ISOLATED OR PURIFIED NUCLEIC ACID SEQUENCE THAT CODES A CHEMICAL ANTIGEN (CAR) RECEPTOR AND ITS USE,

专利摘要:
chimeric antigen receptors targeting b cell maturation antigen. the invention provides an isolated and purified nucleic acid sequence that encodes a chimeric antigen (car) receptor directed against b cell maturation antigen (bcma). the invention also provides host cells, such as t cells, or natural killer cells (nk), which express the car, and methods for destroying multiple myeloma cells.
公开号:BR112014024893B1
申请号:R112014024893-1
申请日:2013-03-15
公开日:2021-03-30
发明作者:James Noble Kochenderfer
申请人:The United States Of America, As Represented By The Secretary, Department Of Health And Human Services；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED REQUESTS
[0001] This patent application claims the benefit of United States Provisional Patent Application No. 61 / 622,600, filed on April 11, 2012, which is incorporated by reference in its entirety here. REFERENCE FOR INCORPORATION OF MATERIAL ELECTRONICALLY SUBMITTED
[0002] Incorporated by reference in its entirety here is a nucleotide / amino acid sequence listing, computer readable, submitted concurrently together and identified as follows: A file of 42,589 Byte ASCII (Text) named "712361_ST25.TXT", created in March 14, 2013. BACKGROUND OF THE INVENTION
[0003] Multiple myeloma (MM) is a malignancy characterized by an accumulation of clonal plasma cells (see, for example, Palumbo et al., New England J. Med., 364 (11): 1046-1060 (2011) , and Lonial et al., Clinical Cancer Res., 17 (6): 1264-1277 (2011)). Current therapies for MM often cause remissions, but almost all patients eventually relapse and die (see, for example, Lonial et al., Supra, and Rajkumar, Nature Rev. Clinical Oncol., 8 (8): 479-491 (2011)). Allogeneic hematopoietic stem cell transplantation has been shown to induce immune mediated elimination of myeloma cells; however, the toxicity of this approach is high, and few patients are cured (see, for example, Lonial et al., supra, and Salit et al., Clin. Lymphoma, Myeloma, and Leukemia, 11 (3): 247-252 (2011)). Currently, there are no FDA-approved monoclonal antibody or autologous T cell therapies that are clinically effective for MM (see, for example, Richardson et al., British J. Haematology, 154 (6): 745-754 (2011), and Yi, Cancer Journal, 15 (6): 502-510 (2009)).
[0004] The adoptive transfer of genetically modified T cells to recognize antigens associated with malignancy is showing promise as a new approach to cancer treatment (see, for example, Morgan et al., Science, 314 (5796): 126129 (2006 ); Brenner et al., Current Opinion in Immunology, 22 (2): 251257 (2010); Rosenberg et al., Nature Reviews Cancer, 8 (4): 299-308 (2008), Kershaw et al., Nature Reviews Immunology, 5 (12): 928-940 (2005); and Pule et al., Nature Medicine, 14 (11): 1264-1270 (2008)). T cells can be genetically engineered to express chimeric antigen receptors (CARs), which are fusion proteins comprised of an antigen recognition fraction and T cell activation domains (see, for example, Kershaw et al., Supra, Eshhar et al., Proc. Natl. Acad. Sci. USA, 90 (2): 720-724 (1993), and Sadelain et al., Curr. Opin. Immunol., 21 (2): 215-223 (2009 )).
[0005] For malignancies of B cell lineage, extensive progress has been made in the development of adoptive T cell approaches that use anti-CD19 CARs (see, for example, Jensen et al., Biology of Blood and Marrow Transplantation, 16 : 1245-1256 (2010); Kochenderfer et al., Blood, 116 (20): 4099-4102 (2010); Porter et al., The New England Journal of Medicine, 365 (8): 725-733 (2011) ; Savoldo et al., Journal of Clinical Investigation, 121 (5): 1822-1826 (2011), Cooper et al., Blood, 101 (4): 1637-1644 (2003); Brentjens et al., Nature Medicine, 9 (3): 279-286 (2003); and Kalos et al., Science Translational Medicine, 3 (95): 95ra73 (2011)). T cells transduced by anti-CD19-CAR adoptively transferred have cured leukemia and lymphoma in mice (see, for example, Cheadle et al., Journal of Immunology, 184 (4): 1885-1896 (2010); Brentjens et al. , Clinical Cancer Research, 13 (18 Pt 1): 5426-5435 (2007); and Kochenderfer et al., Blood, 116 (19): 3875-3886 (2010)). In previous clinical trials, adoptively transferred T cells transduced with normal eradicated anti-CD19 CARs and malignant B cells in patients with leukemia and lymphoma (see, for example, Kochenderfer et l., Blood, 116 (20): 40994102 (2010 ); Porter et al., Supra, Brentjens et al., Blood, 118 (18): 48174828 (2011); and Kochenderfer et al., Blood, December 8, 2011 (epublication ahead of print (2012)). however, it is only rarely expressed in malignant multiple myeloma plasma cells (see, for example, Gupta et al., Amer. J. Clin. Pathology, 132 (5): 728-732 (2009); and Lin et al ., Amer. J. Clin. Pathology, 121 (4): 482-488 (2004)).
[0006] Thus, there remains a need for compositions that can be used in methods to treat multiple myeloma. This invention provides such compositions and methods. BRIEF SUMMARY OF THE INVENTION
[0007] The invention provides an isolated and purified nucleic acid sequence encoding a chimeric antigen receptor (CAR), in which the CAR comprises an antigen recognition fraction and a T cell activation fraction, and in which the fraction Antigen recognition is directed against B cell Maturation Antigen (BCMA). BRIEF DESCRIPTION OF THE VARIOUS VIEWS OF THE DESIGN (S)
[0008] Figures 1A and 1B are graphs that represent the experimental data illustrating the pattern of BCMA expression across a variety of human cell types, as determined using quantitative PCR. The results are expressed as the number of BCMA cDNA copies per 105 copies of actin cDNA.
[0009] Figures 2A to 2L are graphs that represent experimental data that illustrate cell surface BCMA expression was detected in multiple myeloma cell lines, but not in the other cell types, as described in Example 1. For all graphs, the solid line represents staining with anti-BCMA antibodies, and the dashed line represents staining with combined isotype control antibodies. All graphics were surrounded in living cells.
[00010] Figure 3A is a diagram representing a nucleic acid construct that encodes an anti-BCMA CAR. From the N-terminal to the C-terminal, the anti-BCMA CAR includes an anti-BCMA scFv, the articulation and transmembrane regions of the CD8α molecule, the cytoplasmic portion of the CD28 molecule, and the cytoplasmic portion of the molecule of CD3Z.
[00011] Figures 3B to 3D are graphs representing the experimental data that illustrate that the CAR anti-bcma1 CAR, CAR anti-bcma2, and CAR SP6 (described in Example 2), are expressed on the T cell surface. Minimal anti-Fab staining occurred in non-transduced (UT) cells. The graphs are surrounded by CD3 + lymphocytes. The numbers in the graphs are the percentages of cells in each quadrant.
[00012] Figures 4A to 4C are graphs that represent the experimental data that illustrate the T cells that express the anti-BCMA CARs that remove the granulation of the T cells in a BCMA specific manner, as described in Example 3. The graphs they are surrounded by live CD3 + lymphocytes. The numbers in the graphs are the percentages of cells in each quadrant.
[00013] Figures 5A to 5D are graphs that represent experimental data that illustrate that T cells expressing anti-BCMA CARs remove the granulation of T cells in a BCMA specific manner, as described in Example 3. The graphs they are surrounded by live CD3 + lymphocytes. The numbers in the graphs are the percentages of cells in each quadrant.
[00014] Figures 6A to 6C are graphs that represent experimental data illustrating that T cells expressing anti-BCMA CARs produce the cytokines IFNY, IL-2, and TNF in a BCMA specific manner, as described in Example 3. The graphs are surrounded by live CD3 + lymphocytes. The numbers in the graphs are the percentages of cells in each quadrant.
[00015] Figure 7A is a graph representing experimental data that illustrates that T cells expressing the anti-bcma2 CAR proliferate specifically in response to BCMA. Figure 6B is a graph representing experimental data that illustrates that T cells expressing CAR SP6 do not specifically proliferate in response to BCMA.
[00016] Figures 7C and 7D are graphs that represent experimental data illustrating that Donor A T cells expressing the anti-bcma2 CAR specifically kill H929 multiple myeloma cell lines (Figure 6C) and RPMI8226 (Figure 6D) in a four-hour cytotoxicity assay in various ratios of effector cell: target cell. T cells transduced with the negative control P6 CAR induce much lower levels of cytotoxicity in all effector ratios: targets. For all effector ratios: targets, cytotoxicity was determined in duplicate, and the results are revealed as the mean +/- the standard error of the mean.
[00017] Figure 8A is a graph representing the experimental data that illustrates that BCMA is expressed on the surface of multiple myeloma cells of Myeloma Patient 3 primary bone marrow, as described in Example 5. The graph is surrounded by cells of CD38 + CD56 + plasma, which make up 40% of bone marrow cells.
[00018] Figure 8B is a graph representing experimental data that illustrates that allogeneic T cells transduced with Donor C anti-bcma2 CAR produce IFNY after co-culture with Myeloma Patient 3 unhandled bone marrow cells , as described in Example 5. Figure 7B also illustrates that T cells from the same allogeneic donor that expresses anti-bcma2 CAR produce much less IFNY when they were cultured with Patient 3 peripheral blood mononuclear cell (PBMC) of Myeloma. In addition, Donor C T cells expressing CAR SP6 do not specifically recognize Myeloma Patient 3 bone marrow.
[00019] Figure 8C is a graph representing experimental data that a plasmacytoma resected from Myeloma Patient 1 consists of 93% plasma cells, and these primary plasma cells express BCMA, as revealed by BCMA flow cytometry ( solid line) and control coloring compatible with the isotype (dashed line). The graph is surrounded by plasma cells.
[00020] Figure 8D is a graph representing experimental data illustrating that Myeloma Patient 1 T cells expressing anti-bcma2 CAR produce IFNy specifically in response to autologous plasmacytoma cells.
[00021] Figure 8E is a graph representing experimental data illustrating that Myeloma Patient 1 T cells expressing anti-bcma2 CAR specifically kill autologous plasmacytoma cells at low effector to target ratios. In contrast, Myeloma Patient 1 T cells expressing CAR SP6 exhibit low levels of cytotoxicity against autologous plasmacytoma cells. For all effector: target ratios, cytotoxicity was determined in duplicate, and the results are revealed as the mean +/- the standard error of the mean.
[00022] Figure 9A is a graph representing experimental data that illustrates that T cells transduced with the anti-bcma2 CAR can destroy multiple myeloma tumors established in mice. Figure 9B is a graph depicting the survival of tumor-bearing mice treated with T cells that express the anti-bcma2 CAR, as compared to controls. DETAILED DESCRIPTION OF THE INVENTION
[00023] The invention provides an isolated and purified nucleic acid sequence encoding a chimeric antigen (CAR) receptor, in which the CAR comprises an antigen recognition fraction and a T cell activation fraction. A chimeric antigen receptor (CAR) is an artificially constructed hybrid protein, or a polypeptide containing an antigen binding domain of an antibody (for example, a single chain variable fragment (scFv)) linked to the T cell signaling domain or the activation domain of T cell. CARs have the ability to redirect T cell specificity and reactivity towards a selected target in an unrestricted MHC- way, exploring the antigen binding properties of monoclonal antibodies. The recognition of an unrestricted MHC antigen gives T cells that express CARs the ability to recognize an antigen independent of antigen processing, thereby avoiding a greater tumor escape mechanism. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with alpha and beta chains of endogenous T cell receptor (TCR).
