Chimeric antigen receptors targeting b-cell maturation antigen

Abstract

To provide compositions and methods that can be used in methods to treat multiple myeloma.SOLUTION: The invention provides an isolated or purified nucleic acid sequence encoding a chimeric antigen receptor (CAR) directed against B-cell Maturation Antigen (BCMA). The invention also provides host cells, such as T-cells or natural killer (NK) cells, expressing the CAR and methods for destroying multiple myeloma cells.SELECTED DRAWING: None

Description

[Background technology]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This patent application claims the benefit of U.S. Provisional Application No. 61 / 622,600, filed April 11, 2012. The entire application is incorporated herein by reference.

[0002] [INCORPORATION BY REFERENCE OF ELECTRONICALLY SUBMITTED MATERIALS INTO THIS APPLICATION] The following computer readable nucleic acid / amino acid sequences are submitted concurrently with this application: The listings are incorporated herein by reference in their entirety: A 42,589 byte ASCII (text) file named "712361#ST25.TXT," created on March 14, 2013.

[0003] [Background of the invention] Multiple myeloma (MM) is a malignant tumor characterized by the accumulation of clonal plasma cells (see, e.g., 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 treatments for this disease often result in remission, but most patients eventually relapse and die (see, e.g., Lonial et al., supra, and Rajkumar, Nature Rev. Clinical Oncol., 8(8): 479-491 (2011)). Allogeneic hematopoietic stem cell transplantation can induce immune-mediated myeloma cell elimination. Although this approach has been shown to be effective, it is highly toxic and few patients are cured (see, e.g., Lonial et al., supra, and Salit et al., Clin. Lymphoma, Myeloma, and Leukemia, 11(3): 247-252 (2011)). Currently, there are no clinically effective FDA-approved monoclonal antibodies for MM. There are no cloned antibody or autologous T cell therapies (see, e.g., Richardson et al., British J. Haematology, 154(6): 745-754 (2011) and Yi, Cancer Journal, 15(6): 502-510 (2009)).

[0004] Adoptive transfer of T cells genetically modified to recognize paraneoplastic antigens It has shown promise as a new approach to cancer therapy (see, e.g., Morgan et al., Science, 314(5796): 126-129 (2006); Brenner et al., Current Opinion in Immunology, 22(2):251-257 (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 have been genetically engineered to express chimeric antigen receptors (CARs), which are fusion proteins consisting of an antigen-recognition moiety and a T-cell activation domain. (See, e.g., 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] Adoptive T cell transfer approaches utilizing anti-CD19 CARs for B cell lineage malignancies Substantial progress has been made in the development of new therapeutic approaches (see, e.g., 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)). Adoptively transferred anti-CD19CAR-transduced T cells have cured leukemia and lymphoma (e.g., Cheadle et al., Journal of Immunology, 184(4): 1885-1896 (2010); Brentjens (See Kochenderfer et al., Clinical Cancer Research, 13(18 Pt 1): 5426-5435 (2007) and Kochenderfer et al., Blood, 116(19): 3875-3886 (2010). In early clinical trials, anti-CD19 CARs Adoptive cell transfer of transduced T cells has been shown to positively affect patients with leukemia and lymphoma. eradicated normal and malignant B cells (e.g., Kochenderfer et al., Blood, 116(20): 4099-4102 (2010); Porter et al., supra; Brentjen et al., Blood, 118(18): 4817-4828 (2011); and Kochenderfer et al., Blood, December 8, 2011 (see epublication ahead of print (2012)). However, CD19 is rarely expressed on malignant plasma cells in multiple myeloma (see, e.g., 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)). Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there remains a need for compositions that can be used in the treatment of multiple myeloma. The present invention provides such compositions and methods of treatment. [Means for solving the problem]

[0007] [Brief summary of the invention] The present invention provides an isolated or purified nucleic acid encoding a chimeric antigen receptor (CAR) comprising an antigen recognition portion and a T cell activation portion, wherein the antigen recognition portion is directed against B cell maturation antigen (BCMA). Provide the sequence. [Brief explanation of the drawings]

[0008] [Figure 1] 1A and 1B are graphs depicting experimental data showing the expression pattern of BCMA in various human cell types as determined using quantitative PCR. Results are expressed as BCMA cDNA copies per 10 actin cDNA copies. [Figure 2]2A-2L are graphs depicting experimental data demonstrating that cell surface BCMA expression was detected on multiple myeloma cell lines, but not on other cell types, as described in Example 1. For all plots, the solid line represents staining with an anti-BCMA antibody, and the dashed line represents staining with an isotype-matched control antibody. All plots were captured on viable cells. [Figure 3] Figure 3A is a schematic diagram depicting a nucleic acid construct encoding an anti-BCMA CAR. From N- to C-terminus, the anti-BCMA CAR comprises an anti-BCMA scFv, the hinge and transmembrane region of a CD8α molecule, the cytoplasmic portion of a CD28 molecule, and the cytoplasmic portion of a CD3ζ molecule. Figures 3B-3D are graphs depicting experimental data demonstrating that anti-bcma1 CAR, anti-bcma2 CAR, and SP6 CAR (described in Example 2) were expressed on the surface of T cells. Minimal anti-Fab (antigen-binding fragment) staining occurred on untransduced (UT) cells. Plots were captured on viable CD3+ lymphocytes. The numbers on the plot indicate the percentage of cells in each quadrant. [Figure 4] 4A-4C are graphs depicting experimental data showing that T cells expressing an anti-BCMA CAR degranulate T cells in a BCMA-specific manner, as described in Example 3. Plots were captured on viable CD3+ lymphocytes. Numbers on the plot indicate the percentage of cells in each quadrant. [Figure 5] 5A-5D are graphs depicting experimental data showing that T cells expressing an anti-BCMA CAR degranulate T cells in a BCMA-specific manner, as described in Example 3. Plots were captured on viable CD3+ lymphocytes. Numbers on the plot indicate the percentage of cells in each quadrant. [Figure 6] 6A-6C are graphs depicting experimental data showing that T cells expressing anti-BCMA CARs produce the cytokines IFNγ, IL-2, and TNF in a BCMA-specific manner, as described in Example 3. Plots were captured on viable CD3+ lymphocytes. Numbers on the plot indicate the percentage of cells in each quadrant. [Figure 7]Figure 7A is a graph showing experimental data demonstrating that T cells expressing the anti-bcma2 CAR proliferated specifically in response to BCMA. Figure 6B is a graph showing experimental data demonstrating that T cells expressing the SP6 CAR did not proliferate specifically in response to BCMA. Figures 7C and 7D are graphs showing experimental data demonstrating that T cells from donor A expressing the anti-bcma2 CAR specifically killed multiple myeloma cell lines H929 (Figure 6C) and RPMI8226 (Figure 6D) in a 4-hour cytotoxicity assay at various effector:target cell ratios. T cells transduced with the negative control SP6 CAR produced much lower levels of cytotoxicity at all effector:target ratios. At all effector:target ratios, cytotoxicity was measured in duplicate, and results are shown as the mean + / - standard error of the mean. [Figure 8]Figure 8A is a graph representing experimental data showing that BCMA is expressed on the surface of primary bone marrow multiple myeloma cells harvested from myeloma patient 3, as described in Example 5. The plot captures CD38highCD56+ plasma cells, which accounted for 40% of bone marrow cells. Figure 8B is a graph representing experimental data showing that allogeneic T cells transduced with the anti-bcma2CAR from donor C, as described in Example 5, produced IFNγ after co-culture with non-genetically engineered bone marrow cells from myeloma patient 3. Figure 7B shows that T cells from the same allogeneic donor also expressing the anti-bcma2CAR produced much less IFNγ when cultured with peripheral blood mononuclear cells (PBMCs) from myeloma patient 3. Furthermore, T cells from donor C expressing the SP6 CAR did not specifically recognize the bone marrow of myeloma patient 3. Figure 8C is a graph showing experimental data demonstrating that a plasmacytoma resected from myeloma patient 1 consisted of 93% plasma cells, and these primary plasma cells expressed BCMA, as shown by flow cytometry analysis of BCMA (solid line) and isotype-matched control staining (dashed line). Figure 8D is a graph showing that T cells from myeloma patient 1 expressing the anti-bcma2 CAR produced IFNγ specifically in response to autologous plasmacytoma cells. Figure 8E is a graph showing that T cells from myeloma patient 1 expressing the anti-bcma2 CAR specifically killed autologous plasmacytoma cells at low effector-to-target ratios. In contrast, T cells from myeloma patient 1 expressing the SP6 CAR showed low levels of cytotoxicity against autologous plasmacytoma cells. At all effector:target ratios, cytotoxicity was measured in duplicate, and results are expressed as the mean + / - standard error. [Figure 9] Figure 9A is a graph depicting experimental data showing that T cells transduced with anti-bcma2CAR can destroy multiple myeloma tumors in mice, and Figure 9B is a graph depicting the survival rate of tumor-bearing mice treated with T cells expressing anti-bcma2CAR compared to controls. DETAILED DESCRIPTION OF THE INVENTION

[0009] [Detailed Description of the Invention] The present invention provides an isolated or purified nucleic acid sequence encoding a chimeric antigen receptor (CAR). The present invention provides a chimeric antigen receptor (CAR), which comprises an antigen recognition portion and a T cell activation portion. A chimeric antigen receptor (CAR) is a molecule that recognizes an antigen of an antibody bound to a T cell signaling or T cell activation domain. CARs are artificially constructed hybrid proteins or polypeptides that contain a binding domain (e.g., a single-chain variable fragment (scFv)). CARs are designed to bind to monoclonal antibodies in an MHC-independent manner. Use the antigen-binding properties of clonal antibodies to target T cells with specificity and reactivity to selected targets. This MHC-independent antigen recognition gives CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thus Furthermore, CARs can be used to target T cells and bypass a major mechanism of tumor evasion. It also has the advantage that when expressed it does not form dimers with the endogenous T cell receptor (TCR) alpha and beta chains.

