Anti b-cell maturation antigen chimeric antigen receptor having human domain

Abstract

To provide a pharmaceutical composition for treating or preventing cancer.SOLUTION: A pharmaceutical composition formulated for parenteral administration use contains a pharmaceutically acceptable carrier, and a host cell population including a vector including a nucleic acid including a nucleotide sequence which encodes a chimeric antigen receptor (CAR) having antigen specificity to a B-cell maturation antigen (BCMA).SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 527,556, filed June 30, 2017, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support from the National Cancer Institute, National Institutes of Health, under Project No. ZIABC01143905. The government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Submitted Documents The computer-readable nucleotide / amino acid sequence listing, submitted concurrently herewith and identified as follows, is incorporated herein by reference in its entirety: 67,061 byte ASCII (text) file entitled "739534_ST25.TXT", dated June 25, 2018. [Background technology]

[0004] Cancer is a public health concern. Despite advances in treatment, such as chemotherapy, many cancers have poor prognoses. For example, while treatment of multiple myeloma (MM) can result in remission, many patients ultimately relapse and die. Therefore, there is an unmet need for additional cancer treatments. Summary of the Invention

[0005] An embodiment of the present invention provides a chimeric antigen receptor (CAR) comprising an antigen recognition domain, a transmembrane (TM) domain, and a T cell activation domain, wherein the CAR has antigen specificity for B cell maturation antigen (BCMA), and the antigen recognition domain comprises the amino acid sequence of (a) SEQ ID NOs: 1-3, (b) SEQ ID NOs: 4-6, (c) SEQ ID NOs: 7-9, or (d) SEQ ID NOs: 10-12.

[0006] Further embodiments of the invention provide related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions related to the CARs of the invention.

[0007] Additional embodiments of the present invention provide related methods of treating or preventing cancer in a mammal. [Brief explanation of the drawings]