[00024] "Nucleic acid sequence" is intended to involve a DNA or RNA polymer, that is, a polynucleotide, which may be single stranded or double stranded, and which may contain unnatural or altered nucleotides. The terms "nucleic acid" and "polynucleotide", as used herein, refer to a polymeric form of nucleotides of any length, or ribonucleotides (RNA), or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double-stranded and single-stranded DNA, and double-stranded and single-stranded RNA. The terms include, as equivalents, analogs of, or RNA, or DNA, produced from modified nucleotide and polynucleotide analogs, such as, but not limited to, methylated and / or limited polynucleotides.
[00025] By "isolated" is the removal of a nucleic acid from its natural environment. By "purified" it is significant that a given nucleic acid, if one that has been removed from nature (including genomic DNA and mRNA), or synthesized (including, cDNA), and / or amplified, under laboratory conditions, has increased in purity, in which "purity" is a relative term, not "absolute purity". It is to be understood, however, that nucleic acids and proteins can be formulated with diluents or adjuvants, and yet, for practical purposes, be isolated. For example, nucleic acids are typically mixed with an acceptable carrier or diluent when used for introduction into cells.
[00026] The nucleic acid sequence of the invention encoding a CAR comprising an antigen recognition fraction that is directed against B cell Maturation Antigen (BCMA, also known as CD269). BCMA is a member of the tumor necrosis factor receptor superfamily (see, for example, 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 cell binding factor B (BAFF) and a proliferation-inducing ligand (APRIL) (see, for example, Mackay et al., Supra, and Kalled et al., Immunological Reviews, 204: 43 -54 (2005)). Among non-malignant cells, BCMA has been reported to be expressed primarily in plasma cells and subsets of mature B cells (see, for example, Laabi et al., EMBO J., 11 (11): 3897-3904 (1992) ; Laabi et al., Nucleic acids Res., 22 (7): 1147-1154 (1994); Kalled et al., Supra; O'Connor et al., J. Exp. Medicine, 199 (1): 91- 97 (2004); and Ng et al., J. Immunol., 173 (2): 807-817 (2004)). BCMA-deficient mice are healthy, and have normal numbers of B cells, but the survival of long-lived plasma cells is impaired (see, for example, O'Connor et al, supra; Xu et al., Mol. Cell . Biol., 21 (12): 4067-4074 (2001); and Schiemann et al., Science, 293 (5537): 2111-2114 (2001)). BCMA RNA has been universally detected in multiple myeloma cells, and BCMA protein has been detected on the surface of plasma cells in patients with multiple myeloma by several investigators (see, for example, Novak et al., Blood, 103 (2 ): 689-694 (2004); Neri et al., Clinical Cancer Research, 13 (19): 5903-5909 (2007); Bellucci et al., Blood, 105 (10): 3945-3950 (2005); and Moreaux et al., Blood, 103 (8): 3148-3157 (2004)).
[00027] The nucleic acid sequence of the invention encoding a CAR comprising an antigen recognition fraction contains a monoclonal antibody directed against BCMA, or an antigen binding portion thereof. The term "monoclonal antibodies", as used herein, refers to antibodies that are produced by a single B cell clone, and bind to the same epitope. In contrast, "polyclonal antibodies" refer to a population of antibodies that are produced by different B cells, and bind to different epitopes on the same antigen. The CAR antigen recognition fraction encoded by the nucleic acid sequence of the invention can be a complete antibody or an antibody fragment. A complete antibody typically consists of four polypeptides: two identical copies of a heavy chain (H) polypeptide, and two identical copies of a light chain (L) polypeptide. Each of the heavy chains contains an N-terminal variable region (VH) and three C-terminal constant regions (CH1, CH2 and CH3), and each light chain contains an N-terminal variable region (VL) and a C- constant region terminal (CL). The variable regions of each light and heavy chain pair form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising four structure regions, whose sequences are relatively conserved. The structure regions are linked by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of an antibody, which is responsible for antigen binding.
[00028] The terms "antibody fragment", "antibody fragment", "functional antibody fragment", and "antigen binding portion", are used interchangeably here to mean one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23 (9): 1126-1129 (2005)). The CAR antigen recognition fraction encoded by the nucleic acid sequence of the invention can contain any BCMA-binding antibody fragment. The antibody fragment desirably comprises, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the domains of VL, VH, CL, and CH1; (ii) an F (ab ') 2 fragment, which is a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (iv) a single-chain Fv (scFv), which is a monovalent molecule consisting of the two domains of the Fv fragment (ie, VL and VH) linked by a synthetic articulator that enables the two domains to be synthesized as a polypeptide chain simple (see, for example, Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778 (1998)) and (v) a diabody, which is a dimer of polypeptide chains, in which each polypeptide chain comprises a VH linked to a VL by a peptide articulator that it is too short to allow pairing between VH and VL on the same polypeptide chain, thereby triggering pairing between complementary domains on different VH-VL polypeptide chains, to generate a dimeric molecule having two antigen binding sites functional. Antibody fragments are known in the art, and are described in more detail in, for example, United States Patent Application Publication 2009/0093024 A1. In a preferred embodiment, the CAR antigen recognition fraction encoded by the nucleic acid sequence of the invention comprises an anti-BCMA Fv (scFv) single strand.
[00029] An antigen binding portion, or fragment of a monoclonal antibody, can be of any size, considering that the portion binds to BCMA. In this regard, an antigen-binding portion, or fragment of the monoclonal antibody directed against BCMA (also referred to herein as an "anti-BCMA monoclonal antibody"), desirably comprises between about 5 and 18 amino acids (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or a range defined by any two of the preceding values).
[00030] In one embodiment, the nucleic acid sequence of the invention encodes an antigen recognition fraction that comprises a variable region of an anti-BCMA monoclonal antibody. In this regard, the antigen recognition fraction comprises a light chain variable region, a heavy chain variable region, or both a light chain variable region and a heavy chain variable region of an anti-BCMA monoclonal antibody. Preferably, the CAR antigen recognition fraction encoded by a nucleic acid sequence of the invention comprises a light chain variable region and a heavy chain variable region of an anti-BCMA monoclonal antibody. The amino acid sequences of heavy and light chain monoclonal antibody that bind to BCMA are disclosed in, for example, International Patent Application Publication WO 2010/104949.
[00031] In another embodiment, the nucleic acid sequence of the invention encodes a CAR that comprises a signal sequence. The signal sequence can be positioned at the amino terminal of the antigen recognition fraction (for example, the variable region of the anti-BCMA antibody). The signal sequence can comprise any suitable signal sequence. In one embodiment, the signal sequence is a human granulocyte-macrophage colony stimulating factor (GM-CSF) sequence, or a CD8α signal sequence.
[00032] In another embodiment, the CAR comprises an articulation sequence. One skilled in the art will appreciate that a hinge sequence is a short sequence of amino acids that facilitates the flexibility of the antibody (see, for example, Woof et al., Nat. Rev. Immunol., 4 (2): 89-99 (2004 )). The hinge sequence can be positioned between the antigen recognition fraction (for example, an anti-BCMA scFv) and the T cell activation fraction. The hinge sequence can be any suitable sequence derived from or obtained from any suitable molecule. In one embodiment, for example, the hinge sequence is derived from the human CD8α molecule, or from a CD28 molecule.
[00033] The nucleic acid sequence of the invention encodes a CAR comprising a T cell activation fraction. The T cell activation fraction can be any suitable fraction derived from any suitable molecule. In one embodiment, for example, the T cell activation fraction comprises a transmembrane domain. The transmembrane domain can be any transmembrane domain derived from or obtained from any molecule known in the art. For example, the transmembrane domain can be obtained from or derived from a CD8α molecule, or from a CD28 molecule. CD8 is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR), and is expressed primarily on the surface of cytotoxic T cells. The most common form of CD8 exists as a dimer composed of a chain of CD8α and CD8β. CD28 is expressed on T cells, and provides costimulatory signals required for T cell activation. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2). In a preferred embodiment, CD8α and CD28 are human.
[00034] In addition to the transmembrane domain, the T cell activation fraction further comprises the intracellular (i.e., cytoplasmic) T cell signaling domain. The intracellular T cell signaling domain can be obtained from or derived from a CD28 molecule, a CD3 zeta (Z) molecule, or modified versions thereof, a human Fc receptor gamma chain (FCRY), a CD27 molecule, a OX40 molecule, a 4-1BB molecule, or other intracellular signaling molecules known in the art. As discussed above, CD28 is an important T cell marker in T cell costimulation. CD3Z associates with TCRs to produce a signal and to contain tyrosine immunoreceptor-based activation motifs (ITAMs). 4-1BB, also known as CD137, transmits a potent co-stimulatory signal to T cells, promoting differentiation and enhancing long-term survival of T lymphocytes. In a preferred embodiment, CD28, CD3 zeta, 4-1BB, OX40, and CD27, are human.
The CAR T cell activation domain encoded by a nucleic acid sequence of the invention can comprise any of the aforementioned transmembrane domains and any one or more of the aforementioned intracellular T cell signal domains in any combination. For example, the nucleic acid sequence of the invention can encode a CAR comprising a CD28 transmembrane domain and CD28 and CD3 zeta intracellular T cell signaling domains. Alternatively, for example, the nucleic acid sequence of the invention can encode a CAR comprising a CD8α transmembrane domain and CD28, CD3 zeta intracellular T cell signaling domains, the Fc receptor gamma chain (FCRY), and / or 4-1BB.
[00036] In one embodiment, the nucleic acid sequence of the invention encodes a CAR comprising, from 5 'to 3', a granulocyte-macrophage colony stimulating factor receptor (GM-CSF receptor) signal sequence , an anti-BCMA scFv, the hinge and transmembrane regions of the human CD8α molecule, the cytoplasmic T cell signaling domain of the human CD28 molecule, and the T cell signaling domain of the human CD3Z molecule. In another embodiment, the nucleic acid sequence of the invention encodes a CAR comprising, from 5 'to 3', a human CD8α signal sequence, an anti-BCMA scFv, the hinge and transmembrane regions of the human CD8α molecule , the cytoplasmic T cell signaling domain of the human CD28 molecule, and the T cell signaling domain of the human CD3Z molecule. In another embodiment, the nucleic acid sequence of the invention encodes a CAR comprising, from 5 'to 3', a human CD8α signal sequence, an anti-BCMA scFv, the hinge and transmembrane regions of the human CD8α molecule , the cytoplasmic T cell signaling domain of the human 4-1BB molecule, and / or the cytoplasmic T cell signaling domain of the human OX40 molecule, and the T cell signaling domain of the human CD3Z molecule. For example, the nucleic acid sequence of the invention comprises or consists of the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
[00037] The invention further provides an isolated and purified chimeric antigen receptor (CAR) encoded by a nucleic acid sequence of the invention.
[00038] The nucleic acid sequence of the invention can encode a CAR of any length, that is, the CAR can comprise any number of amino acids, provided that the CAR retains its biological activity, for example, the ability to specifically bind to the antigen , detect sick cells in a mammal, or treat or prevent disease in a mammal, etc. For example, the CAR can comprise 50 or more (for example, 60 or more, 100 or more, or 500 or more) amino acids, but less than 1,000 (for example, 900 or less, 800 or less, 700 or less, or 600 or less) amino acids. Preferably, CAR is about 50 to about 700 amino acids (e.g., about 70, about 80, about 90, about 150, about 200, about 300, about 400, about 550, or about 650 amino acids), about 100 to about 500 amino acids (e.g., about 125, about 175, about 225, about 250, about 275, about 325, about 350, about 375, about 425, about 450, or about 475 amino acids), or a range defined by any two of the preceding values.