[0010] A "nucleic acid sequence" is intended to include a polymer of DNA or RNA, i.e., a polynucleotide, which can be single- or double-stranded and which includes non-natural or altered nucleotides. The terms "nucleic acid" and "polynucleotide," as used herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule. The term refers to a structure and includes double- and single-stranded DNA, as well as double- and single-stranded RNA. The term also encompasses, as equivalents, homologs of either RNA or DNA derived from nucleotide analogs and modified polynucleotides, such as, but not limited to, methylated and / or capped polynucleotides.

[0011] "Isolated" means that the nucleic acid has been removed from its natural environment. "Purified" means that any nucleic acid is of high purity, whether removed from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and / or amplified under laboratory conditions. "Purity" means that the nucleic acids and proteins are purified to a high degree of purity, where "purity" is a relative term, not "absolute purity." However, nucleic acids and proteins may be in compositions with diluents or adjuvants, and may be isolated for practical purposes. For example, nucleic acids are typically mixed with an acceptable carrier or diluent when used to introduce them into cells.

[0012] The nucleic acid sequences of the present invention encode a CAR that includes an antigen recognition moiety directed against B-cell maturation antigen (BCMA, also known as CD269). BCMA is a tumor necrosis factor receptor superfamily. BCMA is a member of the BCMA family (see, e.g., Thompson et al., J. Exp. Medicine, 192(1): 129-135 (2000) and Mackay et al., Annu. Rev. Immunol., 21: 231-264 (2003)). BCMA binds to B cell-activating factor (BAFF) and proliferation-inducing ligand (APRIL) (see, e.g., 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 mostly on plasma cells and a subset of mature B cells. (See, e.g., 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 have impaired survival of long-lived plasma cells. (See, e.g., 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 is expressed in several It has been detected on the surface of plasma cells from multiple myeloma patients by researchers (e.g., 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).

[0013] The nucleic acid sequences of the present invention encode a CAR comprising an antigen recognition portion or antigen-binding portion thereof comprising a monoclonal antibody directed against BCMA. The term "antibody" refers to antibodies produced by a single clone of B cells that bind to the same epitope. On the other hand, "polyclonal antibodies" are antibodies produced by various B cells and target the same antigen. It is a population of antibodies that bind to various epitopes. The original recognition moiety may be a whole antibody or an antibody fragment. A whole antibody typically consists of four polypeptides, namely two identical chains of heavy (H) polypeptides. Each heavy chain contains two identical copies of a heavy (L) and a heavy (H) chain polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding portion of an antibody. The VH and VL regions have the same general structure, and each region contains four framework regions whose sequences are relatively conserved. The complementarity determining region is bounded by three CDRs: CDR1, CDR2, and CDR3. The three CDRs, known as CDRs, form a "hypervariable region" that is responsible for antigen binding.

[0014] The terms "antibody fragment," "antibody fragment," "functional fragment of an antibody," and "antigen-binding portion" are used interchangeably herein to refer to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see, e.g., generally, Holliger et al., Nat. Biotech, 23(9): 1126-1129 (2005)). Nucleic Acid Sequences of the Invention The antigen recognition portion of the CAR encoded by the antibody includes any BCMA-binding antibody fragment. The antibody fragment preferably contains, for example, one or more CDRs, a variable region (or portions thereof). Examples of antibody fragments include (i) a monovalent fragment consisting of a VL, VH, CL and CH1 domains, a constant region (or a portion thereof), a constant region (or a portion thereof), or a combination thereof. (ii) a Fab fragment, which is a bivalent fragment containing two Fab fragments linked by disulfide bridges at the hinge region; (iii) an Fv fragment, which consists of the VL and VH domains of a single arm of an antibody; (iv) a single fragment, which consists of two domains. Single-chain Fv (scFv) is a monovalent molecule consisting of two domains of an Fv fragment (i.e., VL and VH) joined by a synthetic linker, which allows the Fv fragment to be synthesized as a polypeptide chain of the same structure (see, e.g., 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)). (v) bispecific antibodies that are dimers of polypeptide chains (in which each (The polypeptide chain comprises a VH connected to a VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby inducing pairing between complementary domains on different VH-VL polypeptide chains to generate a dimeric molecule with two functional antigen-binding sites.) Antibody fragments are known in the art and are described, for example, in U.S. Patent Publication 2009 / 0093024 A1. In a preferred embodiment, the antigen recognition portion of the CAR encoded by the nucleic acid sequence of the present invention comprises an anti-BCMA single-chain Fv (scFv).

[0015] The antigen-binding portion or monoclonal antibody fragment may be of any size, so long as the portion binds to anti-BCMA. In this regard, antigen-binding portions or monoclonal antibody fragments directed against BCMA (sometimes referred to herein as "anti-BCMA monoclonal antibodies") desirably have a molecular weight of about 5 to 18 (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 foregoing numbers). It contains amino acids ranging from 0 to 100.

[0016] In one embodiment, the nucleic acid sequence of the present invention encodes an antigen recognition portion comprising a variable region of an anti-BCMA monoclonal antibody. In this regard, the antigen recognition portion comprises a light chain variable region, a heavy chain variable region, or both a light chain variable region and a heavy chain variable region. Preferably, the antigen recognition portion of a CAR encoded by the nucleic acid sequence of the present invention comprises a light chain variable region and a heavy chain variable region of an anti-BCMA monoclonal antibody. Heavy and light chain monoclonal antibody amino acid sequences that bind to BCMA, including variable regions, are disclosed, for example, in International Patent Application Publication WO 2010 / 104949.

[0017] In another embodiment, the nucleic acid sequence of the invention encodes a CAR comprising a signal sequence. The signal sequence may be present at the amino terminus of the antigen recognition portion (e.g., the variable region of an anti-BCMA antibody). The signal sequence may include any suitable signal sequence. In one embodiment, the signal sequence is the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor sequence or the CD8α signal sequence.

[0018] In another embodiment, the CAR comprises a hinge sequence. Those skilled in the art will recognize that hinge sequences are a part of the structure of antibodies. It will be understood that the hinge sequence is a short sequence of amino acids that increases the flexibility of the antigen-recognizing portion (see, for example, Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)). For example, a hinge sequence may be present between the anti-BCMA scFv and the T cell activation moiety. The hinge sequence may be any suitable sequence derived or obtained from any suitable molecule. For example, in one embodiment, the hinge sequence is derived from the human CD8α molecule or the CD28 molecule. do.

[0019] The nucleic acid sequences of the present invention encode a CAR comprising a T cell activation moiety. It may be any suitable moiety derived or obtained from any suitable molecule. In one embodiment, for example, the T cell activation moiety comprises a transmembrane domain. The transmembrane domain may be any transmembrane domain derived or obtained from any molecule known in the art. For example, the transmembrane domain may be derived from a CD8α molecule or a CD28 molecule. CD8 acts as a co-receptor for the T cell receptor (TCR). It is a transmembrane glycoprotein that is primarily expressed on the surface of T cells and has cytotoxic (damaging) properties. The most common form of CD8 exists as a dimer consisting of CD8α and CD8β chains. CD28 is expressed on T cells and provides a costimulatory signal necessary 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.

[0020] In addition to the transmembrane domain, the T cell activation moiety further comprises an intracellular (i.e., cytoplasmic) T cell signaling domain. The intracellular T cell signaling domain may be derived from a CD28 molecule, a CD3 zeta (ζ) molecule or modified versions thereof, a human Fc receptor gamma (FcRγ) chain, a CD27 molecule, an OX40 molecule, a 4-1BB molecule, or other intracellular signaling molecule known in the art. As mentioned above, the CD28 molecule plays a key role in T cell costimulation. CD3ζ is an important T cell marker for T cell proliferation. CD3ζ signals in conjunction with TCRs and contains immunoreceptor tyrosine-based activation motifs (ITAMs). 4-1BB, also known as CD137, is a potent In a preferred embodiment, CD28, CD3 zeta, 4-1BB, OX40, and CD27 are human.

[0021] The T cell activation domain encoded by the nucleic acid sequence of the present invention is the same as the above-mentioned transmembrane domain. Any one of these and any one or more of the above-mentioned intracellular T cell signaling domains in combination For example, a nucleic acid sequence of the invention can encode a CAR comprising a CD28 transmembrane domain and the intracellular T cell signaling domains of CD28 and CD3 zeta. Alternatively, a nucleic acid sequence of the invention can encode a CAR comprising a CD8α transmembrane domain and the intracellular T cell signaling domains of CD28, CD3 zeta, the Fc receptor gamma (FcRγ) chain, and / or 4-1BB.

[0022] In one embodiment, the nucleic acid sequence of the invention comprises, from 5' to 3', a granulocyte-macrophage colony-stimulating factor (GM-CSF receptor) signal sequence, an anti-BCMA scFv, the hinge and transmembrane region of a human CD8α molecule, the cytoplasmic T cell signaling domain of a human CD28 molecule, and the T cell signaling domain of a CD3ζ molecule. In another embodiment, the nucleic acid sequences of the invention encode CARs comprising, from 5' to 3', a human CD8α signal sequence, an anti-BCMA scFv, a hinge and membrane domain of the human CD8α molecule. In another embodiment, the nucleic acid of the invention encodes a CAR comprising a transmembrane region, a cytoplasmic T cell signaling domain of a human CD28 molecule, and a cytoplasmic T cell signaling domain of a human CD3ζ molecule. The sequence comprises, from 5' to 3', a human CD8α signal sequence, an anti-BCMA scFv, a hinge and transmembrane region of a human CD8α molecule, a cytoplasmic T cell signaling domain of a human 4-1BB molecule and / or a cytoplasmic T cell signaling domain of a human OX40 molecule and a T cell signaling domain of a human CD3ζ molecule. For example, the nucleic acid sequences of the present invention may be those of SEQ ID NO: 1, SEQ ID NO: 2, or comprising or consisting of the nucleic acid sequence of SEQ ID NO: 3.

