 143 to 146, comprising one or more light chain CDRs described in any one of SEQ ID NOs. 33 to 35 and / or one or more heavy chain CDRs described in any one of SEQ ID NOs. 36 to 38. (Item 152) The antibody or antigen-binding fragment according to any one of items 143 to 146, comprising one or more light chain CDRs described in any one of sequence numbers 41 to 43 and / or one or more heavy chain CDRs described in any one of sequence numbers 44 to 46. (Item 153) The antibody or antigen-binding fragment according to any one of items 143 to 146, wherein the antibody or antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in any one of SEQ ID NOs: 7, 15, 23, 31, 39, or 47, and / or comprises a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in any one of SEQ ID NOs: 8, 16, 24, 32, 40, or 48. (Item 154) The antibody or antigen-binding fragment according to any one of items 143 to 146, wherein the antibody or antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 7, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 8. (Item 155) The antibody or antigen-binding fragment according to any one of items 143 to 146, wherein the antibody or antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 15, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 16. (Item 156) The antibody or antigen-binding fragment according to any one of items 143 to 146, wherein the antibody or antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 23, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 24. (Item 157) The antibody or its antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 31, and / or at least 85%, 90%, 95%, 96%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 32. An antibody or antigen-binding fragment according to any one of items 143 to 146, comprising a variable heavy chain containing an amino acid sequence having 7%, 98%, or 99% identity. (Item 158) The antibody or antigen-binding fragment according to any one of items 143 to 146, wherein the antibody or antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 39, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 40. (Item 159) The antibody or antigen-binding fragment according to any one of items 143 to 146, wherein the antibody or antigen-binding fragment comprises a variable light chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable light chain amino acid sequence described in SEQ ID NO: 47, and / or a variable heavy chain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the variable heavy chain amino acid sequence described in SEQ ID NO: 48. (Item 160) The antibody or antigen-binding fragment according to any one of items 143 to 146, wherein the anti-BCMA antibody or its antigen-binding fragment comprises a variable light chain sequence described in any one of SEQ ID NOs: 7, 15, 23, 31, 39, or 47 and / or a variable heavy chain sequence described in any one of SEQ ID NOs: 8, 16, 24, 32, 40, or 48. (Item 161) The antibody or antigen-binding fragment according to any one of items 143 to 146, wherein the antibody or antigen-binding fragment comprises the variable light chain sequence described in SEQ ID NO: 7 and / or the variable heavy chain sequence described in SEQ ID NO: 8. (Item 162) The antibody or antigen-binding fragment according to any one of items 143 to 146, wherein the antibody or antigen-binding fragment comprises the variable light chain sequence described in SEQ ID NO: 15 and / or the variable heavy chain sequence described in SEQ ID NO: 16. (Item 163) The antibody or antigen-binding fragment according to any one of items 143 to 146, wherein the antibody or antigen-binding fragment comprises the variable light chain sequence described in SEQ ID NO: 23 and / or the variable heavy chain sequence described in SEQ ID NO: 24. (Item 164) The antibody or antigen-binding fragment according to any one of items 143 to 146, wherein the antibody or antigen-binding fragment comprises the variable light chain sequence described in SEQ ID NO: 31 and / or the variable heavy chain sequence described in SEQ ID NO: 32. (Item 165) The antibody or antigen-binding fragment according to any one of items 143 to 146, wherein the antibody or antigen-binding fragment comprises a variable light chain sequence described in SEQ ID NO: 39 and / or a variable heavy chain sequence described in SEQ ID NO: 40. (Item 166) The antibody or antigen-binding fragment according to any one of items 143 to 146, wherein the antibody or antigen-binding fragment comprises the variable light chain sequence described in SEQ ID NO: 47 and / or the variable heavy chain sequence described in SEQ ID NO: 48. (Item 167) The antibody or antigen-binding fragment according to any one of items 153 to 166, wherein the antibody or antigen-binding fragment is scFv, and the variable light chain is located c-terminus relative to that of the variable heavy chain. (Item 168) The antibody or antigen-binding fragment according to any one of items 153 to 166, wherein the antibody or antigen-binding fragment is scFv, and the variable heavy chain is c-terminal to that of the variable light chain.

Claims

[Claim 1] The invention described in the specification.

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