[00039] Included within the scope of the invention are nucleic acid sequences that encode functional portions of the CAR described herein. The term "functional portion", when used in reference to a CAR, refers to any part or fragment of the CAR of the invention, the part or fragment of which retains the biological activity of the CAR of which it is a part (the CAR of origin). Functional portions involve, for example, those parts of a CAR that retain the ability to recognize target cells, or detect, treat, or prevent a disease, to a similar extent, to the same extent, or to a higher extent, such as the CAR of origin. In reference to a nucleic acid sequence that encodes the parent CAR, the nucleic acid sequence that encodes a functional portion of the CAR may encode a protein comprising, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the original CAR.
[00040] The nucleic acid sequence of the invention can encode a functional portion of a CAR that contains additional amino acids at the amino or carboxy terminus of the portion, or both ends, whose additional amino acids are not found in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, for example, recognizing target cells, detecting cancer, treating or preventing cancer, etc. More desirably, the additional amino acids enhance the biological activity of CAR, as compared to biological activity of the CAR of origin.
[00041] The invention also provides nucleic acid sequences that encode functional variants of the aforementioned CAR. The term "functional variant", as used herein, refers to a CAR, a polypeptide, or a protein having substantial or significant sequence identity, or similarity to the CAR encoded by a nucleic acid sequence of the invention, whose functional variant retains the biological activity of the CAR of which it is a variant. Functional variants involve, for example, those CAR variants described here (the CAR of origin) that retain the ability to recognize target cells to a similar extent, to the same extent, or to a higher extent, such as the CAR of origin . In reference to a nucleic acid sequence that encodes the source CAR, the nucleic acid sequence that encodes a functional variant of the CAR may, for example, be about 10% identical, about 25% identical, about 30% identical , about 50% identical, about 65% identical, about 80% identical, about 90% identical, about 95% identical, or about 99% identical, to the nucleic acid sequence encoding the source CAR.
[00042] A functional variant may, for example, comprise the amino acid sequence of the CAR encoded by a nucleic acid sequence of the invention with at least one conservative amino acid substitution. The phrase "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and Schirmer, R. H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such an analysis, groups of amino acids can be defined where the amino acids within a group preferentially exchange with each other, and, therefore, closely resemble each other in their impact on the total protein structure (Schulz, GE and Schirmer, RH, supra). Examples of conservative mutations include amino acid substitution of amino acids within the above subgroups, for example, lysine with arginine, and vice versa, such that a positive charge can be maintained by glutamic acid with aspartic acid, and vice versa, such that a charge negative can be maintained; serine by threonine, such that a free -OH can be maintained; and asparagine glutamine, such that a free -NH2 can be maintained.
[00043] Alternatively or in addition, functional variants may comprise the amino acid sequence of the original CAR with at least one hand-conservative amino acid substitution. "Non-conservative mutations" involve substitution of amino acids between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc. In this case, it is preferable for the non-conservative amino acid substitution not to interfere with, or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the original CAR.
[00044] The nucleic acid sequence of the invention can encode a CAR (including functional portions and functional variants thereof) that comprises amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, n-decanoic α-amino acid, homoserine, S-acetylaminomethylcytin, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tquinichydro-3-hydroxy acid , aminomalonic acid, monoamide aminomalonic acid, N'-benzyl-N'-methyl-lysine, N ', N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, a-aminocyclopentane carboxylic acid, a-aminocyclohexane carboxylic acid, a -aminocycloheptane carboxylic acid, a- (2-amino-2-norbomano) -carboxylic acid, α, y-diaminobutyric acid, α, β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.
[00045] The nucleic acid sequence of the invention can encode a CAR (including functional portions and functional variants thereof) that is glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, for example, a disulfide bridge, or converted to an acid addition salt and / or, optionally, dimerized or polymerized, or conjugated.
[00046] In a preferred embodiment, the nucleic acid sequence of the invention encodes a CAR that comprises or consists of the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 , SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.
[00047] The nucleic acid sequence of the invention can be generated using methods known in the art. For example, nucleic acid sequences, polypeptides, and proteins, can be recombinantly produced using standard recombinant DNA methodology (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994). In addition, a synthetically produced nucleic acid sequence encoding the CAR can be isolated and / or purified from a source, such as a plant, a bacterium, an insect, or a mammal, for example, a rat, a human, etc. Isolation and purification methods are well known in the art. Alternatively, the nucleic acid sequences described herein can be commercially synthesized. In this regard, the nucleic acid sequence of the invention can be synthetic, recombinant, isolated, and / or purified.
[00048] The invention also provides a vector comprising the nucleic acid sequence encoding the CAR of the invention. The vector can be, for example, a plasmid, a cosmid, a viral vector (for example, retroviral or adenoviral), or a phage. Suitable vectors and methods of vector preparation are well known in the art (see, for example, Sambrook et al., Supra, and Ausubel et al., Supra).
[00049] In addition to a nucleic acid sequence of the invention encoding the CAR, the vector preferably comprises expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES ), and the like, which provide expression of the nucleic acid sequence in a host cell. Exemplary expression control sequences are known in the art and described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (nineteen ninety).
[00050] A large number of promoters, including constitutive, inducible, and repressible promoters, from a variety of different sources, are well known in the art. Representative sources of promoters include, for example, viruses, mammals, insects, plants, yeast, and bacteria, and suitable promoters from these sources are readily available, or can be produced synthetically, based on the publicly available sequences, for example, of depositaries, such as such as the ATCC, as well as other commercial or individual sources. Promoters can be unidirectional (i.e., transcription initiated in one direction) or bi-directional (i.e., transcription initiated in either a 3 'or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, pBAD bacterial expression system (araA), the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Inducible promoters include, for example, the Tet system (United States Patents 5,464,758 and 5,814,618), the Ecdysone inducible system (No et al., Proc. Natl. Acad. Sci., 93: 3346-3351 (1996)), the T-REXTM system (Invitrogen, Carlsbad, CA), LACSWITCH ™ System (Stratagene, San Diego, CA), and the inducible recombinase system Cre-ERT tamoxifen (Indra et al., Nuc. Acid. Res., 27: 4324-4327 (1999); Nuc. Acid. Res., 28: e99 (2000); United States Patent 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol., 308: 123-144 (2005)).
[00051] The term "enhancer", as used herein, refers to a DNA sequence that increases the transcription of, for example, the nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases distant from the coding region of the nucleic acid sequence, and can mediate the binding of regulatory factors, DNA methylation patterns, or changes in DNA structure. A large number of enhancers from different sources from a variety of different sources are well known in the art, and are available as, or within, cloned polynucleotides (from, for example, depositaries, such as ATCC, as well as other commercial or individual sources ). A number of polynucleotides comprising promoters (such as the commonly used CMV promoter) also comprise enhancer sequences. The intensifiers can be located upstream, within, or downstream of the coding sequences. The term "Ig enhancers" refers to enhancer elements derived from enhancer regions mapped within the immunoglobulin (Ig) site (such enhancers include, for example, 5 'heavy chain (mu) enhancers, 5' chain enhancers mild (kappa), kappa and intronic mu intensifiers, and 3 'intensifiers (see, generally, Paul WE (ed), Fundamental Immunology, 3rd Edition, Raven Press, New York (1993), pages 353-363; and United States Patent States 5,885,827).
[00052] The vector can also comprise a "selectable marker gene". The term "selectable marker gene", as used herein, refers to a nucleic acid sequence that allows cells that express the nucleic acid sequence to be specifically selected for, or against, in the presence of a corresponding selective agent. Selectable marker genes are known in the art and described in, for example, International Patent Application Publications WO 1992/08796 and WO 1994/28143; Wigler et al., Proc. Natl. Acad. Sci. USA, 77: 3567 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78: 1527 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78: 2072 (1981); Colberre-Garapin et al., J. Mol. Biol., 150: 1 (1981); Santerre et al., Gene, 30: 147 (1984); Kent et al., Science, 237: 901-903 (1987); Wigler et al., Cell, 11: 223 (1977); Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48: 2026 (1962); Lowy et al., Cell, 22: 817 (1980); and United States Patents 5,122,464 and 5,770,359.
[00053] In some embodiments, the vector is an episomal expression vector "or" episome ", which is capable of replicating in a host cell, and persists as an extrachromosomal segment of DNA within the host cell in the presence of appropriate selective pressure ( see, for example, Conese et al., Gene Therapy, 11: 17351742 (2004)). Commercially representative episomal expression vectors include, but are not limited to, episomal plasmids using Epstein Barr Nuclear Antigen 1 (EBNA1) and origin of Epstein Barr Virus (EBV) of replication (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, CA) and pBK-CMV from Stratagene (La Jolla, CA) represent non-limiting examples of an episomal vector that uses T antigen and the SV40 origin of replication in view of EBNA1 and oriP.
[00054] Other suitable vectors include integration expression vectors, which may randomly integrate into the host cell's DNA, or may include a recombination site to enable specific recombination between the expression vector and the host cell's chromosome. Such integration expression vectors can use the endogenous expression control sequences of the host cell's chromosomes to effect expression of the desired protein. Examples of vectors that integrate in a specific way include, for example, components of the Invitrogen flp-in system (Carlsbad, CA) (for example, pcDNA ™ 5 / FRT), or the cre-lox system, as can be found in pExchange-6 Core Vectors by Stratagene (La Jolla, CA). Examples of vectors that randomly integrate into host cell chromosomes include, for example, pcDNA3.1 (when introduced in the absence of T antigen) from Invitrogen (Carlsbad, CA), and pCI or pFN10A (ACT) FLEXI ™ from Promega (Madison , WI).
[00055] Viral vectors can also be used. Representative viral expression vectors include, but are not limited to, adenovirus-based vectors (for example, the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands)), vectors based on lentivirus (for example, Life Technologies' lentiviral pLP1 (Carlsbad, CA)), and retroviral vectors (for example, Stratagene's pFB-ERV plus pCFB-EGSH (La Jolla, CA)). In a preferred embodiment, the viral vector is a lentivirus vector.
[00056] The vector comprising the nucleic acid of the invention encoding the CAR can be introduced into a host cell that is capable of expressing the CAR thus encoded, including any suitable prokaryotic or eukaryotic cell. Preferred host cells are those that can be easily and safely grown, have reasonably rapid growth rates, have well-characterized expression systems, and can be transformed or transfected easily and efficiently.
[00057] As used herein, the term "host cell" refers to any type of cell that may contain the expression vector. The host cell can be a eukaryotic cell, for example, plant, animal, fungi, or algae, or it can be a prokaryotic cell, for example, bacteria or protozoa. The host cell can be a cultured cell, or a primary cell, that is, directly isolated from an organism, for example, a human. The host cell can be an adherent cell, or a suspending cell, that is, a cell that grows in suspension. Suitable host cells are known in the art, and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For the purpose of amplifying or replicating the recombinant expression vector, the host cell can be a prokaryotic cell, for example, a DH5a cell. For the proposed production of a recombinant CAR, the host cell can be a mammalian cell. The host cell is preferably a human cell. The host cell can be of any type of cell, it can originate from any type of tissue, and it can be of any stage of development. In one embodiment, the host cell can be a peripheral blood lymphocyte (PBL), a peripheral blood mononuclear cell (PBMC), or a natural killer (NK). Preferably, the host cell is a natural killer cell (NK). More preferably, the host cell is a T cell. Methods for selecting suitable mammalian host cells and methods for cell transformation, culture, amplification, classification, and purification are known in the art.