[0023] The present invention further provides an isolated or purified chimeric antigen receptor (CAR) encoded by a nucleic acid sequence of the present invention.

[0024] The nucleic acid sequences of the present invention may be used to provide a CAR with biological activity, e.g., specific antigen binding and targeting of a mammal. A CAR of any length can be encoded, i.e., containing any number of amino acids, so long as it retains the ability to detect diseased cells, treat or prevent disease in a mammal, etc. For example, the CAR can be 50 or more (e.g., 60 or more, 100 or more). 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 Preferably, the CAR comprises 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 sequence defined by any two of the foregoing values. This is the range that can be achieved.

[0025] Nucleic acid sequences encoding functional portions of the CARs described herein are also within the scope of the present invention. The term "functional portion," when used in reference to a CAR, refers to any portion or fragment of a CAR of the invention that retains the biological activity of the CAR of which it is a part (parent CAR). A functional portion can be, for example, a portion that is similar to the parent CAR. The term "CAR" encompasses portions of a CAR that retain the ability to recognize target cells and detect, treat, or prevent disease to the same extent, the same extent, or a greater extent. A nucleic acid sequence encoding a functional portion of the parent CAR can encode a protein comprising, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.

[0026] The nucleic acid sequences of the present invention may contain, at the amino or carboxy terminus or at both termini, the amino acid sequence of the parent CAR. It is also possible that additional amino acids not found in the amino acid sequence encode functional portions of CAR. Preferably, the additional amino acids do not interfere with the biological function of the functional moiety, such as recognizing target cells, detecting cancer, treating or preventing cancer, etc. More preferably, the additional amino acids enhance the biological activity of the CAR relative to the biological activity of the parent CAR.

[0027] The present invention also provides nucleic acid sequences encoding functional variants of the above-mentioned CARs. In this context, the term "functional variant" refers to a CAR encoded by a nucleic acid sequence of the present invention. CAR, polypeptide or protein having substantial or significant sequence identity or similarity to A functional mutant of CAR is a protein that retains the biological activity of the original CAR. For example, a variant of the CAR described herein (parent CAR) that is similar, identical, or similar to the parent CAR. These include those that retain the ability to recognize target cells to a greater extent. With respect to a nucleic acid sequence encoding a functional variant of a CAR, the nucleic acid sequence encoding the functional variant of a CAR can have, for example, about 10% identity, about 25% identity, about 30% identity, about 50% identity, about 65% identity, about 80% identity, about 90% identity, about 95% identity, or about 99% identity to the nucleic acid sequence encoding the parent CAR. It may be something like that.

[0028] A functional variant is, for example, an amino acid sequence of a CAR encoded by a nucleic acid sequence of the present invention, , and those containing at least one conservative amino acid substitution. "Conservative amino acid substitution" and "conservative mutation" mean the replacement of one amino acid with another amino acid that shares common properties. One functional method for defining common properties between individual amino acids is to analyze the frequency with which amino acid exchanges between corresponding proteins of related organisms are normalized (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Such an analysis reveals that amino acids within the same group exchange predominantly with each other, and therefore allows the definition of groups of amino acids that are most similar to each other in their effects on the overall structure of proteins (Schulz, GE and Schirmer, RH, supra). Conservative mutations Examples include, for example, substitution of lysine with arginine or vice versa to maintain a positive charge; substitution of serine with threonine to maintain a free OH; and substitution of hydroxyl groups with amino acids to maintain a free NH. Examples of amino acid substitutions within the above subgroups include substitutions of amino acids within the above subgroups, such as substitution of glutamine with asparagine.

[0029] Alternatively or additionally, a functional variant may comprise the amino acid sequence of the parent CAR with at least one non-conservative amino acid substitution. A "non-conservative mutation" is, for example, a substitution at lysine Non-conservative amino acid substitutions include substitutions of amino acids between different groups, such as substitution of tryptophan with phenylalanine or serine with phenylalanine, in which case the non-conservative amino acid substitutions may be used to improve the biological activity of the functional variant. Non-conservative amino acid substitutions may enhance the biological activity of the functional variant, even if the biological activity of the functional variant is increased relative to the parent CAR. good.

[0030] The nucleic acid sequences of the invention can encode CARs (including functional portions and functional variants thereof) that contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl- Stein, trans-3 and trans-4 hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, Indian Examples of aminoglycosides include phosphorus-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0031] The nucleic acid sequences of the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, e.g., disulfide-bridged, etc. cyclized or converted into an acid addition salt by (including

[0032] In a preferred embodiment, the nucleic acid sequence of the invention is 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: It encodes a CAR comprising or consisting of a 12 amino acid sequence.

[0033] The nucleic acid sequences of the present invention can be produced using methods known in the art. For example, nucleic acid sequences, polypeptides, and proteins can be produced using standard recombinant DNA methods (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd CARs can be produced recombinantly using recombinant DNA technology (see, for example, "Carbohydrates of the Human Genome," 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). The nucleic acid sequences can be isolated and / or purified from sources such as plants, bacteria, insects, mammals (e.g., rats, humans, 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 sequences of the present invention can be synthetic, recombinant, or isolated and purified.

[0034] The present invention also provides a vector comprising a nucleic acid sequence encoding a CAR of the present invention. The vector may be, for example, a plasmid, a cosmid, a viral vector (e.g., a retrovirus or adenovirus), or a phage. Suitable vectors and methods for preparing vectors are well known in the art (see, e.g., Sambrook et al., supra, and Ausubel et al., supra). (see).

[0035] In addition to the nucleic acid sequence of the invention encoding the CAR, the vector preferably comprises a nucleic acid sequence that is capable of expressing the CAR in the host cell. These include expression control sequences such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), etc. that regulate the expression of a nucleic acid sequence. Examples of modern regulatory sequences are known in the art and are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

[0036] Numerous promoters are well known in the art, including constitutive, inducible, and repressible promoters, from a variety of sources. Representative sources of promoters include, for example, viral, mammalian, insect, plant, yeast, and bacterial, and suitable promoters are readily available from these sources or can be produced synthetically, for example, based on publicly available sequences from depositories such as the ATCC, as well as other commercial or private sources. Promoters can be unidirectional (i.e., those that initiate transcription in one direction) or bidirectional (i.e., those that initiate transcription in either the 3' or 5' direction). Examples of promoters include, but are not limited to, the T7 bacterial expression system, the pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Examples of inducible promoters include, for example, the Tet system (U.S. 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-REX ... TM System (Invitrogen, Carlsbad, CA), LACSWITCH TM The Cre-ERT tamoxifen-inducible recombinase system (Stratagene, San Diego, CA) was used. Stem (Indra et al., Nuc. Acid. Res., 27: 4324-4327 (1999); Nuc. Acid. Res., 28: e99 (2000); U.S. Patent No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol., 308: 123-144 (2005)).

[0037] As used herein, the term "enhancer" refers to, for example, a DNA sequence that increases the transcription of an operably linked nucleic acid sequence. Enhancers are known to increase the transcriptional activity of many genes from the codon region of a nucleic acid sequence. Enhancers may be located at positions spaced apart by a base and may mediate the binding of regulatory factors, changes in DNA methylation patterns, or changes in DNA structure. Numerous enhancers from a variety of sources are well known in the art and are available as or contained within cloned polynucleotides (e.g., from depositories such as the ATCC, as well as other commercial or private sources). Many polynucleotides containing promoters (such as the commonly used CMV promoter) are available. It also contains enhancer sequences. Enhancers may be located upstream, within, or downstream of a codon sequence. The term "Ig enhancer" refers to an enhancer element derived from an enhancer region located within an immunoglobulin (Ig) gene locus (such as the heavy chain (mu) 5' enhancer). enhancer, light chain (kappa) 5' enhancer, kappa and mu intron enhancer and and 3' enhancers (generally, see Paul WE (ed.), Fundamental Immunology, 3rd Edition, Raven Press, New York (1993), pp. 353-363 and British Patent No. 5,885,827 See ).

[0038] The vector may also contain a "selection marker gene." The term "selection marker gene," as used herein, refers to a nucleic acid sequence that allows cells expressing the nucleic acid sequence to be specifically selected or not selected for in the presence of a corresponding selection agent. Suitable selection marker genes are well known in the art and are described, for example, in International Patent Applications WO 1992 / 08796 and 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. 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 U.S. Patents 5,122,464 and 5,770,359.

[0039] In some embodiments, the vector is capable of replicating in the host cell and, under appropriate selective pressure, persisting as an extrachromosomal segment of DNA in the host cell. An episomal expression vector is an "episomal expression vector" or "episome" (see, e.g., Conese et al., Gene Therapy, 11: 1735-1742 (2004)). Representative commercially available episomal expression vectors include Epstein-Barr Nuclear Antigen 1 (EBNA1) and EBNA2 Episomal plasmids that utilize the Epstein Barr Virus (EBV) origin of replication (oriP) include, but are not limited to, the vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, CA). A representative example of an episomal vector that uses T antigen and SV40-derived replication instead of EBNA1 and oriP includes, but is not limited to, pBK-CMV from Stratagene (La Jolla, CA).

[0040] Other suitable vectors include those that randomly integrate into the DNA of a host cell or are expression vectors. An example of such an integrating expression vector is an integrating expression vector that contains a recombination site that allows specific recombination between the vector and the chromosome of the host cell. Such an integrating expression vector can utilize the endogenous expression control sequence of the host cell chromosome to effect expression of the desired protein. Examples of vectors that integrate in a site-specific manner include the flp-in system (e.g., pcDNA 1.0) from Invitrogen (Carlsbad, CA). TM Examples of such components include the cre-lox system found in the pExchange-6 Core Vectors from Stratagene (La Jolla, CA) or the pExchange-5 Core Vectors from Stratagene (La Jolla, CA). Examples of vectors for integrating into the genome include pcDNA3.1 from Invitrogen (Carlsbad, CA) (when introduced in the absence of T antigen) and pCI or pFN10A (ACT) FLEXI from Promega (Madison, WI). TM Examples include:

[0041] Viral vectors can also be used. Representative viral vectors include adenovirus-based vectors (e.g., the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands)), lentivirus-based vectors (e.g., the lentivirus-based pLP1 from Life Technologies (Carlsbad, CA)), and retrovirus vectors ( Examples include pFB-ERV plus pCFB-EGSH from Stratagene (La Jolla, CA). Without being limited thereto, in a preferred embodiment, the viral vector is a lentiviral vector.

[0042] A vector containing a nucleic acid sequence encoding a CAR of the invention can be introduced into a host cell (including a suitable prokaryotic or eukaryotic cell) capable of expressing the encoded CAR. Preferred host cells are those that can be grown easily and reliably, have a reasonably fast growth rate, have a well-characterized expression system, and are easily and efficiently transformed or transfected.

[0043] As used herein, the term "host cell" refers to any type of cell that can contain an expression vector. Host cells can be eukaryotic cells, such as, for example, plant, animal, fungal, or algal cells, or prokaryotic cells, such as, for example, bacteria or protozoan cells. Host cells can be cultured cells or primary cells, i.e., cells immediately isolated from an organism, such as, for example, a human. Host cells can be adherent cells or suspended cells, i.e., cells that grow in suspension. Suitable host cells are well known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For amplifying or replicating recombinant expression vectors, host cells can be prokaryotic cells, such as, for example, DH5α cells. For producing recombinant CARs, In particular, the host cell may be a mammalian cell. Preferably, the host cell is a human cell. The host cell may be of any type, may be derived from any type of tissue, and may be at any stage of development. In one embodiment, the host cell The cells may be peripheral blood lymphocytes (PBLs), peripheral blood mononuclear cells (PBMCs), or natural killer (NK) cells. Preferably, the host cells are natural killer (NK) cells. More preferably, it is a cell. Methods for selecting suitable mammalian host cells and for transforming, culturing, amplifying, screening and purifying the cells are well known in the art.

[0044] The present invention provides host cells expressing the nucleic acid sequences of the present invention encoding the CARs described herein. In one embodiment, the host cell is a T cell. The T cells of the present invention can be cultured T cells, such as primary T cells or T cells obtained from a cultured T cell line, or The T cells may be any T cells, such as T cells obtained from a mammal. If the T cells are obtained from a mammal, the T cells may be obtained from a variety of sources, including, but not limited to, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells may be enriched or purified. The T cells are preferably human T cells (e.g., isolated from a human). The T cells may be CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells (e.g., Th1 and Th2 cells), CD8 + T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, The T cells may be at any stage of development, including, but not limited to, memory T cells, naive T cells, etc. In one embodiment, the T cells are CD8 + T cells is CD4 + T cell lines are, for example, those listed in the American Type Culture Collection. Cells from the American Type Culture Collection (ATCC, Manassas, VA) and the German Collection of Microorganisms and Cell Cultures (DSMZ) are available, such as Jurkat cells (ATCC TIB-152), Sup-T1 cells (ATCC CRL-1942), RPMI 8402 cells (DSMZ ACC-290), Karpas 45 cells (DSMZ ACC-545), and their respective endonucleases. Derivatives are included.

[0045] In another embodiment, the host cell is a natural killer (NK) cell. NK cells are cytotoxic (destructive) lymphocytes that play an important role in the innate immune system. NK cells are defined as large granular lymphocytes and constitute a third type of cell differentiated from conventional lymphoid precursors that give rise to B and T lymphocytes (see, e.g., Immunobiology, 5 th NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus. After maturation, NK cells enter the circulation as large lymphocytes with characteristic cytotoxic granules. NK cells can recognize and kill abnormal cells, such as tumor cells and virus-infected cells, and are thought to be important in the innate immune defense against intracellular pathogens. As discussed above with respect to T cells, NK cells can be cultured NK cells, e.g., primary NK cells or NK cells from cultured NK cell lines, or mammalian NK cells. The NK cells may be any NK cells, such as NK cells obtained from a mammal. If the NK cells are obtained from a mammal, the NK cells may be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The NK cells may be enriched or purified. The NK cells are preferably human NK cells (e.g., isolated from a human). NK cell lines are available, for example, from the American Type Culture Collection (ATCC, Manassas, VA), and include, for example, NK-92 cells (ATCC CRL-2407), NK92MI cells (ATCC CRL-2408, and their derivatives). do.

[0046] The nucleic acid sequence encoding the CAR of the present invention may be "transfected," "transformed," or "transduced." As used herein, "transfection," "transformation," or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell by physical or chemical methods. Many transfection techniques are known in the art, including, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection. Transfection; tungsten particle-enhanced microbombardment (Johnston, Nature, 346: 776-777 (1990) ) and strontium phosphate DNA coprecipitation (Brash, Mol. Cell Biol., 7:2031-2034 (1987)) etc. After growing the infectious particles in suitable packaging cells, many of which are commercially available, the phage or virus can be introduced into host cells.

[0047] Without being bound by any particular theory or mechanism, it is believed that CARs encoding the nucleic acid sequences of the present invention elicit an antigen-specific response against BCMA, resulting in one or more of the following: targeting and destroying BCMA-expressing cancer cells, reducing or eliminating cancer cells, promoting immune cell entry into tumor sites, and enhancing / expanding anti-cancer responses. The present invention also provides a method for producing a CAR by contacting one or more of the isolated T cells or natural killer cells described above with a population of multiple myeloma cells expressing BCMA, thereby producing a CAR and targeting BCMA on the multiple myeloma. and destroying the multiple myeloma cells. As mentioned above, multiple myeloma, also known as plasma cell myeloma or Kahler's disease, is a cancer of plasma cells, a type of white blood cell normally responsible for antibody production (Raab et al., Lancet, 374: 324-329 (2009)). Approximately 100,000 people die annually. Multiple myeloma affects 1-4 people in every 100 women. The disease affects men more often, for reasons that are still unclear. Although multiple myeloma is a rare disease, it is twice as common among African Americans as it is among white Americans. It is the least common hematologic cancer (14%) and accounts for 1% of all cancers (Raab et al., supra). Treatment of multiple myeloma typically involves high-dose chemotherapy followed by chemotherapy. This is followed by hematopoietic stem cell transplantation (either allogeneic or autologous), but a significant proportion of multiple myeloma patients who undergo such treatment typically relapse. As noted above, BCMA is expressed to a significant extent by multiple myeloma cells (see, e.g., Novak et al., supra; Neri et al., supra; Bellucci et al., supra; and Moreaux et al., supra).

[0048] One or more of the isolated T cells expressing a nucleic acid sequence encoding an anti-BCMA CAR of the invention described herein may be cultured ex vivo, in vivo, or in vitro. Ex vivo contact with a BCMA-expressing multiple myeloma cell population can be performed. "In vivo" refers to a process carried out in or on cells in an artificial, extra-organismal environment that minimally alters their natural conditions. In contrast, "in vivo" refers to a process carried out in or on cells in their normal, intact state. "In vitro" refers to a method performed within a living organism, while "in vitro" refers to a method performed using components of an organism that are free from their normal biological environment. The methods of the present invention preferably include both ex vivo and in vivo components. In this regard, for example, the isolated T cells described above can be cultured ex vivo under conditions that result in expression of a nucleic acid sequence encoding an anti-BCMA CAR of the present invention, followed by subsequent culture of the T cells in vivo. Alternatively, the cells can be directly transplanted into a mammal (preferably a human) suffering from multiple myeloma. This cell transplantation method is known in the art as "adoptive cell transfer (ACT)." In this method, the immune-induced cells are passively transplanted into a new recipient host, transferring the function of the donor's immune-induced cells to the new host. Adoptive cell transfer methods for the treatment of various types of cancer, including hematological cancers such as myeloma, are known in the art and are disclosed, for example, in 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).

[0049] The present invention also provides a method for destroying Hodgkin's lymphoma cells. Hodgkin's lymphoma (formerly known as Hodgkin's disease) is a cancer of the immune system characterized by the presence of multinucleated cell types called Reed-Sternberg cells. The two major types of Hodgkin's lymphoma include classical Hodgkin's lymphoma and nodular lymphocyte-predominant Hodgkin's lymphoma. Hodgkin's lymphoma is currently treated with radiation therapy, chemotherapy, and hematopoietic stem cell transplantation, with treatment options depending on the patient's age and gender, as well as the stage, size, and histological subtype of the disease. BCMA expression is detected on the surface of Hodgkin's lymphoma cells (see, e.g., Chiu et al., Blood, 109(2): 729-739 (2007)).

[0050] When T cells or NK cells are administered to a mammal, the cells may be allogeneic or autologous. In "autologous" administration methods, cells (e.g., hematopoietic stem cells or lymphocytes) are removed from a mammal, stored (optionally modified), and returned to the same mammal. In "allogeneic" administration methods, a mammal receives cells (e.g., hematopoietic stem cells or lymphocytes) from a genetically similar, but not identical, donor. Preferably, the cells are autologous to the mammal.

[0051] The T cells or NK cells are administered to a human in the form of a composition, such as a pharmaceutical composition. Alternatively, a nucleic acid sequence encoding a CAR of the invention or a vector comprising a nucleic acid sequence encoding a CAR can be formulated into a composition, such as a pharmaceutical composition, and then administered to a human. The pharmaceutical composition of the invention can include a population of T cells or NK cells expressing a CAR of the invention. The pharmaceutical composition can also include a host cell expressing a nucleic acid sequence of the invention or a CAR of the invention, as well as It may contain other pharmaceutically active agents or drugs, such as, for example, chemotherapeutic agents such as 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 expressing a CAR of the invention, more preferably a population of T cells or NK cells expressing a CAR of the invention.