[00058] The invention provides an isolated host cell that expresses the nucleic acid sequence of the invention encoding the CAR described herein. In one embodiment, the host cell is a T cell. The T cell of the invention can be any T cell, such as a cultured T cell, for example, a primary T cell, or a T cell of a cultured T cell line, or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from numerous sources, including, but not limited to, blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched or purified. The T cell is preferably a human T cell (for example, isolated from a human). The T cell can be of any stage of development, including, but not limited to, a CD4 + / CD8 + double positive T cell, a CD4 + helper T cell, for example, Th1 and Th2 cells, a CD8 + T cell (for example, example, a cytotoxic T cell), a tumor infiltrating cell, a memory T cell, a naive T cell, and the like. In one embodiment, the T cell is a CD8 + T cell, or a CD4 + T cell. T cell lines are available from, for example, the American Type Culture Collection (ATCC, Manassas, VA), and the German Collection of Microorganisms and Cell Cultures (DSMZ), and include, for example, Jurkat cells (ATCC TIB -152), Sup-T1 cells (ATCC CRL-1942), RPMI 8402 cells (DSMZ ACC-290), Karpas 45 cells (DSMZ ACC-545), and derivatives thereof.
[00059] In another embodiment, the host cell is a natural killer cell (NK). NK cells are a type of cytotoxic lymphocyte that plays a role in the innate immune system. NK cells are defined as large granular lymphocytes, and constitute the third type of cells differentiated from the common lymphoid progenitor that also gives rise to B and T lymphocytes (see, for example, Immunobiology, 5th ed., Janeway et al. , eds., Garland Publishing, New York, NY (2001)). NK cells differentiate and mature in the bone marrow, lymph node, spleen, tonsils, and thymus. Following maturation, NK cells enter the circulation as large lymphocytes with distinctive cytotoxic granules. NK cells are able to recognize and kill some abnormal cells, such as, for example, some tumor cells and virus-infected cells, and are thought to be important in innate immune defense against intracellular pathogens. As described above with respect to T cells, the NK cell can be any NK cell, such as a cultured NK cell, for example, a primary NK cell, or an NK cell from a cultured NK cell line, or an obtained NK cell of a mammal. If obtained from a mammal, the NK cell can be obtained from numerous sources, including, but not limited to, blood, bone marrow, lymph node, thymus, or other tissues or fluids. NK cells can also be enriched or purified. The NK cell, preferably, is a human NK cell (for example, isolated from a human). NK cell lines are available from, for example, the American Type Culture Collection (ATCC, Manassas, VA), and include, for example, NK-92 cells (ATCC CRL-2407), NK92MI cells (ATCC CRL-2408) , and derivatives thereof.
[00060] The nucleic acid sequence of the invention encoding a CAR can be introduced into a cell by "transfection", "transformation", or "transduction". "Transfection", "transformation", or "transduction", as used herein, refers to the introduction of one or more exogenous polynucleotides into a host cell by the use of physical or chemical methods. Many transfection techniques are known in the art and include, for example, DNA co-precipitation with calcium phosphate (see, for example, Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols , Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; microparticle bombardment facilitated by tungsten particle (Johnston, Nature, 346: 776-777 (1990)); and co-precipitation of DNA with strontium phosphate (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors can be introduced into host cells, after growth of infectious particles in suitable packaging cells, many of which are commercially available.
[00061] Without being linked to a particular theory or mechanism, it is believed that by inducing a specific antigen response against BCMA, CARs encoded by a nucleic acid sequence of the invention provide one or more of the following: targeting and destroying cancer cells that express BCMA, reduction or elimination of cancer cells, facilitating infiltration of immune cells to the tumor site (s), and intensification / extension of anti-cancer responses. Thus, the invention provides a method of destroying multiple myeloma cells, which comprises contacting one or more of the aforementioned isolated T cells, or natural killer cells, with a population of multiple myeloma cells that express BCMA, whereby CAR it is produced and binds to BCMA in multiple myeloma cells, and multiple myeloma cells are destroyed. As discussed here, multiple myeloma, also known as plasma cell myeloma or Kahler's disease, is a cancer of plasma cells, which are a type of white blood cell normally responsible for the production of antibodies (Raab et al., Lancet , 374: 324329 (2009)). Multiple myeloma affects 1-4 per 100,000 people a year. The disease is more common in men, and for reasons still unknown, it is twice as common in African Americans, as it is in Caucasian Americans. Multiple myeloma is the least common hematological malignancy (14%), and constitutes 1% of all cancers (Raab et al., Supra). Treatment of multiple myeloma typically involves high-dose chemotherapy, followed by hematopoietic stem cell transplantation (allogeneic or autologous); however, a high rate of recurrence is common in multiple myeloma patients who have endured such treatment. As discussed above, BCMA is highly expressed by multiple myeloma cells (see, for example, Novak et al., Supra; Neri et al., Supra; Bellucci et al., Supra; and Moreaux et al., Supra).
[00062] One or more isolated T cells expressing a nucleic acid sequence of the invention encoding the anti-BCMA CAR described herein can be contacted with a population of multiple myeloma cells that express BCMA ex vivo, in vivo, or in vitro . "Ex vivo" refers to methods conducted inside or on cells or tissue in an artificial environment outside an organism with minimal change in natural conditions. In contrast, the term "in vivo" refers to a method that is conducted within living organisms in their normal, intact state, while an "in vitro" method is conducted using components of an organism that have been isolated from their biological context usual. The method of the invention preferably involves components ex vivo and in vivo. In this regard, for example, the isolated T cells described herein can be cultured ex vivo under conditions to express a nucleic acid sequence of the invention encoding the anti-BCMA CAR, and then directly transferred to a mammal (preferably, a affected by multiple myeloma. Such a cell transfer method is referred to in the art as "adoptive cell transfer (ACT)", in which immune derived cells are passively transferred in a new recipient host to transfer the functionality of the donor immune derived cells to the new host. Adoptive cell transfer methods to treat various types of cancers, including hematological cancers, such as myeloma, are known in the art, and disclosed in, for example, Gattinoni et al., Nat. Rev. Immunol., 6 (5) : 383-393 (2006); June, CH, J. Clin. Invest., 117 (6): 1466-76 (2007); Rapoport et al., Blood, 117 (3): 788-797 (2011); and Barber et al., Gene Therapy, 18: 509-516 (2011)).
[00063] The invention also provides a method of destroying Hodgkin's lymphoma cells. Hodgkin's lymphoma (first known as Hodgkin's disease) is a cancer of the immune system that is marked by the presence of a type of multinucleated cell called Reed-Sternberg cells. The two major types of Hodgkin's lymphoma include classic Hodgkin's lymphoma and predominant nodular lymphocyte Hodgkin's lymphoma. Hodgkin's lymphoma is currently treated with radiation therapy, chemotherapy, or hematopoietic stem cell transplantation, with the choice of treatment depending on the patient's age and sex, and the stage, volume, and histological subtype of the disease. BCMA expression was detected on the surface of Hodgkin's lymphoma cells (see, for example, Chiu et al., Blood, 109 (2): 729-739 (2007)).
[00064] When T cells or NK cells are administered to a mammal, the cells can be allogeneic or autologous to the mammal. In "autologous" methods of administration, cells (e.g., blood-forming stem cells, or lymphocytes) are removed from a mammal, stored (and, optionally, modified), and returned back to the same mammal. In "allogeneic" delivery methods, a mammal receives cells (for example, blood-forming stem cells, or lymphocytes) from a genetically similar, but not identical, donor. Preferably, the cells are autologous to the mammal.
[00065] T cells or NK cells are desirably administered to a human in the form of a composition, such as a pharmaceutical composition. Alternatively, the nucleic acid sequence of the invention encoding the CAR, or a vector comprising the nucleic acid sequence encoding the CAR, can be formulated into a composition, such as a pharmaceutical composition, and administered to a human. The pharmaceutical composition of the invention can comprise a population of T cells of NK cells expressing the CAR of the invention. In addition to a nucleic acid sequence of the invention, or host cells expressing the CAR of the invention, the pharmaceutical composition can comprise other pharmaceutically active agents or drugs, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin , doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In a preferred embodiment, the pharmaceutical composition comprises an isolated T cell or NK cell that expresses the CAR of the invention, more preferably, a population of T cells or NK cells that express the CAR of the invention.
[00066] The T cells or NK cells of the invention can be provided in the form of a salt, for example, a pharmaceutically acceptable salt. Pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric acids, hydrobromic acids, phosphoric acids, metaphosphoric acids, and sulfuric acids, and organic acids, such as tartaric acid, acetic acid, citric acid, malic acid , lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acid, for example, p-toluenesulfonic acid.
[00067] The choice of carrier will be determined by the nucleic acid sequence of the particular invention, vector, or host cells, which express the CAR, as well as by the particular method used to administer a nucleic acid sequence of the invention, vector, or host cells , which express the CAR. Consequently, there are a variety of suitable formulations of the pharmaceutical composition of the invention. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. A mixture of two or more preservatives can optionally be used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition.
[00068] In addition, buffering agents can be used in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. A mixture of two or more buffering agents can optionally be used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition.
[00069] Methods for preparing administrable compositions (for example, parenterally administrable) are known to those skilled in the art and are described in greater detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st ed. (May 1, 2005).
[00070] The composition comprising the nucleic acid sequence of the invention encoding the CAR, or host cells expressing the CAR, can be formulated as an inclusion complex, such as a cyclodextrin inclusion complex, or as a liposome. Liposomes can serve to target host cells (e.g., T cells or NK cells), or the nucleic acid sequence of the invention to a particular tissue. Liposomes can also be used to increase the half-life of a nucleic acid sequence of the invention. Many methods are available for preparing liposomes, such as those described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and United States Patents 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
[00071] The composition may employ time-released, delayed-release, and sustained-release delivery systems, such that the distribution of the composition of the invention occurs before, and with sufficient time to cause sensitization of, the site to be treated. Many types of delivery delivery systems are available and known to those skilled in the art. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the individual and the physician, and may be particularly suitable for certain embodiments of the composition of the invention.
[00072] The composition desirably comprises host cells that express the nucleic acid sequence of the invention encoding a CAR, or a vector comprising the nucleic acid sequence of the invention, in an amount that is effective to treat or prevent multiple myeloma, or Hodgkin's lymphoma. As used herein, the terms "treatment", "treating", and the like, refer to obtaining an appropriate pharmacological and / or physiological effect. Preferably, the effect is therapeutic, that is, the effect partially or completely cures a disease and / or adverse symptom attributable to the disease. For this purpose, the method of the invention comprises administering a "therapeutically effective amount" of the composition comprising the host cells expressing the nucleic acid sequence of the invention encoding a CAR, or a vector comprising the nucleic acid sequence of the invention. A "therapeutically effective amount" refers to an effective amount, in dosages, and for periods of time necessary, to achieve a desired therapeutic result. The therapeutically effective amount can vary according to factors, such as the disease stage, age, sex, and weight of the individual, and the ability of the CAR to induce a desired response in the individual. For example, a therapeutically effective amount of CAR of the invention is an amount that binds to BCMA in multiple myeloma cells, and destroys them.
[00073] Alternatively, the pharmacological and / or physiological effect can be prophylactic, that is, the effect completely or partially prevents a disease or symptom thereof. In this regard, the method of the invention comprises administering a "prophylactic effective amount" of the composition comprising the host cells expressing a nucleic acid sequence of the invention encoding a CAR, or a vector comprising the nucleic acid sequence of the invention, to a mammal who is predisposed to multiple myeloma or Hodgkin's lymphoma. A "prophylactically effective amount" refers to an effective amount, in dosages and for periods of time necessary, to achieve a desired prophylactic result (for example, prevention of disease onset).
[00074] A typical amount of host cells administered to a mammal (for example, a human) can be, for example, in the range of one million to 100 billion cells; however, amounts below or above this exemplary range are within the scope of the invention. For example, the daily dose of host cells of the invention can be about 1 million to about 50 billion cells (for example, about 5 million cells, about 25 million cells, about 500 million cells, about of 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the preceding values), preferably about 10 million to about 100 billion cells (for example, about 20 billion cells, about 30 billion cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a defined range by any two of the preceding values), more preferable about 100 million cells to about 50 billion cells (for example, about 120 million cells, about 250 million cells, about 450 million cells, about 800 million cells, about 350 million of cells, about 650 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells, or a range defined by any of the preceding values).