[0052] The T cells or NK cells of the present invention may be provided in the form of a salt, e.g., a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid, as well as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, p-toluic acid, and the like. Examples include salts derived from organic acids such as arylsulfonic acids, such as benzenesulfonic acid.

[0053] In selecting a carrier, one consideration is the ability to express the particular nucleic acid sequence, vector, or CAR of the present invention. The nucleic acid sequences, vectors, and CARs of the present invention are determined by the host cell in which they are expressed. The amount of preservatives used will also depend on the particular method used to administer the host cells. Therefore, there are many suitable compositions for the pharmaceutical compositions of the present invention. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. Two or more preservatives may be used optionally. The preservative or mixtures thereof typically comprise about 0.0001% to about 2% by weight of the total composition.

[0054] Additionally, a buffering agent may 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. Two or more buffering agents may be used optionally. The buffering agent or mixture thereof typically comprises about 0.001% to about 4% by weight of the total composition.

[0055] Methods for preparing administrable (e.g., parenterally administrable) compositions are well known to those skilled in the art and are further described in detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0056] Compositions containing a nucleic acid sequence encoding a CAR of the invention or host cells expressing a CAR can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or as liposomes. Liposomes can be used to inject host cells (e.g., T cells or NK cells) or the nucleic acid of the invention. Liposomes can also be used to increase the half-life of the nucleic acid sequences of the present invention. Liposomes can be prepared using methods such as those described in Szoka et al., Ann. Rev. Biophys. Bioeng., 9:467 (1980) and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837, Many methods are available, including those described in US Pat. No. 5,019,369 and US Pat. No. 5,028,028 and US Pat. No. 5,019,369.

[0057] To ensure that the compositions of the present invention reach the treatment site prior to or in sufficient time to detect the site, the compositions can employ timed-release, delayed-release, and sustained-release drug delivery systems. Many forms of delivery systems are available and are well known to those skilled in the art. Such systems are particularly suitable for some embodiments of the compositions of the present invention because they avoid repeated administration of the composition, thereby increasing convenience for patients and physicians.

[0058] The composition may comprise a host cell expressing a nucleic acid sequence encoding a CAR of the invention or a nucleic acid sequence of the invention. Preferably, the vector contains an amount of a vector containing a sequence effective to treat or prevent multiple myeloma or Hodgkin's lymphoma. As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. Preferably, the effect is therapeutic, i.e., an effect that partially or completely cures the disease and / or undesirable symptoms resulting from the disease. To this end, the methods of the invention involve the use of a "therapeutically effective amount" of a host cell expressing a nucleic acid sequence encoding a CAR of the invention or a vector containing a nucleic acid sequence of the invention. The present invention includes administering a composition containing the vector. A "therapeutically effective amount" refers to an amount effective, at a dosage and for a period of time necessary, to achieve the desired therapeutic result. The therapeutically effective amount may vary depending on the individual patient's disease state, age, sex, and weight, as well as the ability of the CAR to elicit the desired response in the patient. For example, a therapeutically effective amount of the present invention may be The amount is sufficient to bind to BCMA on primary myeloma cells and destroy the cells.

[0059] Alternatively, the pharmacological and / or physiological effect is prophylactic, i.e., an effect that completely or partially prevents a disease or condition. In this regard, the methods of the invention involve administering a nucleic acid encoding a CAR of the invention to a subject. This includes administering a "prophylactically effective amount" of a composition comprising a host cell expressing a nucleic acid sequence or a vector comprising a nucleic acid sequence of the invention to a mammal predisposed to developing multiple myeloma or Hodgkin's lymphoma. By "prophylactically effective amount" is meant an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result (e.g., prevention of disease onset).

[0060] A typical amount of host cells administered to a mammal (e.g., a human) is, for example, about 1 million to In the range of about 100 billion cells, although amounts below or above this exemplary range are also within the scope of the invention. For example, a daily dose of host cells of the invention may range from about 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 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 foregoing values). range), preferably about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million 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 any of the foregoing values. a range defined by any two of the values ​​of (a) to (c), more preferably from about 100 million cells to Approximately 50 billion cells (e.g., approximately 120 million cells, approximately 250 million cells, approximately 350 million cells, approximately 450 million cells, approximately 650 million cells) cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells, or a range defined by any two of the foregoing values).

[0061] Therapeutic or prophylactic effectiveness can be monitored by periodic evaluation of the patient receiving treatment. Depending on the condition, repeated administrations may be given over several days or longer, in which case the treatment is repeated until the desired suppression of disease symptoms is achieved. However, other administration regimens may be useful and are within the scope of the invention. Preferred modes of administration include administering the composition in a single bolus dose or administering the composition by continuous infusion.

[0062] Host cells expressing nucleic acid sequences encoding CARs of the invention or nucleic acids encoding CARs of the invention Compositions containing vectors containing the sequences can be administered to mammals using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, nasal, buccal, sublingual, or suppository administration. Preferably, the compositions are suitable for parenteral administration. The term "parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the compositions are administered to mammals using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0063] A host cell expressing a nucleic acid sequence encoding a CAR of the invention or a composition comprising a vector comprising a nucleic acid sequence encoding a CAR of the invention may be administered with one or more additional therapeutic agents that can be co-administered to a mammal. "Co-administration" refers to the administration of one or more additional therapeutic agents in addition to a host cell of the invention or a composition comprising a vector of the invention, to enhance the effect of the one or more additional therapeutic agents. "The term 'administration' means administration close enough in time to allow the effect of the composition of the invention to be enhanced or to enhance the effect of the composition of the invention. In this regard, 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 can be administered simultaneously with the one or more additional therapeutic agents. One example of a therapeutic agent that can be used in combination with the host cells of the invention or the composition comprising the vector of the invention is IL-2.

[0064] Once a host cell expressing a nucleic acid sequence encoding a CAR of the invention or a composition comprising a vector comprising a nucleic acid sequence encoding a CAR of the invention is administered to a mammal (e.g., a human), the biological activity of the CAR can be measured by any suitable method known in the art. According to the method of the present invention, the CAR binds to BCMA on multiple myeloma cells and induces the proliferation of the multiple myeloma. The tumor cells are destroyed. Binding of the CAR to BCMA on the surface of multiple myeloma cells can be analyzed using any suitable method known in the art, such as, for example, ELISA or flow cytometry. The ability of the CAR to destroy multiple myeloma cells can be measured using any suitable method known in the art, such as, for example, the cytotoxicity assays described in 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 the CAR can also be measured by measuring the activity of cytokines such as CD107a, IFNγ, IL-2, and TNF. It can also be measured by analyzing the expression of genes.

[0065] Those skilled in the art will readily appreciate that the nucleic acid sequences encoding the CARs of the present invention can be modified in a variety of ways such that the therapeutic or prophylactic efficacy of the CAR can be further enhanced by the modification. For example, the CAR can be linked directly to a targeting moiety or indirectly via a linker. The compound, e.g., CAR, can be conjugated to a targeting moiety. Techniques for performing such targeting are known in the art. See, for example, Wadwa et al., J. Drug Targeting 3: 111 (1995) and U.S. Patent 5,087,616.

[0066] The present invention is further illustrated by the following examples, but it goes without saying that these examples should not be construed as in any way limiting the scope of the invention. [Example]

[0067] Example 1 This example demonstrates the expression pattern of BCMA in human cells.

[0068] BCMA-specific primer and probe sets (Life Technologies, Carlsbad, CA) were used. Quantitative polymerase chain reaction (qPCR) was performed on a panel of cDNA samples from a wide range of normal tissues included in the Human Primary Tissue qPCR Panel II (Origine Technologies, Rockville, MD). cDNA from plasmacytoma cells resected from a patient with advanced multiple myeloma was analyzed as a positive control. RNA was analyzed using the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA). BCMA cDNA was extracted from plasmacytoma cells using Origine Technologies, Rockville, MD, and cDNA was synthesized using standard methods. A BCMA qPCR standard curve was generated by diluting a plasmid encoding full-length BCMA cDNA (Origine Technologies, Rockville, MD) in carrier DNA. qPCR was performed using 10 mAb per reaction. 2 From 10 9 The copy number of BCMA was accurately detected from the β-actin cDNA in the same tissues. The β-actin cDNA copy number in the same tissues was quantified using the Taqman β-actin primer and probe kit (Life Technologies, Carlsbad, CA). A standard curve was generated by amplifying serial dilutions of β-actin plasmid. All qPCR reactions were performed on a Roche LightCycler 480 instrument (Roche Applied Sciences, Indianapolis, IN).

[0069] The results of the qPCR analysis are shown in Figures 1A and 1B. As measured by flow cytometry, plasma cells 93% of the cells from the tumor samples were plasma cells. BCMA expression in plasmacytoma samples was significantly higher than in other tissues. The expression of BCMA cDNA was significantly higher than that in human tissues. BCMA cDNA was detected in several blood tissues, such as peripheral blood mononuclear cells (PBMCs), bone marrow, spleen, lymph nodes, and tonsils. Low levels of BCMA cDNA were detected in most gastrointestinal organs, such as the duodenum, rectum, and stomach. BCMA expression in gastrointestinal organs may be a result of the presence of plasma cells and B cells in gut-associated lymphoid tissues, such as the lamina propria and Peyer's patches (see, e.g., Brandtzaeg, Immunological Investigations, 39(4-5): 303-355 (2010)). Low levels of BCMA cDNA were also detected in the testis and trachea. The low level of BCMA cDNA detected in the trachea may be due to the presence of plasma cells and B cells in the gut-associated lymphoid tissues, such as the lamina propria and Peyer's patches (see, e.g., Brandtzaeg, Immunological Investigations, 39(4-5): 303-355 (2010)). This may be due to the presence of plasma cells in the lamina propria (see, e.g., Soutar, Thorax, 31(2):158-166 (1976)).