[00075] Therapeutic or prophylactic efficacy can be monitored by periodic assessment of treated patients. For repeated administrations for several days or longer, depending on the condition, treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful, and are within the scope of the invention. The desired dosage can be delivered by single mass administration of the composition, by multiple mass administration of the composition, or by administration by continuous infusion of the composition.
[00076] The composition comprising the host cells expressing the CAR encoding nucleic acid sequence of the invention, or a vector comprising the CAR encoding nucleic acid sequence of the invention, can be administered to a mammal using standard administration techniques, including, oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. The composition is preferably suitable for parenteral administration. The term "parenteral", as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to a mammal using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
[00077] The composition comprising the host cells expressing the CAR encoding nucleic acid sequence of the invention, or a vector comprising the CAR encoding nucleic acid sequence of the invention, can be administered with one or more additional therapeutic agents, which can be co-administered. -administered to the mammal. By "co-administration" it is significant to administer one or more additional therapeutic agents and the composition comprising the host cells of the invention, or the vector of the invention, sufficiently close in time that the CAR of the invention can enhance the effect of one or more agents additional therapeutic options, or vice versa. In this particular, the composition comprising the host cells of the invention, or the vector of the invention, can be administered first, and the one or more additional therapeutic agents can be administered second, or vice versa. Alternatively, the composition comprising the host cells of the invention, or the vector of the invention, and the one or more additional therapeutic agents can be administered simultaneously. An example of a therapeutic agent that can be co-administered with the composition comprising the host cells of the invention, or the vector of the invention, is IL-2.
[00078] Since the composition comprising host cells expressing the CAR encoding nucleic acid sequence of the invention, or a vector comprising the CAR encoding nucleic acid sequence of the invention, is administered to a mammal (e.g., human), the biological activity of CAR can be measured by any suitable method known in the art. According to the method of the invention, CAR binds to BCMA in multiple myeloma cells, and multiple myeloma cells are destroyed. The binding of CAR to BCMA on the surface of multiple myeloma cells can be assayed using any suitable method known in the art, including, for example, ELISA and flow cytometry. The ability of CAR to destroy multiple myeloma cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32 (7): 689-702 ( 2009), and Herman et al. J. Immunological Methods, 285 (1): 25-40 (2004). The biological activity of CAR can also be measured by assaying the expression of certain cytokines, such as CD107a, IFNY, IL-2, and TNF.
[00079] One skilled in the art will readily appreciate that the CAR encoding nucleic acid sequence of the invention can be modified in any number of ways, such that the therapeutic or prophylactic efficiency of the CAR is increased through modification. For example, the CAR can be combined, either directly, or indirectly, through an articulator to a target fraction. The practice of conjugating compounds, for example, CAR, for targeting fractions, is known in the art. See, for example, Wadwa et al., J. Drug Targeting 3: 111 (1995), and United States Patent 5,087,616.
[00080] The following examples further illustrate the invention, but, of course, should not be construed as in any way limiting its scope. EXAMPLE 1
[00081] This example demonstrates the pattern of BCMA expression in human cells.
[00082] Quantitative polymerase chain reaction (qPCR) was performed on a panel of cDNA samples from a wide range of normal tissues included in panel II of Human Larger tissue qPCR (Origine Technologies, Rockville, MD) using a primer BCMA-specific probe set (Life Technologies, Carlsbad, CA). The cDNA of the cells of a plasmacytoma that was resected from a patient with advanced multiple myeloma was analyzed as a positive control. RNA was extracted from plasmacytoma cells with an RNeasy mini kit (Qiagen, Inc., Valencia, CA), and cDNA was synthesized using standard methods. A standard curve for the BCMA qPCR was created by diluting a plasmid encoding the full-length BCMA cDNA (Origine Technologies, Rockville, MD) in carrier DNA. The qPCR accurately detects copy numbers from 102 to 109 copies of BCMA per reaction. The number of β-actin cDNA copies in the same tissues was quantified with a Taqman β-actin primer and probe kit (Life Technologies, Carlsbad, CA). A β-actin standard curve was created by amplifying serial dilutions of a β-actin plasmid. All qPCR reactions were performed on the Roche LightCycler480 machine (Roche Applied Sciences, Indianapolis, IN).
[00083] The results of the qPCR analysis are shown in Figures 1A and 1B. 93% of the cells from the plasmacytoma sample were plasma cells, as determined by flow cytometry. BCMA expression in the plasmacytoma sample was dramatically higher than BCMA expression in any other tissue. BCMA cDNA was detected in several hematological tissues, such as peripheral blood mononuclear cells (PBMC), bone marrow, spleen, lymph node, and amygdala. Low levels of BCMA cDNA have been detected in many gastrointestinal organs, such as the duodenum, rectum, and stomach. BCMA expression in gastrointestinal organs may be the result of plasma cells and B cells present in lymphoid tissues associated with the intestine, such as lamina propria and Peyer's Patches (see, for example, Brandtzaeg, Immunological Investigations, 39 (4-5 ): 303-355 (2010)). Low levels of BCMA cDNA have also been detected in the tonsils and trachea. The low levels of BCMA cDNA detected in the trachea may be due to the presence of plasma cells in the lamina propria of the trachea (see, for example, Soutar, Thorax, 31 (2): 158-166 (1976)).
[00084] BCMA expression on the surface of various cell types was further characterized using flow cytometry (see Figures 2A-2L), including multiple myeloma cell lines H929, U266, and RPMI8226. Multiple myeloma cell lines H929, U266, and RPMI8226 all express cell surface BCMA. In contrast, the TC71 sarcoma cell line, T-cell leukemia line CCRF-CEM, and the anti-BCMA cell line 293T-17 do not express cell surface BCMA. Primary CD34 + hematopietic cells, primary small airway epithelial cells, primary bronchial epithelial cells, and primary intestinal epithelial cells, all lack serial surface BCMA expression.
[00085] The results of this example demonstrate that BCMA is expressed on the surface of multiple myeloma cells, and has a restricted expression pattern in normal tissues. EXAMPLE 2
[00086] This example describes the construction of a nucleic acid sequence of the invention that encodes anti-BCMA chimeric antigen receptors (CARs).
[00087] The antibody sequences of two mouse-anti-human-BCMA antibodies designated as "C12A3.2" and "C11D5.3" were obtained from International Patent Application Publication WO 2010/104949 (Kalled et al.). The amino acid sequences of the heavy chain variable and light chain variable regions of these antibodies were used to designate single chain variable fragments (scFvs) having the following general structure:
[00088] variable region of light chain - articulator - variable region of heavy chain.
[00089] The articulator has the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 7) (see, for example, Cooper et al., Blood, 101 (4): 1637-1644 (2003)).
[00090] DNA sequences encoding two chimeric antigen receptors have been designated, each of which contains the following elements from 5 'to 3': the CD8α signal sequence, the scFv, articulation, and transmembrane regions of anti-BCMA previously mentioned from the human CD8α molecule, the cytoplasmic portion of the CD28 molecule, and the cytoplasmic portion of the CD3Z molecule. A schematic of these CAR encoding nucleic acid sequences is placed in Figure 3A. CARs that incorporate the variable regions of C12A3.2 and C11D5.3 have been designated anti-bcma1 and anti-bcma2, respectively.
[00091] DNA sequences encoding five additional chimeric antigen receptors based on the anti-bcma2 CAR described above have been designated, each of which contains different signal sequences and T cell activation domains. In this regard, the anti-bcma2 8ss-CAR contains the following elements from 5 'to 3: the CD8α signal sequence, scFv, articulation and transmembrane regions of the human CD8α molecule, the cytoplasmic portion of the CD28 molecule, and the cytoplasmic portion of the CD3Z molecule. The anti-bcma2 G-CAR contains the following elements from 5 'to 3': the human GM-CSF receptor signal sequence, the scFv, hinge, and transmembrane regions of the human CD8α molecule, the cytoplasmic portion of the CD28 molecule, and the cytoplasmic portion of the CD3Z molecule. The anti-bcma2-BB CAR contains the following elements from 5 'to 3': the CD8α signal sequence, the scFv, articulation, and transmembrane regions of the human CD8α molecule, the cytoplasmic portion of the 4- molecule 1BB, and the cytoplasmic portion of the CD3Z molecule. The anti-bcma2-OX40 CAR contains the following elements from 5 'to 3': the CD8α signal sequence, the scFv, articulation, and transmembrane regions of the human CD8α molecule, the cytoplasmic portion of the OX40 molecule ( see, for example, Latza et al., European Journal of Immunology, 24: 677-683 (1994)), and the cytoplasmic portion of the CD3Z molecule. The anti-bcma2-BBOX40 contains the following elements from 5 'to 3': the CD8α signal sequence, the scFv, joint, and transmembrane regions of the human CD8α molecule, the cytoplasmic portion of the 4-1BB molecule , the cytoplasmic portion of the OX40 molecule, and the cytoplasmic portion of the CD3Z molecule. The elements present in each of the seven CAR sequences are placed in Table 1.

[00092] The sequences used for CD8α, CD28, CD3Z, 4-1BB (CD137), and OX40 (CD134) were obtained from the databases of the publicly available National Center for Biotechnology Information (NCBI).
[00093] The nucleic acid sequences encoding CAR were generated using methods known in the art, such as those described in, for example, Kochenderfer et al., J. Immunology, 32 (7): 689-702 (2009), and Zhao et al., J. Immunology, 183 (9): 5563-5574 (2009). The nucleic acid sequence encoding each CAR was codon-optimized and synthesized using GeneArt ™ technology (Life Technologies, Carlsbad, CA) with appropriate restriction sites.
[00094] The sequences encoding the anti-bcma1 CAR and the anti-bcma2s CAR were ligated into a lentiviral vector plasmid called pRRLSIN.cPPT.MSCV.coDMF5.oPRE (see, for example, Yang et al., J. Immunotherapy , 33 (6): 648-658 (2010)). The coDMF5 portion of this vector was replaced with the nucleic acid sequences encoding CAR using standard methods. The two resulting anti-BCMA CAR vectors were denoted pRRLSIN.cPPT.MSCV.anti- bcma1.oPRE and pRRLSIN.cPPT.MSCV.anti-bcma2.oPRE. A negative control CAR containing the SP6 scFv that recognizes the 2,4,6-trinitrophenyl hapten has also been constructed (see, for example, Gross et al., Proc. Natl. Acad. Sci. USA, 86 (24): 10024 -10028 (1989)). This CAR was referred to as SP6. CAR SP6 has been cloned into the same lentiviral vector as anti-BCMA CARs, and contains the same signaling domains as anti-bcma1 and anti-bcma2. The lentivirus-containing supernatant encoding each CAR was produced by the protocol described in Yang et al., Supra. Specifically, 293T-17 cells (ATCC CRL-11268) were transfected with the following plasmids: pMDG (which encodes the vesicular stomatitis virus envelope protein), pMDLg / pRRE (which encodes HIV Gag and Pol proteins), pRSV- Rev (which encode the RSV Rev protein), and plasmids encoding anti-BCMA CARs (see, for example, Yang et al., Supra).
[00095] The sequences encoding the G-anti-bcma2, 8ss-anti-bcma2, anti-bcma2-BB, anti-bcma2-OX40, and anti-bcma2-BBOX40 CARs were each ligated into a gamma-vector vector plasmid called MSGV (mouse stem cell virus-based splice-gag vector) using standard methods, such as those described in, for example, Hughes et al., Human Gene Therapy, 16: 457-472 (2005). After the CAR encoding gamma-retroviral plasmids were generated, the incompetent retrovirus replication with the RD114 envelope was produced by the transient transfection of 293 base packaging cells, as described in Kochenderfer et al., J. Immunotherapy, 32 (7): 689 -702 (2009).