[0070] Cell surface BCMA expression in various cell types was observed in multiple myeloma cell lines H929, U266, and RPMI8226. These were further characterized using flow cytometry (see Figures 2A-2L). In contrast, H929, U266, and RPMI8226 all expressed cell surface BCMA. The sarcoma cell line TC71, the T-cell leukemia line CCRF-CEM, and the kidney cell line 293T-17 did not express cell surface BCMA. Primary CD34 + Hematopoietic cells, primary small airway epithelial cells, primary bronchial epithelial cells, and primary intestinal epithelial cells all lacked cell surface BCMA expression.

[0071] The results of this example demonstrate that BCMA is expressed on the surface of multiple myeloma cells and exhibits a restricted expression pattern in normal tissues.

[0072] Example 2 This example describes the construction of nucleic acid sequences encoding anti-BCMA chimeric antigen receptors (CARs) of the invention.

[0073] Two murine anti-human BCMA antibodies, designated "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 and light chain variable regions of these antibodies were used to design single-chain variable fragments (scFvs) with the following general structure: Used: Light chain variable region-linker-heavy chain variable region.

[0074] The linker has the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 7) (e.g., Cooper et al., Blood, 101(4): 1637-1644 (2003)).

[0075] The DNA sequences encoding two chimeric antigen receptors each containing the following elements in the 5' to 3' regions: Designed: CD8α signal sequence, the anti-BCMA scFv, the hinge and transmembrane region of the human CD8α molecule, CD28 The cytoplasmic portion of the molecule and the cytoplasmic portion of the CD3ζ molecule. A schematic diagram of the nucleic acid sequence encoding the CAR is shown in Figure 3 A. CARs containing the variable regions from C12A3.2 and C11D5.3 were designated anti-bcma1 and anti-bcma2, respectively.

[0076] The above-mentioned anti-bcma2 CARs each have a different signal sequence and T cell activation domain. DNA sequences encoding five additional chimeric antigen receptors based on the 8ss-anti-bcma2 CAR were designed. In this regard, the 8ss-anti-bcma2 CAR contained the following elements from 5' to 3': CD8α signal sequence, scFv, hinge and transmembrane regions of the human CD8α molecule, the cytoplasmic portion of the CD28 molecule, and the cytoplasmic portion of the CD3ζ molecule. The G-anti-bcma2 CAR contained the following elements from 5' to 3': Human GM-CSF receptor signal sequence, scFv, hinge and transmembrane region of human CD8α molecule, cytoplasmic portion of CD28 molecule, and cytoplasmic portion of CD3ζ molecule. The anti-bcma2-BB CAR contained the following elements from 5' to 3': CD8α signal sequence, scFv, hinge and transmembrane region of the human CD8α molecule, cytoplasmic portion of the 4-1BB molecule and cytoplasmic portion of the CD3ζ molecule. The anti-bcma2-OX40 CAR contained the following elements from 5' to 3': CD8α signal sequence, scFv, hinge and transmembrane domains of human CD8α molecule, OX40 molecule (See, e.g., Latza et al., European Journal of Immunology, 24: 677-683 (1994) ) and the cytoplasmic portion of the CD3ζ molecule. Anti-bcma2-BBOX40 contained the following elements from 5' to 3': CD8α signal sequence, scFv, hinge 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 CD3ζ molecule. The elements present in each of the seven CAR sequences are shown in Table 1.

[0077] [Table 1]

[0078] The sequences used for CD8α, CD28, CD3 zeta, 4-1BB (CD137), and OX40 (CD134) were publicly available. The sequences were obtained from the available databases of the National Center for Biotechnology Information (NCBI).

[0079] Nucleic acid sequences encoding CARs are described, for example, in Kochenderfer et al., J. Immunology, 32(7): 689-7 02 (2009) and Zhao et al., J. Immunology, 183(9): 5563-5574 (2009), The nucleic acid sequence encoding each CAR was prepared using well-known methods. GeneArt Technology TM technology) (Life Technologies, Carlsbad, CA) The codons were optimized and synthesized.

[0080] The sequences encoding the anti-bcma1 and anti-bcma2 CARs were ligated into a lentiviral vector plasmid designated pRRLSIN.cPPT.MSCV.coDMF5.oPRE (see, e.g., Yang et al., J. Immunotherapy, 33(6): 648-658 (2010)). The coDMF5 portion of this vector was transfected using standard methods. The nucleic acid sequence encoding the CAR was replaced with the CAR-encoding nucleic acid sequence. The resulting anti-BCMA CAR vectors were designated pRRLSIN.cPPT.MSCV.anti-bcma1.oPRE and pRRLSIN.cPPT.MSCV.anti-bcma2.oPRE. A negative control CAR containing the SP6 scFv, which recognizes the hapten 2,4,6-trinitrophenyl, was also constructed (see, e.g., Gross et al., Proc. Natl. Acad. Sci. USA, 86(24): 10024-10028 (1989)). This CAR was designated SP6. The SP6 CAR was cloned into the same lentiviral vector as the anti-BCMA CARs. It contained the same signaling domains as anti-bcma1 and anti-bcma2. The supernatant containing the lentivirus encoding each CAR was then transferred to the protease inhibitors described in Yang et al., supra. Specifically, 293T-17 cells (ATCC CRL-11268) were transfected with the following plasmids: pMDG (encoding the vesicular stomatitis virus envelope protein); pMDLg / pRRE (encoding HIV Gag and Pol proteins), pRSV-Rev (encoding RSV Rev proteins), and plasmids encoding anti-bcma CARs (see, e.g., Yang et al., supra).

[0081] For example, G-anti-bcma2, 8ss-anti-bcma2, anti-bcma2-BB, anti-bcma2-OX40, and anti-bcma2-OX40 antibodies can be isolated using standard methods, such as those described in Hughes et al., Human Gene Therapy, 16: 457-472 (2005). The sequences encoding the anti-bcma2-BBOX40 CARs were ligated together and expressed in MSGV (murine stem cell virus). Gamma retroviral vectors called gamma retroviral vectors (based splice-gag vectors) After creating a gammaretroviral plasmid encoding the CAR, the RD114 vector was used. Enveloped, replication-defective retroviruses are transiently transfected into 293-based packaging cells as described in Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009). It was generated by sexual transfection.

[0082] The replication-deficient lentivirus and retroviruses encoding the CARs were used to transduce human T cells. For anti-bcma1 and anti-bcma2, T cells were cultured as previously described ( See, e.g., Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009)), 5% human AB AIM V containing serum (Valley Biomedical, Winchester, VA) TM T cells were stimulated with anti-CD3 monoclonal antibody OKT3 (Ortho-Biotech, Horsham, PA) and 300 international units (IU) / mL of interleukin-2 (Novartis Diagnostics, Emeryville, CA) in medium (Life Technologies, Carlsbad, CA). After 36 hours of culture, activated T cells were suspended in lentiviral supernatant containing protamine sulfate and 300 IU / mL IL-2. The cells were centrifuged at 1200 x g for 1 hour. The T cells were then cultured at 37°C for 3 hours. The supernatant was then resuspended in RPMI medium (Mediatech, Inc., Manassas, VA) plus 10% fetal bovine serum (Life Technologies, Carlsbad, CA) and IL-2. T cells were cultured overnight in this diluted supernatant and then incubated with IL-2-supplemented AIM V. TM The T cells were then reconstituted in a culture medium containing 5% human AB serum (Life Technologies, Carlsbad, CA). To detect anti-BCMA CARs, we stained with biotin-labeled polyclonal goat anti-mouse F(ab)2 antibody (Jackson Immunoresearch Laboratories, Inc., West Grove, PA). As shown in Figures 3B-3D, transduced T cells were stained with anti-bcma1 CAR, anti-bcma2 CAR, and anti-bcma3 CAR. and high levels of cell surface expression of SP6 CAR were observed.

[0083] For G-anti-bcma2, 8ss-anti-bcma2, anti-bcma2-BB, anti-bcma2-OX40, and anti-bcma2-BBOX40 CARs, 1x10 peripheral blood mononuclear cells per mL were used. 6 At the cell division, 50 ng / mL of anti-CD3 monoclonal antibody was used. in T cell medium containing the clonal antibody OKT3 (Ortho, Bridgewater, NJ) and 300 IU / mL of IL-2. RETRONECTIN, a recombinant polypeptide fragment of human fibronectin that binds viral and cell surface proteins, was suspended in PBS. TM (RetroNectin TM ) Polypeptide (Ta (Carabio Inc., Shiga, Japan) was dissolved in phosphate buffered saline (PBS) at a concentration of 11 μg / mL, and 2 mL of RETRONECTIN TM A PBS solution of the polypeptide was added to each well of a non-tissue culture coated 6-well plate (BD Biosciences, Franklin Lakes, New Jersey). The cells were incubated at room temperature (RT) for 2 hours. After incubation, TM melt Aspirate the liquid and add 2 mL of blocking solution consisting of Hank's Balanced Salt Solution (HBSS) plus 2% bovine serum albumin (BSA) to each retrone sample. TMThe blocking solution was then aspirated and the wells were filled with HBSS + 2.5% (4-(2-hydroxybenzoates)). The solution was washed with a solution of (hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The supernatant was quickly liquified and diluted 1:1 in T cell medium, and then 2 mL of diluted supernatant was added to each RETRONECTIN TM After the addition of the supernatant, the plate was centrifuged at 2000 x g for 2 hours at 32°C. The supernatant was then aspirated from the wells and transferred to 2 x 10 cells that had been previously cultured with OKT3 antibody and IL-2 for 2 days. 6 T cells were added to each well. When adding T cells to the retrovirus-coated plate, the T cells were added at a density of 0.5x10 per mL. 6 The cells were suspended in T cell medium containing 300 IU / mL IL-2 at a concentration of 1000 μg / well. After adding the T cells to each well, the plate was centrifuged at 1000×g for 10 minutes. The plate was incubated at 37°C overnight. The transduction was repeated the next day. After 18-24 hours of incubation, the T cells were removed from the plate. , 0.5x10 per mL 6 Cells were suspended in fresh T cell medium containing 300 IU / mL IL-2 at a concentration of The cells were cultured at 37°C in 5% CO2. High levels of cell surface expression of anti-bcma2-BBOX40, anti-bcma2-BB, and 8ss-anti-bcma2 were observed on the transduced T cells.