[00096] Incompetent replication of lentiviruses and retroviruses encoding the CARs described above was used to transduce human T cells. For anti-bcma1 and anti-bcma2, T cells were cultured as described previously (see, for example, Kochenderfer et al., J. Immunotherapy, 32 (7): 689-702 (2009)), and were stimulated with OKT3 anti-CD3 monoclonal antibody (Ortho-Biotech, Horsham, PA) in AIM V ™ medium (Life Technologies, Carlsbad, CA) containing 5% human AB serum (Valley Biomedical, Winchester, VA) and 300 international units (UI) / ml interleukin-2 (Novartis Diagnostics, Emeryville, CA). Thirty-six hours after the cultures started, activated T cells were suspended in lentiviral supernatant with protamine sulfate and 300 IU / ml IL-2. The cells were centrifuged for 1 hour at 1200xg. The T cells were then cultured for three hours at 37 ° C. The supernatant was then diluted 1: 1 with RPMI medium (Mediatech, Inc., Manassas, VA) + 10% fetal bovine serum (Life Technologies, Carlsbad, CA) and IL-2. T cells were cultured in the diluted supernatant overnight, and then returned to culture in AIM V ™ medium (Life Technologies, Carlsbad, CA) plus 5% human AB serum with IL-2. T cells were stained with goat anti-mouse F (ab) 2 polyclonal antibodies labeled with biotin (Jackson Immunoresearch Laboratories, Inc., West Grove, PA), to detect anti-BCMA CARs. High levels of cell surface expression of the anti-bcma1 CAR, anti-bcma2 CAR, and CAR SP6 in the transduced T cells were observed, as shown in Figures 3B-3D.
[00097] For G-anti-bcma2, 8ss-anti-bcma2, anti-bcma2-BB, anti-bcma2-OX40, and anti-bcma2-BBOX40 CARs, peripheral blood mononuclear cells were suspended at a concentration of 1x106 cell per ml in T cell medium containing 50 ng / ml of the anti-CD3 monoclonal antibody OKT3 (Ortho, Bridgewater, NJ) and 300 IU / ml of IL-2. RETRONECTIN ™ polypeptide (Takara Bio Inc., Shiga, Japan), which is a recombinant polypeptide of fragments of human fibronectin that binds to viruses and cell surface proteins, has been dissolved at a concentration of 11 μg / mL in saline solution phosphate buffered (PBS), and two mL of RETRONECTIN ™ polypeptide in PBS solution added to each well of 6 well plates coated with nonwoven culture (BD Biosciences, Franklin Lakes, New Jersey). The plates were incubated for two hours at room temperature (RT). After incubation, the RETRONECTIN ™ solution was aspirated, and 2 mL of a blocking solution consisting of Hanks balanced salt solution (HBSS) plus 2% bovine serum albumin (BSA) was added to each well coated with RETRONECTIN ™. The plates were incubated for 30 minutes at room temperature (RT). The blocking solution was aspirated, and the wells were rinsed with a solution of HBSS + 2.5% (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid) (HEPES). The retroviral supernatant was quickly thawed and diluted 1: 1 in T cell medium, and two mL of the diluted supernatant was then added to each well coated with RETRONECTIN ™. After adding the supernatants, the plates were centrifuged at 2000xg for 2 hours at 32 ° C. The supernatant was then aspirated from the wells, and 2x106 T cells that had been cultured with OKT3 and IL-2 antibodies for 2 days were added to each well. When T cells were added to the retrovirus-coated plates, T cells were suspended at a concentration of 0.5 x 10 6 cells per ml in T cell medium plus 300 IU / ml IL-2. After T cells were added to each well, the plates were centrifuged for 10 minutes at 1000xg. The plates were incubated at 37 ° C overnight. The transduction was repeated the next day. After an 18-24 hour incubation, T cells were removed from the plates, and suspended in fresh T cell medium with 300 IU / ml IL-2 at a concentration of 0.5x106 cells per ml, and cultured at 37 ° C and 5% CO2. High levels of cell surface expression of anti-bcma2-BBOX40, anti-bcma2-BB, and 8ss-anti-bcma2 in the translated T cells were observed.
[00098] The results of this example demonstrate a method of producing the nucleic acid sequence encoding the CAR of the invention, and methods of expressing the CAR on the surface of T cells. EXAMPLE 3
[00099] This example describes a series of experiments used to determine the specificity of the CAR of the invention for BCMA. Cells
[000100] NCI-H929, U266, and RPMI8226 are all BCMA + multiple myeloma cell lines that were obtained from ATCC (ATCC Nos. CRL-9068, TIB-196, and CCL-155, respectively). A549 (ATCC No. CCL-185) is a BCMA negative lung cancer cell line. TC71 is a BCMA negative sarcoma cell line. CCRF-CEM is a BCMA negative T cell line (ATCC No. CCL-119). BCMA-K562 are K562 cells (ATCC No. CCL-243) that have been transduced with a nucleic acid sequence encoding full-length BCMA. NGFR-K562 are K562 cells that have been transduced with the gene that encodes low affinity nerve growth factor (see, for example, Kochenderfer et al., J. Immunotherapy., 32 (7): 689-702 (2009) ). Peripheral blood lymphocytes (PBL) from three patients with multiple myeloma (ie, Patient 1 with Myeloma a 3) were used, as were PBL from three other individuals: Donor A, Donor B, and Donor C. C all had melanoma. Primary CD34 + cells were obtained from three healthy normal donors. A sample of plasmacytoma cells was obtained from Patient 1 with Myeloma, and a bone marrow sample was obtained from Patient 3 with Myeloma. All of the aforementioned human samples were obtained from patients enrolled in clinical trials approved by the IRB at the National Cancer Institute. The following primary human epithelial cells were obtained from Lonza, Inc. (Basel, Switzerland): small airway epithelial cells, bronchial epithelial cells, and intestinal epithelial cells. Interferon-Y and TNF ELISA
[000101] BCMA positive cells or BCMA negative cells were combined with CAR transduced T cells in duplicate wells of a 96 well round bottom plate (Corning Life Sciences, Lowell, MA) in AIM V ™ medium (Life Technologies , Carlsbad, CA) + 5% human serum. The plates were incubated at 37 ° C for 18-20 hours. Following incubation, ELISAs for IFNY and TNF were performed using standard methods (Pierce, Rockford, IL).
[000102] T cells transduced with the anti-bcma1 CAR or anti-bcma2 CAR produced large amounts of IFNY when they were grown overnight with the BCMA BCMA-K562 expression cell line, but the T cells transduced by CAR only produced previous levels of IFNY when they were cultured with the NGFR-K562 negative control cell line, as shown in Table 2 (all units are pg / mL of IFNy. Table 2
1 The effector cells were T cells from a patient with multiple myeloma (Patient 2 with Myeloma). T cells were transduced with the indicated CAR, or left untransduced. 2 * The indicated target cells were combined with the effector cells by an overnight incubation, and an IFNY ELISA was performed. Petition 870200153557, of 12/07/2020, p. 54/80
[000103] T cells expressing the 8ss-anti-bcma2, anti-bcma2-BB, and anti-bcma2-OX40 CARs produce IFNY specifically in response to BCMA + target cells when the T cells and target cells were co-cultured overnight, as shown in Table 3 (all units are pg / mL IFNy. Table 3

[000104] T cells transduced with anti-BCMA CARs produced large amounts of IFNy when they were grown overnight with BCMA-expressing multiple myeloma cell lines. In contrast, anti-BCMA CARs produce much lower amounts of IFNy when they were cultured with a variety of BCMA negative cell lines. Compared with the T cells transduced with the anti-bcma1 CAR, the T cells transduced with the anti-bcma2 CAR, and variants thereof (i.e., 8ss-anti-bcma2, anti-bcma2-BB, and anti-bcma2-OX40) , produced more IFNy when cultured with BCMA positive cells, and less IFNy when cultured with BCMA negative cells.
[000105] T cells transduced with anti-bcma2 CAR variants produced TNF specifically in response to BCMA + target cells when T cells and target cells were co-cultured overnight, as shown in Table 4 (all units are pg / mL tumor necrosis factor (TNF)). Table 4

[000106] Because T cells transduced with the anti-bcma2 CAR and variants of it exhibit slightly stronger and more specific recognition of cells expressing BCMA than T cells transduced with the anti-bcma1 CAR, only the anti-bcma2 CAR variants and CAR anti-bcma2 were used in the following experiments. CD107a assay
[000107] Two populations of T cells were prepared in two separate tubes. One tube contained BCMA-K562 cells, and the other tube contained NGFR-K562 cells. Both tubes also contained T cells transduced with the variants of the anti-bcma2 CAR and anti-bcma2 CAR, 1 mL of AIM V ™ medium (Life Technologies, Carlsbad, CA) + 5% human serum, a titrated concentration of one anti-CD107a antibody (eBioscience, Inc., San Diego, CA; eBioH4A3 clone), and 1 μL of Golgi Stop (BD Biosciences, Franklin Lakes, NJ). All tubes were incubated at 37 ° C for four hours and then colored for expression of CD3, CD4, and CD8.
[000108] T cells transduced with CAR from three different individuals upwardly regulated CD107a specifically in response to stimulation with target cells expressing BCMA (see Figures 4A-4C). This indicates the occurrence of BCMA-specific degranulation of T cells, which is a prerequisite for perforin-mediated cytotoxicity (see, for example, Rubio et al., Nature Medicine, 9 (11): 1377-1382 (2003)) . In addition, T cells expressing the anti-bcma2 8ss-anti-bcma2, anti-bcma2-BB, anti-bcma2-OX40 CAR variants degranulate in a BCMA-specific manner when stimulated with target cells in vitro, as shown in Figures 5A-5D. Intracellular cytokine staining assay (ICCS)
[000109] A cell population of BCMA-K562 and a cell population of NGFR-K562 were prepared in two separate tubes, as described above. Both tubes also contained T cells transduced with the anti-bcma2 CAR of Patient 2 with Myeloma, 1 mL of AIM V medium (Life Technologies, Carlsbad, CA) + 5% human serum, and 1 μL of Golgi Stop (BD Biosciences , Franklin Lakes, NJ). All tubes were incubated at 37 ° C for six hours. The cells were stained on the surface with anti-CD3, anti-CD4, and anti-CD8 antibodies. The cells were permeabilized, and intracellular staining was conducted by IFNY (BD Biosciences, Franklin Lakes, NJ, clone B27), IL-2 (BD Biosciences, Franklin Lakes, NJ, clone MQ1-17H12), and TNF (BD Biosciences, Franklin Lakes, NJ, MAb11 clone) by following instructions from Kir Cytofix / Cytoperm (BD Biosciences, Franklin Lakes, NJ).