[0084] The results of this example demonstrate how to generate a nucleic acid sequence encoding a CAR of the present invention and how to express the CAR on the surface of T cells.

[0085] Example 3 This example describes a series of experiments used to determine the specificity of the CAR of the invention for BCMA. Write it down.

[0086] cell NCI-H929, U266, and RPMI8226 are all BCMA+ multiple myeloma cell lines 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 encodes a BCMA full-length gene. K562 cells (ATCC No. CCL-243) transfected with a nucleic acid sequence encoding NGFR-K562 are K562 cells transfected with a gene encoding low-affinity nerve growth factor (see, e.g., Kochenderfer et al., J. Immunotherapy., 32(7):689-702 (2009)). Peripheral blood lymphocytes (PBLs) from three patients (i.e., myeloma patients 1 to 3) were used, as well as PBLs from three other subjects, donors A, B, and C. Donors A to C all had melanoma. CD34+ primary cells were from three normal healthy donors. The plasmacytoma sample was obtained from myeloma patient 1, and the bone marrow sample was obtained from myeloma patient 2. All of the above human samples were obtained from patients enrolled in IRB-approved clinical trials at the National Cancer Institute. Small airway epithelial cells, suprabronchial Primary human epithelial cells, epithelial cells and intestinal epithelial cells, were obtained from Lonza, Inc. (Basel, Switzerland).

[0087] Interferon-gamma and TNF ELISA BCMA-positive or BCMA-negative cells were treated with AIM V TM Duplicate 96-well round-bottom plates (Corning Life Sciences, Lowell, MA) were cultured in medium (Life Technologies, Carlsbad, CA) plus 5% human serum. The plates were incubated at 37°C for 18-20 hours. After incubation, ELISAs for IFNγ and TNF were performed using standard methods (Pierce, Rockford, IL).

[0088] As shown in Table 2 (all units are pg / mL of IFNγ), T cells transduced with anti-bcma1 or anti-bcma2 CARs produced high amounts of IFNγ when cultured overnight with the BCMA-expressing cell line, BCMA-K562, whereas CAR-transduced T cells produced only background levels of IFNγ when cultured with the negative control cell line, NGFR-K562. Ta.

[0089] [Table 2]

[0090] *Effector cells are T cells from a patient with multiple myeloma (Myeloma Patient 2). The T cells are either transduced with the CARs indicated above or non-transduced. **Indicated target cells were incubated with effector cells overnight and subjected to IFNγ ELISA.

[0091] T cells expressing 8ss-anti-bcma2, anti-bcma2-BB, and anti-bcma2-OX40 CARs produced IFNγ specifically in response to BCMA+ target cells when T cells and target cells were co-cultured overnight, as shown in Table 3 (all units are pg / mL of IFNγ).

[0092] [Table 3]

[0093] T cells transduced with anti-BCMA CARs were co-cultured overnight with a BCMA-expressing multiple myeloma cell line. In contrast, when cultured with various BCMA-negative cell lines, they produced large amounts of IFNγ. In contrast to T cells transduced with anti-bcma1 CAR, T cells transduced with anti-bcma2 CAR and their variants (i.e., 8ss-anti-bcma2, anti-bcma2-BB, and anti-bcma2-OX40) produced significantly less IFNγ when cultured with BCMA-positive cells. When cultured with BCMA-negative cells, they produced more IFNγ and less IFNγ.

[0094] T cells transduced with anti-bcma2 CAR variants produced BCMA+ T cells upon overnight co-culture with target cells, as shown in Table 4 (all units are pg / mL of tumor necrosis factor (TNF)). TNF was produced in response to specific target cells.

[0095] [Table 4]

[0096] T cells transduced with anti-bcma2 CAR and its variants outperformed T cells transduced with anti-bcma1 CAR. Since anti-bcma2CAR and its variants recognized BCMA-expressing cells slightly stronger and more specifically than BCMA-expressing cells, only anti-bcma2CAR and its variants were used in the following experiments.

[0097] CD107a analysis The two T cell populations were prepared in two separate tubes: one tube containing BCMA-K562 cells and the other The tubes contained NGFR-K562 cells. Additionally, both tubes contained anti-bcma2 CAR and anti-bcma2 CAR mutants. transduced T cells, 1 mL of AIM V TMCulture medium (Life Technologies, Carlsbad, CA) plus 5% human serum, a predetermined concentration of anti-CD107a antibody (eBioscience, Inc., San Diego, CA; clone eBioH4A3), and 1 μL of Golgi Stop (BD Biosciences, Franklin Lakes, NJ) were added. All tubes were incubated at 37°C for 4 hours and then stained for the expression of CD3, CD4, and CD8.

[0098] CAR-transduced T cells from three different subjects were specifically stimulated by BCMA-expressing target cells In response to erythrocyte proliferation, CD107a expression increased (see Figures 4A-4C). This indicates that degranulation of BCMA-specific T cells, a prerequisite for cytotoxicity, occurs (e.g., (See, e.g., Rubio et al., Nature Medicine, 9(11): 1377-1382 (2003)). In addition, T cells expressing the anti-bcma2 CAR variants 8ss-anti-bcma2, anti-bcma2-BB, and anti-bcma2-OX40 showed increased inflammatory responses, as shown in Figures 5A-5D. As shown in Figure 1, when stimulated with target cells in vitro, they degranulated in a BCMA-specific manner.

[0099] Intracellular cytokine staining assay (ICCS) A population of BCMA-K562 cells and a population of NGFR-K562 cells were prepared in two separate tubes as described above. To both tubes, anti-bcma2 CAR-transduced T cells from myeloma patient 2, 1 mL of AIM V medium (Life Technologies, Carlsbad, CA) + 5% human serum, and 1 μL of Golgi Stop (BD Biosciences, Franklin Lakes, NJ) were added. All tubes were incubated at 37°C for 6 hours. The cells were surface stained with anti-CD3, anti-CD4, and anti-CD8 antibodies. Intracellular staining for IFNγ (BD Biosciences, Franklin Lakes, NJ, clone B27), IL-2 (BD Biosciences, Franklin Lakes, NJ, clone MQ1-17H12), and TNF (BD Biosciences, Franklin Lakes, NJ, clone MAb11) was performed according to the instructions of the Cytofix / Cytoperm kit (BD Biosciences, Franklin Lakes, NJ).

[0100] The majority of the anti-bcma2 CAR-transduced T cell population from myeloma patient 2 is shown in Figures 6A-6C. As shown, after 6 hours of stimulation with BCMA-expressing target cells, BCMA-specific cytokines IFNγ, IL-2, and TNF were produced.

[0101] Proliferation analysis The proliferative capacity of anti-bcma2 CAR-transduced T cells when stimulated with BCMA-expressing target cells was assessed. Specifically, 0.5x10 6 irradiated BCMA-K562 cells or 0.5x10 6 Irradiated with NGFR-K562 cells were transduced with either anti-bcma2 CAR or SP6 CAR at a total of 1x10 6 The T cells were co-cultured with T cells from AIM V. The T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Life Technologies, Carlsbad, CA) as described in Mannering et al., J. Immunological Methods, 283(1-2):173-183 (2003). TMThe medium was medium containing 5% human AB serum (Life Technologies, Carlsbad, CA). IL-2 was not added to the medium. After 4 days, live cells in each co-culture were counted using trypan blue to exclude dead cells. Subsequently, cells were immunoblotted with a polyclonal biotin-labeled goat anti-human BCMA antibody (R&D Systems, Minneapolis, MN), followed by streptavidin (BD Biosciences, Franklin Lakes, NJ), and anti-CD38 antibody (eBioscience, Inc., San Diego, CA). Flow cytometry was performed by staining T cells with anti-CD56 antibody (BD Biosciences, Franklin Lakes, NJ). Flow cytometry data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).

[0102] T cells expressing the anti-bcma2 CAR showed 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 BCMA-expressing target cells. In contrast, there was no significant difference in CFSE dilution when SP6 CAR-expressing T cells were cultured with BCMA-K562 or NGFR-K562 target cells (see Figure 7B). This suggests that BCMA-specific proliferation by SP6 CAR-expressing T cells is not a significant factor. This indicates no reproduction.

[0103] The proliferation assay initially consisted of 0.8x10 cells expressing the anti-bcma2 CAR. 6 T cells were cultured with either BCMA-K562 or NGFR-K562 cells. After 4 days of culture, 2.7x10 cells were cultured in the BCMA-K562 cell-containing medium. 6of anti-bcma2 CAR-expressing T cells were present in the cultures containing NGFR-K562 cells, whereas 0.6x10 6 Only 10 anti-bcma2 CAR-expressing T cells were present. The BCMA-specific increase in absolute numbers of T cells indicates that these T cells proliferated in response to BCMA.

[0104] The results of this example demonstrate that the CAR-expressing T cells of the invention exhibit BCMA-specific cytokine production, degranulation, and proliferation.

[0105] Example 4 This example demonstrates that anti-BCMA CAR-expressing T cells of the present invention are capable of destroying multiple myeloma cell lines.

[0106] Cytotoxicity assays were performed to determine whether T cells transduced with the anti-bcma2 CAR described in Examples 2 and 3 could destroy BCMA-expressing multiple myeloma (MM) cell lines. Specifically, target cell cytotoxicity was measured by comparing the viability of BCMA-expressing target cells (i.e., multiple myeloma cell lines H929 and RPMI8226) relative to the viability of negative control CCRF-CEM cells using assays described, for example, in Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009) and Hermans et al., J. Immunological Methods, 285(1): 25-40 (2004).

[0107] Approximately 50,000 BCMA-expressing target cells and approximately 50,000 CCRF-CEM cells were combined in the same tube with various numbers of CAR-transduced T cells. CCRF-CEM negative control cells were labeled with 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR) (Life Technologies, Carlsbad, CA) fluorescent dye, and BCMA-expressing target cells were labeled with CFSE. In all experiments, the cytotoxicity of effector T cells transduced with the anti-bcma2 CAR was compared with that of negative control effector T cells transduced with the SP6 CAR, obtained from the same subject. Cocultures were performed in duplicate in sterile 5 mL tubes (BD Biosciences, Franklin Lakes, NJ) at T cell:target cell ratios of 20.0:1, 7:1, 2:1, and 0.7:1. Cultures were incubated at 37°C for 4 hours. Immediately after incubation, 7-amino-actinomycin D (7AAD; BD Biosciences, Franklin Lakes, NJ) was added. Viable BCMA-expressing target cells and viable CCRF-CEM negative control cells were determined in each T cell / target cell coculture.

[0108] For each T cell / target cell co-culture, the percentage viability of BCMA-expressing target cells relative to CCRF-CEM negative control cells was determined by dividing the percentage of BCMA-expressing target cells by the percentage of CCRF-CEM negative control cells. The corrected percentage viability of BCMA-expressing target cells was determined by dividing the percentage of viable BCMA-expressing target cells in each T cell / target co-culture by the percentage of viable BCMA-expressing target cells in a test tube containing only BCMA-expressing target cells and CCRF-CEM negative control cells but no effector T cells. The cytotoxicity was calculated by dividing by the ratio of the percentage of cells:CCRF-CEM negative control cells. This correction is necessary to account for variations due to the starting cell number and spontaneous target cell death. Cytotoxicity was calculated as follows: Percent cytotoxicity of BCMA-expressing target cells = 100 - corrected viability percentage of BCMA-expressing target cells.

[0109] Cytotoxicity analysis results are shown in Figures 7C and 7D. T cells transduced with the anti-bcma2 CAR specifically killed the BCMA-expressing multiple myeloma cell lines H929 and RPMI8226. In contrast, T cells transduced with the SP6 CAR exhibited much lower levels of cytotoxicity against these cell lines.

[0110] The results of this example demonstrate that the nucleic acid sequence encoding the anti-BCMA CAR of the present invention inhibits the proliferation and proliferation of multiple myeloma cells. It has been demonstrated that it can be used in line breaking methods.

[0111] Example 5 This example demonstrates that T cells expressing the anti-BCMA CAR of the present invention can destroy primary multiple myeloma cells.

[0112] Primary multiple myeloma cells, as described in Example 2, were analyzed for BCMA expression and BCMA-specific cytotoxicity. Tokine production, degranulation and proliferation were assessed using the methods described above.

[0113] Cell surface BCMA expression was detected on primary bone marrow multiple myeloma cells as well as on four primary multiple myeloma samples from myeloma patient 3 (see Figure 8A). BCMA-expressing plasma cells accounted for 40% of the cells in the bone marrow sample from myeloma patient 3. As shown in Figure 8B, BCMA-expressing plasma cells from donor C Allogeneic T cells transduced with the anti-bcma2 CAR produced IFNγ after co-culture with unmodified bone marrow cells from myeloma patient 3. Anti-bcma2 CAR-transduced T cells from the same allogeneic donor were cultured with peripheral blood mononuclear cells (PBMCs) from myeloma patient 3. Furthermore, SP6-CAR-transduced T cells from donor C produced much less IFNγ. It did not specifically recognize the bone marrow of myeloma patient 3. Normal PBMCs did not contain cells expressing BCMA. It has been previously reported that BCMA expression is a key factor in the development of BCMA (see, e.g., Ng et al., J. Immunology, 173(2): 807-817 (2004)). To confirm this observation, PBMCs from patient 3 were assessed for BCMA expression by flow cytometry. PBMCs from patient 3 were CD56+CD38, which accounted for approximately 0.75% of PBMCs. high Apart from a small population of cells, they did not contain BCMA-expressing cells. This population was likely due to circulating multiple myeloid cells. It is considered to consist of tumor cells.

[0114] The plasmacytoma resected from myeloma patient 1 consisted of 93% plasma cells, and as shown in Figure 8C, these primary plasma cells expressed BCMA. T cells from myeloma patient 2 produced IFNγ when cultured with allogeneic, non-genetically engineered plasmacytoma cells from myeloma patient 1. T cells from myeloma patient 2 did not produce significant amounts of IFNγ when cultured with PBMCs from myeloma patient 1. T cells from myeloma patient 2 transduced with the SP6 CAR produced IFNγ in a manner similar to that of the myeloma patient 2. When cultured with plasmacytoma cells or PBMCs from patient 1, they did not produce significant amounts of IFNγ. PBMCs from bone marrow patient 1 did not express BCMA, as determined by flow cytometry.

[0115] T cells from myeloma patient 1, who had received eight cycles of prior myeloma therapy, were successfully cultured and transduced with a lentiviral vector encoding the anti-bcma2 CAR. Eight days after the start of culture, expression of the anti-bcma2 CAR was confirmed on 65% of T cells. T cells from myeloma patient 1 expressing the anti-bcma2 CAR produced IFNγ in specific response to autologous plasmacytoma cells (Figure 8D). T cells from myeloma patient 1 expressing the SP6 CAR did not recognize autologous plasmacytoma cells. Anti-bcma2 CAR-expressing T cells and SP6 CAR-expressing T cells did not recognize autologous PBMCs. Furthermore, T cells from myeloma patient 1 expressing the anti-bcma2 CAR exhibited a low effector-to-target ratio. In contrast, the SP6 CAR-expressing myeloma patient 1 specifically killed autologous plasmacytoma cells. T cells from the mice showed only low levels of cytotoxicity against autologous plasmacytoma cells. (Figure 8E)

[0116] The results of this example demonstrate that the anti-BCMA CAR of the present invention has a potent inhibitory effect on primary multiple myeloma cells. It has been proven that it can be used in law.

[0117] Example 6 This example demonstrates that T cells expressing the anti-BCMA CARs of the present invention destroy established tumors in mice. This illustrates that it is possible.

[0118] Immunodeficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, Jackson Laboratory) were injected with 8x10 6 RPMI8226 cells were injected intradermally. Tumors were allowed to grow for 17 to 19 days, after which mice were injected with 8x10 6 T cells transduced with anti-bcma2 CAR or SP6 CAR were intravenously infused. Tumors were measured every 3 days using calipers. The longest length and the length perpendicular to the longest length were measured. Multiply and calculate tumor size (area) in mm2 When the maximum length reached 15 mm, Animal studies were performed with the approval of the National Cancer Institute Animal Care and Use Committee. provided.

[0119] The results of this example are shown in Figures 9A and 9B. Around day 6, mice treated with anti-bcma2 transduced T cells showed tumor cell regression, which disappeared by day 15. Furthermore, mice treated with anti-bcma2 transduced T cells showed tumor cell regression. All treated mice survived up to 30 days after T cell infusion.

[0120] The results of this example demonstrate that the anti-BCMA CAR of the present invention can destroy multiple myeloma cells in vivo.

[0121] All references cited in this application, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as if each such reference was individually and specifically indicated to be set forth in its entirety in this application.

[0122] In the context of describing the present invention (particularly in the context of the claims which follow), the articles "a," "an," and "the" and similar demonstratives refer to the same unless otherwise expressly stated in this application. The terms "comprising," "having," "including," and "containing" should be construed as covering both the singular and the plural unless the context clearly contradicts it. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., "including, but not limited to") unless specifically stated otherwise. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless specifically stated otherwise herein, and each separate value is incorporated herein to the same extent as if each separate value were individually set forth herein. All methods described herein can be performed in any suitable order unless specifically stated otherwise or clearly contradicted by the context. The use of any and all examples or exemplary language (e.g., "such as") herein, unless specifically stated otherwise, is intended solely to facilitate understanding of the invention and not to limit the scope of the invention. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0123] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that skilled artisans will make appropriate modifications, and contemplate that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto to the extent permitted by applicable law. Furthermore, any and all combinations of the above-described elements, in any and all possible variations thereof, are encompassed by the invention unless otherwise specified or clearly contradicted by the context.

Claims

1. A pharmaceutical composition for use in a method for treating multiple myeloma, comprising a population of T cells expressing a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-recognizing portion and a T cell-activating portion, and the antigen-recognizing portion is directed toward a B cell maturation antigen (BCMA).

2. The pharmaceutical composition for use according to claim 1, wherein the antigen-recognizing portion comprises a monoclonal antibody or its antigen-binding portion that is directed toward BCMA.

3. The pharmaceutical composition for use according to claim 1 or 2, wherein the CAR comprises a hinge array.

4. The pharmaceutical composition for use according to claim 3, wherein the hinge arrangement is a hinge arrangement of human CD8α molecules or CD28 molecules.

5. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the T cell activating portion comprises a transmembrane domain.

6. The pharmaceutical composition for use according to any one of claims 1 to 5, wherein the T cell activating portion comprises an intracellular T cell signaling domain.

7. The pharmaceutical composition for use according to claim 6, wherein the intracellular T cell signaling domain is a CD28 molecule, a CD3 zeta (ζ) molecule, a human Fc receptor gamma (FcRγ) chain, a CD27 molecule, an OX40 molecule, or a 4-1BB molecule.

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