[000110] Large populations of T cells transduced with the anti-bcma2 CAR of Patient 2 with Myeloma specifically produced the cytokines IFNY, IL-2, and TNF in a BCMA specific manner after six hours of stimulation with target cells expressing BCMA , as shown in Figures 6A-6C. Proliferation assays
[000111] The ability of T cells to transduce with the anti-bcma2 CAR to proliferate when stimulated with target cells that express BCMA, was evaluated. Specifically, 0.5x106 irradiated BCMA-K562 cells, or 0.5x106 irradiated NGFR-K562 cells, were co-cultured with 1x106 total T cells that were transduced with either the anti-bcma2 CAR or SP6 CAR. T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) (Life Technologies, Carlsbad, CA), as described in Mannering et al., J. Immunological Methods, 283 (1-2): 173-183 (2003). The medium used in the co-cultures was AIM V ™ medium (Life Technologies, Carlsbad, CA) + 5% human AB serum. IL-2 was not added to the medium. Four days after initiation, live cells in each co-culture were counted with trypan blue to exclude dead cells. Flow cytometry was then performed by staining T cells with goat anti-human BCMA antibodies labeled with polyclonal biotin (R&D Systems, Minneapolis, MN), followed by streptavidin (BD Biosciences, Franklin Lakes, NJ) , anti-CD38 antibody (eBioscience, Inc., San Diego, CA), and anti-CD56 antibody (BD Biosciences, Franklin Lakes, NJ). Flow cytometry data analysis was performed using FlowJo software (Tree Star, Inc., Ashland, OR).
[000112] T cells expressing the anti-bcma2 CAR exhibited a greater dilution of CFSE when cultured with BCMA-K562 cells than when cultured with negative control NGFR-K562 cells, as shown in Figure 7A. These results indicate that T cells transduced with the anti-bcma2 CAR specifically proliferate when stimulated with target cells that express BCMA. In contrast, there is no significant difference in the dilution of CFSE when T cells expressing CAR SP6 were cultured with either BCMA-K562 target cells or NGFR-K562 target cells (see Figure 7B), which demonstrates a lack of specific BCMA proliferation by T cells expressing CAR SP6.
[000113] At the beginning of the proliferation assays, 0.8x106 T cells expressing the anti-bcma2 CAR were cultured with either BCMA-K562 cells or NGFR-K562 cells. After 4 days of culture, 2.7 x 10 6 T cells expressing anti-bcma 2 CAR were present in cultures containing BCMA-K562 cells, while only 0.6 x 10 6 T cells expressing anti-bcma 2 CAR were present in cultures containing NGFR-K562 cells. This specific increase in BCMA in the absolute number of T cells expressing the anti-bcma2 CAR indicates that these Ts cells proliferate in response to BCMA.
[000114] The results of this example demonstrate that the T cells expressing the CAR of the invention exhibit production, degranulation and proliferation of BCMA-specific cytokion. EXAMPLE 4
[000115] This example demonstrates that the T cells expressing the anti-BCMA CAR of the invention can destroy multiple myeloma cell lines.
[000116] Cytotoxicity assays were performed to determine whether T cells transduced with the anti-bcma2 CAR described in Examples 2 and 3 can destroy BCMA-expressing multiple myeloma (MM) cell lines. Specifically, the cytotoxicity of target cells was measured by comparing the survival of target cells expressing BCMA (i.e., H929 and RPMI8226 multiple myeloma cell lines) relative to the survival of negative control CCRF-CEM cells using an assay described in , for example, Kochenderfer et al., J. Immunotherapy, 32 (7): 689-702 (2009), and Hermans et al., J. Immunological Methods, 285 (1): 25-40 (2004).
[000117] Approximately 50,000 BCMA-expressing target cells and 50,000 CCRF-CEM cells were combined in the same tubes with different numbers of T cells transduced by CAR. The CCRF-CEM negative control cells were labeled with the fluorescent dye 5- (e-6) - (((4-chloromethyl) benzoyl) amino) tetramethyl rhodamine (CMTMR) (Life Technologies, Carlsbad, CA), and the cells targets that express BCMA have been labeled with CFSE. In all experiments, the cytotoxicity of effector T cells that were transduced with anti-bcma2 CAR was compared to the cytotoxicity of negative control effector T cells from the same individuals that were transduced with CAR SP6. Co-cultures were established in 5 ml sterile test tubes (BD Biosciences, Franklin Lakes, NJ) in duplicate at the following T cell: target cell ratios: 20.0: 1, 7: 1, 2: 1, and 0.7: 1. The cultures were incubated for four hours at 37 ° C. Immediately after incubation, 7-amino-actinomycin D (7AAD; BD Biosciences, Franklin Lakes, NJ) was added. The percentages of live target cells expressing BCMA and live CCRF-CEM negative control cells were determined for each T cell / target cell co-culture.
[000118] For each T cell / target cell co-culture, the percentage survival of target cells expressing BCMA relative to CCRF-CEM negative control cells was determined by dividing the percentage of cells expressing BCMA by the percentage of cells negative control of CCRF-CEM. The percentage of corrected survival of target cells expressing BCMA was calculated by dividing the percentage of survival of target cells expressing BCMA in each T cell / target cell co-culture by the ratio of the percentage of target cells expressing BCMA: percentage of cells negative CCRF-CEM control cells in tubes containing only BCMA-expressing target cells and negative CCRF-CEM control cells without effector T cells. This correction was necessary to count the variation at the beginning of the cell numbers, and for spontaneous death of the target cell. Cytotoxicity was calculated as follows:
[000119]% cytotoxicity of target cells expressing BCMA = 100 -% corrected survival of target cells expressing BCMA.
[000120] The results of the cytotoxicity assay are shown in Figures 7C and 7D. T cells transduced with anti-bcma2 CAR specifically kill multiple myeloma cell lines that express BCMA H929 and RPMI8226. In contrast, T cells transduced with CAR SP6 exhibit much lower levels of cytotoxicity against these cell lines.
[000121] The results of this example demonstrate that the nucleic acid sequence of the invention encoding an anti-BCMA CAR can be used in a method of destroying multiple myeloma cell lines. EXAMPLE 5
[000122] This example demonstrates that T cells expressing the anti-BCMA CAR of the invention can destroy primary multiple myeloma cells.
[000123] The primary multiple myeloma cells described in Example 2 were evaluated for BCMA expression, as well as for BCMA-specific cytokine production, degranulation and proliferation using the methods described above.
[000124] BCMA expression from the cell surface was detected in four primary multiple myeloma samples, as well as in primary myeloma cells from patient 3 with myeloma (see Figure 8A). BCMA expression plasma cells produce 40% of the cells in the bone marrow sample of Patient 3 with Myeloma. Allogeneic T cells transduced with Donor C's anti-bma2 CAR produced IFNY after co-culture with Patient 3 cells with unmanipulated bone marrow myeloma, as shown in Figure 8B. T cells transduced with anti-bcma2 CAR from the same allogeneic donor produced much less IFNY when they were cultured with peripheral blood mononuclear cell (PBMC) from Patient 3 with Myeloma. In addition, T cells transduced with CAR SP6 from Donor C do not specifically recognize the bone marrow of Patient 3 with Myeloma. It has been previously reported that normal PBMC does not contain cells that express BCMA (see, for example, Ng et al., J. Immunology, 173 (2): 807-817 (2004)). To confirm this observation, the PBMC of Patient 3 was assessed for BCMA expression by flow cytometry. The PBMC of Patient 3 does not contain cells that express BCMA, apart from a small population of CD56 + CD38high cells that produce approximately 0.75% of the PBMC. This population possibly consists of circulation of multiple myeloma cells.
[000125] A plasmacytoma resected from Patient 1 with Myeloma consisted of 93% of plasma cells, and these primary plasma cells express BCMA, as shown in Figure 8C. The T cells of Patient 2 with Myeloma produced IFNY when cultured with the allogeneic unmanipulated plasmacytoma cells of Patient 1 with Myeloma. T cells from Patient 2 with Myeloma did not produce significant amounts of IFNy when cultured with PBMC from Patient 1 with Myeloma. The T cells of Patient 2 with Myeloma that were transduced with CAR SP6 do not produce significant amounts of IFNy when they were cultured with, or plasmacytoma cells, or PBM of Patient 1 with Myeloma. The PBMC of Patient 1 with Myeloma does not express BCMA, as measured by flow cytometry.
[000126] T cells from Patient 1 with Myeloma, who received eight previous cycles of myeloma therapy, were successfully cultured and transduced with a lentivirus vector encoding the anti-bcma2 CAR. Eight days after the cultures started, the expression of anti-bcma2 CAR was detected in 65% of T cells. Myeloma 1 T cells expressing anti-bcma2 produce IFNY specifically in response to autologous plasmacytoma cells (Figure 8D). The T cells of Patient 1 with Myeloma that express CAR SP6 do not recognize autologous plasmacytoma cells. T cells expressing anti-bcma2 CAR and T cells expressing CAR SP6 do not recognize autologous PBMC. Myeloma Patient 1 T cells that express the anti-bcma2 CAR also specifically kill autologous plasmacytoma cells at low effector-to-target ratios. In contrast, the T cells of Patient 1 with Myeloma that express CAR SP6 exhibited low levels of cytotoxicity against autologous plasmacytoma cells (Figure 8E).
[000127] The results of this example demonstrate that the anti-BCMA CAR of the invention can be used in a method of destroying primary multiple myeloma cells. EXAMPLE 6
[000128] This example demonstrates that T cells expressing the anti-BCMA CARs of the invention can destroy established tumors in mice.
[000129] Immunodeficient NDG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, Jackson Laboratory) were injected intradermally with 8x106 cells of RPMI8226. The tumors were allowed to grow for 17 to 19 days, and then the mice received intravenous infusions of 8x106 human T cells that were transduced with either CAR anti-bcma2 or CAR SP6. Tumors were measured with calipers every 3 days. The longest length and the length perpendicular to the longest length were multiplied to obtain the tumor size (area) in mm2. When the longest length reached 15 mm, the mice were sacrificed. The animals studied were approved by the National Cancer Institute Animal Care and Use Committee.
[000130] The results of this example are shown in Figures 9A and 9B. Around day 6, mice treated with T-cells transduced by anti-bcma2 showed a reduction in tumor size, and the tumors were eradicated on day 15. In addition, all mice treated with T cells transduced by anti-bcma2 survived 30 days after T cell infusion.
[000131] The results of this example demonstrate that the anti-BMCA CAR of the invention can destroy multiple myeloma cells in vivo.
[000132] All references, including publications, patent applications, and patents, cited herein, are therefore incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference, and have been placed in its entirety here.
[000133] The use of the terms "one" and "one" and "o", and similar referents, in the context of describing the invention (especially in the context of the following claims), are to be constructed to cover both the singular and the plural , unless otherwise indicated here, or clearly contradicted by the context. The terms "comprising", "having", "including", and "containing", are to be constructed as open terms (that is, meaning "including, but not limited to"), unless otherwise noted. The recitation of ranges of values here is merely intended to serve as an abbreviated method of referring individually to each separate value that falls within the range, unless otherwise indicated here, and each separate value is incorporated into the specification as if were individually recited here. All of the methods described herein can be performed in any suitable order, unless otherwise indicated herein, or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (for example, "such as") provided herein, is intended merely to better clarify the invention, and does not impose a limitation on the scope of the invention, unless otherwise , claimed. No language in the specification should be constructed as indicating any element not claimed as essential to the practice of the invention.
[000134] Preferred embodiments of this invention are described herein, including the best way known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those skilled in the art after reading the preceding description. The inventors, except those skilled in the art, employ such variations as appropriate, and the inventors intend the invention to be practiced in another way than as specifically described herein. Consequently, this invention includes all modifications and equivalents of the subject matter recited in the claims hereby attached, as permitted by applicable law. In addition, any combination of the elements described above in all possible variations of these is involved by the invention, unless otherwise indicated herein, or otherwise clearly contradicted by the context.

权利要求:
Claims (55)
[0001]
1. Isolated or purified nucleic acid sequence, characterized by the fact that it encodes a chimeric antigen (CAR) receptor, in which the CAR comprises (a) an antibody or antigen binding fragment directed against the B cell maturation antigen (BCMA); (b) a transmembrane domain selected from the group consisting of: a CD28 transmembrane domain and a CD8α transmembrane domain; and (c) an intracellular T cell signaling domain isolated from a protein selected from the group consisting of: CD8 alpha, CD28, CD27, OX40 and 4-1BB; and (d) an intracellular CD3Z or FcRY T cell signaling domain.
[0002]
An isolated or purified nucleic acid sequence according to claim 1, characterized in that the antibody or antigen binding fragment comprises a monoclonal antibody directed against BCMA, or an antigen binding fragment thereof.
[0003]
An isolated or purified nucleic acid sequence according to claim 2, characterized in that the antibody or antigen binding fragment comprises a variable region of a monoclonal antibody directed against BCMA.
[0004]
An isolated or purified nucleic acid sequence according to any one of claims 1 to 3, characterized in that one or more T cell signaling domains are derived from a protein selected from the group consisting of: a CD8 protein -human alpha, a human CD28 protein, a human CD3-zeta protein, a human FCRY protein, a CD27 protein, an OX40 protein, a human 4-1BB protein, modified versions of any of the foregoing, or any combination of the foregoing.
[0005]
An isolated or purified nucleic acid sequence according to any one of claims 1 to 4, characterized in that the one or more T cell signaling domains are derived from a human 4-1BB protein.
[0006]
An isolated or purified nucleic acid sequence according to any one of claims 1 to 5, characterized in that the one or more T cell signaling domains are derived from a human 4-1BB protein and a CD3- human zeta.
[0007]
An isolated or purified nucleic acid sequence according to any one of claims 1 to 6, characterized in that the antibody or antigen binding fragment comprises: (a) the (i) heavy chain complementarity determining region (CDR) 1, (ii) CDR2 of the heavy chain, (iii) CDR3 of the heavy chain, (iv) CDR1 of the light chain, (v) CDR2 of the light chain, and (vi) CDR3 of the light chain of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12; or (b) a (i) variable region of the heavy chain and (ii) variable region of the light chain of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 , SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.
[0008]
An isolated or purified nucleic acid sequence according to any one of claims 1 to 4, characterized in that it comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 .
[0009]
9. Vector, characterized by the fact that it comprises the isolated or purified nucleic acid sequence, as defined in any one of claims 1 to 8.
[0010]
10. Isolated or purified CAR, characterized by the fact that it is encoded by the nucleic acid sequence, as defined in any one of claims 1 to 8.
[0011]
11. Isolated or purified CAR according to claim 9, characterized by the fact that it comprises the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.
[0012]
12. CAR according to claim 10 or 11, characterized in that the antigen-binding domain comprises a variable region of the heavy chain of a monoclonal antibody directed against BCMA, or an antigen-binding fragment thereof.
[0013]
13. CAR according to any one of claims 10 to 12, characterized in that the antibody or antigen-binding fragment comprises an antigen-binding fragment of a monoclonal antibody directed against BCMA.
[0014]
14. CAR according to claim 13, characterized by the fact that the antigen-binding fragment is selected from the group consisting of: a Fab fragment, an F (ab ') 2 fragment, an Fv fragment, a variable fragment single chain (scFv) and a diabody.
[0015]
15. CAR according to claim 14, characterized by the fact that the antigen-binding fragment comprises an scFv.
[0016]
16. CAR according to any one of claims 10 to 15, characterized in that the one or more domains of intracellular signaling of T cells are isolated from a protein selected from the group consisting of: CD28, CD3Z, FcRY, CD27 , OX40 and protein 4-1BB.
[0017]
17. CAR according to any one of claims 10 to 16, characterized in that the one or more intracellular T cell signaling domains comprise an intracellular CD28 T cell signaling domain.
[0018]
18. CAR according to claim 17, characterized in that one or more intracellular T cell signaling domains further comprise an intracellular CD3Z T cell signaling domain.
[0019]
19. CAR according to claim 18, characterized in that the one or more intracellular T cell signaling domains comprise an intracellular T cell signaling domain OX40.
[0020]
20. CAR according to any one of claims 10 to 16, characterized in that the one or more intracellular T cell signaling domains comprises an intracellular FCRY T cell signaling domain.
[0021]
21. CAR according to claim 20, characterized in that one or more intracellular T cell signaling domains further comprise an intracellular CD3Z T cell signaling domain.
[0022]
22. CAR according to any one of claims 10 to 16, characterized in that the one or more intracellular T cell signaling domains comprise an intracellular CD27 T cell signaling domain.
[0023]
23. CAR according to claim 22, characterized in that the one or more intracellular T cell signaling domains further comprise an intracellular CD3Z T cell signaling domain.
[0024]
24. CAR according to any one of claims 10 to 16, characterized in that the one or more intracellular T cell signaling domains comprise an intracellular T cell signaling domain OX40.
[0025]
25. CAR according to claim 24, characterized in that the one or more intracellular T cell signaling domains further comprise an intracellular CD3Z T cell signaling domain.
[0026]
26. CAR according to claim 25, characterized in that the one or more intracellular T cell signaling domains further comprise an intracellular 4-1BB T cell signaling domain.
[0027]
27. CAR according to any one of claims 10 to 16, characterized in that the one or more intracellular T cell signaling domains comprise an intracellular 4-1BB T cell signaling domain.
[0028]
28. CAR according to claim 27, characterized in that one or more intracellular T cell signaling domains further comprise an intracellular CD3Z T cell signaling domain.
[0029]
29. CAR according to any one of claims 10 to 28, characterized in that it further comprises a signal sequence polypeptide.
[0030]
30. CAR according to claim 29, characterized in that the signal sequence polypeptide is a macrophage and granulocyte colony stimulating factor (GM-CSF) signal polypeptide or a CD8α signal sequence polypeptide .
[0031]
31. CAR according to any one of claims 10 to 30, characterized by the fact that it further comprises a hinge domain.
[0032]
32. CAR according to claim 31, characterized by the fact that the hinge domain is a CD8α hinge domain or a CD28 hinge domain.
[0033]
33. Method for destroying cancer cells, characterized by the fact that it comprises the contact of cancer cells that express BCMA with one or more isolated T cells that express CAR, as defined in any one of claims 10 to 32, in which the CAR is produced and binds to BCMA in the cancer cells and the cancer cells are destroyed, in which the cancer cells are in vitro.
[0034]
34. Method for destroying cancer cells, characterized by the fact that it comprises contacting cancer cells that express BCMA with one or more isolated NK cells that express CAR, as defined in any one of claims 10 to 32, wherein CAR is produced and binds to BCMA in the cancer cells and the cancer cells are destroyed, in which the cancer cells are in vitro.
[0035]
35. Use of the nucleic acid sequence, as defined in any of claims 1 to 8, of the vector, as defined in claim 9, or of the CAR, as defined in any of claims 10 to 32, characterized in that it is for the manufacture of a medicine for the destruction of cancer cells.
[0036]
36. Method according to claim 33 or 34, or use according to claim 35, characterized in that the cancer cells are multiple myeloma cells or Hodgkin's lymphoma cells.
[0037]
37. CAR, characterized by the fact that it comprises: a signal sequence polypeptide; an antibody or its antigen-binding fragment directed against BCMA; a CD8α hinge domain or a CD28 hinge domain; a CD8α transmembrane domain or a CD28 transmembrane domain; a 4-1BB intracellular T cell signal domain and / or an OX40 intracellular T cell signal domain and / or a CD28 intracellular T cell signal domain; and a CD3Z T cell intracellular signaling domain.
[0038]
38. CAR according to claim 37, characterized in that it comprises the signal sequence polypeptide; the antibody or its antigen-binding fragment directed against BCMA; the CD8α hinge domain; the CD8α transmembrane domain; the 4-1BB intracellular T cell signaling domain; and the CD3Z T cell intracellular signaling domain.
[0039]
39. CAR according to claim 37, characterized in that it comprises the signal sequence polypeptide; the antibody or its antigen-binding fragment directed against BCMA; the CD8α hinge domain; the CD8α transmembrane domain; the OX40 T cell intracellular signaling domain; and the CD3Z T cell intracellular signaling domain.
[0040]
40. CAR according to claim 37, characterized in that it comprises the signal sequence polypeptide; the antibody or its antigen-binding fragment directed against BCMA; the CD8α hinge domain; the CD8α transmembrane domain; the CD28 T cell intracellular signaling domain; and the CD3Z T cell intracellular signaling domain.
[0041]
41. CAR according to claim 37, characterized in that it comprises the signal sequence polypeptide; the antibody or its antigen-binding fragment directed against BCMA; the CD8α hinge domain; the CD28 transmembrane domain; the CD28 T cell intracellular signaling domain; and the CD3Z T cell intracellular signaling domain.
[0042]
42. CAR according to claim 37, characterized in that it comprises the signal sequence polypeptide; the antibody or its antigen-binding fragment directed against BCMA; the CD28 hinge domain; the CD28 transmembrane domain; the CD28 T cell intracellular signaling domain; and the CD3Z T cell intracellular signaling domain.
[0043]
43. CAR, characterized by the fact that it comprises: a CD8α signal sequence polypeptide; a scFv directed against BCMA; a CD8α hinge domain or a CD28 hinge domain; a CD8α transmembrane domain or a CD28 hinge domain; a 4-1BB intracellular T cell signal domain and / or an OX40 intracellular T cell signal domain and / or a CD28 intracellular T cell signal domain; and a CD3Z T cell intracellular signaling domain.
[0044]
44. CAR according to claim 43, characterized by the fact that it comprises: the CD8α signal sequence polypeptide; scFv directed against BCMA; the CD8α hinge domain; the CD8α transmembrane domain; a 4-1BB T cell intracellular signaling domain; and the CD3Z T cell intracellular signaling domain.
[0045]
45. CAR according to claim 43, characterized by the fact that it comprises: the CD8α signal sequence polypeptide; scFv directed against BCMA; the CD8α hinge domain; the CD8α transmembrane domain; the OX40 T cell intracellular signaling domain; and the CD3Z T cell intracellular signaling domain.
[0046]
46. CAR according to claim 43, characterized by the fact that it comprises: the CD8α signal sequence polypeptide; scFv directed against BCMA; the CD8α hinge domain; the CD8α transmembrane domain; the CD28 T cell intracellular signaling domain; and the CD3Z T cell intracellular signaling domain.
[0047]
47. CAR according to claim 43, characterized by the fact that it comprises: the CD8α signal sequence polypeptide; scFv directed against BCMA; the CD8α hinge domain; the CD28 transmembrane domain; the CD28 T cell intracellular signaling domain; and the CD3Z T cell intracellular signaling domain.
[0048]
48. CAR according to claim 43, characterized by the fact that it comprises: the CD8α signal sequence polypeptide; scFv directed against BCMA; the CD28 hinge domain; the CD28 transmembrane domain; the CD28 T cell intracellular signaling domain; and the CD3Z T cell intracellular signaling domain.
[0049]
49. Polynucleotide, characterized by the fact that it encodes the CAR, as defined in any of claims 37 to 48.
[0050]
50. Polynucleotide according to claim 49, characterized by the fact that it comprises ribonucleotides.
[0051]
51. Vector, characterized by the fact that it comprises a polynucleotide that encodes the CAR, as defined in any one of claims 37 to 48.
[0052]
52. Vector, according to claim 51, characterized by the fact that it is a viral vector.
[0053]
53. Vector according to claim 52, characterized by the fact that the viral vector is a retroviral vector.
[0054]
54. Vector according to claim 53, characterized by the fact that the retroviral vector is a retroviral gamma vector.
[0055]
55. Vector according to claim 53, characterized by the fact that the retroviral vector is a lentiviral vector.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-11-05| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-09-08| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-02-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-03-30| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 15/03/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201261622600P| true| 2012-04-11|2012-04-11|

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US61/622,600|2012-04-11|

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PCT/US2013/032029|WO2013154760A1|2012-04-11|2013-03-15|Chimeric antigen receptors targeting b-cell maturation antigen|

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