[0008] [Figure 1-1] Figures 1A-1D are diagrams depicting CARs containing the fully human heavy chain-only antigen recognition domains of FHVH74 (A), FHVH32 (B), FHVH33 (C), or FHVH93 (D) combined with the hinge and transmembrane regions of the CD8α molecule, the cytoplasmic portion of the CD28 costimulatory molecule, and the cytoplasmic portion of the CD3ζ T cell activation domain. [Figure 1-2] Figures 1E-1H are diagrams depicting CARs containing the fully human heavy chain-only antigen recognition domains of FHVH74 (E), FHVH32 (F), FHVH33 (G), or FHVH93 (H) combined with the hinge and transmembrane regions of the CD8α molecule, the cytoplasmic portion of the 4-1BB costimulatory molecule, and the cytoplasmic portion of the CD3ζ T cell activation domain. [Figure 1-3] Figures 1I-1L are diagrams depicting CARs containing the fully human heavy chain-only antigen recognition domains of FHVH74 (I), FHVH32 (J), FHVH33 (K), or FHVH93 (L) combined with the hinge and transmembrane regions of the CD8α molecule, the cytoplasmic portion of the inducible T cell costimulatory protein (ICOS), and the cytoplasmic portion of the CD3ζ T cell activation domain. [Figure 2]Figure 2 is a series of graphs depicting experimental data demonstrating that the four indicated FHVH CARs were expressed by primary human T cells, as described in Example 2. Untransduced (UT) T cells were included as a negative control, and 11D5-3-CD828Z served as a positive control CAR. T cells were transduced on day 2 of culture and stained with BCMA-Fc protein reagent on day 7 of culture. Plots are gated live lymphocytes. Numbers on the plots are the percentage of CD3+ cells expressing the CAR (top) or not expressing the CAR (bottom). [Figure 3] Figure 3 is a series of graphs depicting experimental data showing BCMA-specific degranulation by T cells expressing FHVH CARs. These graphs show the results of primary human T cells transduced with one of four FHVH CARs in a CD107a degranulation assay to assess antigen-specific function. As described in Example 3, T cells expressing each CAR degranulated to a greater extent when cultured with BCMA+ target cells (BCMA-K562) compared with BCMA-negative target cells (NGFR-K562). UT cells were included as a negative control, and 11D5-3-CD828Z served as a positive control CAR. Plots represent gated CD3+ lymphocytes. Numbers on the plots represent the percentage of CD3+ cells that upregulate CD107a (top) or do not upregulate CD107a (bottom). [Figure 4-1] Figure 4A is a graph showing experimental data demonstrating that 11D5-3-CD828Z CARs expanded in a BCMA-specific manner. The plots are gated live CD3+ lymphocytes. The white histogram represents CAR+ T cells stimulated with BCMA-K562 (BCMA-expressing) target cells, and the black histogram represents CAR+ T cells stimulated with NGFR-K562 (BCMA-negative) cells. All results were obtained simultaneously using cells from the same patient. Figures 4B-4C are graphs showing experimental data demonstrating that FHVH74-CD828Z (B) or FHVH32-CD828Z (C) CARs expanded in a BCMA-specific manner, as described in Example 5. [Figure 4-2] Figures 4D-4E are graphs depicting experimental data demonstrating BCMA-specific proliferation of FHVH33-CD828Z(D) or FHVH93-CD828Z(E) CARs, as described in Example 5. Figure 4F is a graph depicting experimental data demonstrating that the absolute number of CAR+ T cells increased when T cells transduced with the indicated CARs were cultured with BCMA+ target cells. When CAR T cells were cultured with BCMA-K562 cells, the number of CAR+ T cells increased for T cells expressing all CARs. The Y-axis represents the number of CAR+ T cells (x106). The X-axis represents the number of days T cells were cultured with BCMA+ target cells. [Figure 5] Figure 5A is a graph representing experimental data showing the ability of FHVH33-CD828Z CAR to kill BCMA+ target cells compared to the ability of UT cells to kill BCMA+ target cells. T cells expressing FHVH33-CD828Z CAR were cultured with RPMI8226 target cells in vitro for 4 hours at the indicated effector-to-target ratios. Cytotoxicity was determined in duplicate. Results are presented as + / - standard error of the mean. The Y-axis represents the % cytotoxicity of the CAR. The X-axis represents the ratio of T cells to target cells. Figure 5B is a graph representing experimental data showing the ability of FHVH33-CD8BBZ CAR to kill BCMA+ target cells compared to the ability of UT cells to kill BCMA+ target cells. T cells expressing FHVH33-CD8BBZ CAR were cultured with RPMI8226 target cells in vitro for 4 hours at the indicated effector-to-target ratios. Cytotoxicity was determined in duplicate. Results are presented as + / - standard error of the mean. The Y-axis represents % cytotoxicity of the CAR. The X-axis represents the ratio of T cells to target cells. [Figure 6]Figure 6 is a series of graphs showing experimental data demonstrating that CARs with a 4-1BB costimulatory domain are expressed on the surface of primary human T cells, with FHVH33-CD8BBZ showing the highest expression. The plots show BCMA-Fc staining of four FHVH CARs, staining of the 11D5-3-CD828Z control CAR, and staining of UT cells. The plots are gated live lymphocytes. The numbers on the plots are the percentage of cells that are BCMA-Fc stained (upper number) or not (lower number). [Figure 7-1] Figures 7A-7B show the expression of this CAR in UT cells (A) compared to T cells expressing FHVH33-CD8BBZ (B). Plots are gated live CD3+ lymphocytes. Numbers in the plots are the percentages of BCMA-PE+ (top) and BCMA-PE- (bottom). [Figure 7-2] Figures 7C-7F show experimental data demonstrating that FHVH33-CD8BBZ-transduced T cells degranulated BCMA-specifically, as assessed by CD107a staining. The data show upregulation of CD107a in UT+BCMA-K562 (C) and UT+NGFR-K562 cells (D) compared with upregulation of CD107a in FHVH33+BCMA-K562 (E) and FHVH33+NGFR-K562 cells (F). The same T cell cultures were used as shown in Figures 7A-7B. Plots show gated live CD3+ lymphocytes. Numbers in the plots indicate the percentage of CD107a+ (top) and CD107a- (bottom). [Figure 7-3]Figures 7C-7F show experimental data demonstrating that FHVH33-CD8BBZ-transduced T cells degranulated BCMA-specifically, as assessed by CD107a staining. The data show upregulation of CD107a in UT+BCMA-K562 (C) and UT+NGFR-K562 cells (D) compared with upregulation of CD107a in FHVH33+BCMA-K562 (E) and FHVH33+NGFR-K562 cells (F). The same T cell cultures were used as shown in Figures 7A-7B. Plots show gated live CD3+ lymphocytes. Numbers in the plots indicate the percentage of CD107a+ (top) and CD107a- (bottom). [Figure 7-4] Figure 7G shows experimental data demonstrating that CAR-expressing T cells produced IFNγ in a BCMA-specific manner. When T cells were cultured with the BCMA+ cell lines BCMA-K562 and RPMI8226, large amounts of IFNγ were released. The Y-axis represents the amount of IFNγ (pg / mL). The X-axis represents the target cells used in the experiment. [Figure 8] Figure 8 is a graph showing the amount of IFNγ (pg / mL) secreted by untransduced (UT) or FHVH33-CD828Z or FHVH33-CD8BBZ CAR-transduced T cells when co-cultured with target primary human myeloma bone marrow cells (black bars) or control target PBMCs (gray bars). [Figure 9-1] Figure 9A is a schematic diagram showing the titration of FHVH33-CD8BBZ T cell doses in mice. Female (F) 7-8 week (wk)-old NSG mice were injected intradermally (id) with 8 million (M) RPMI8226 cells, and tumors were allowed to grow for 10 days. On day 0, mice were intravenously (IV) injected with various numbers of FHVH33-CD8BBZ-expressing T cells. Tumors were measured every 3 days (d). [Figure 9-2]Figure 9B is a graph showing tumor volume (mm3) measured in mice treated as shown in Figure 9A with 0.2 x 106 (filled triangles), 0.7 x 106 (filled circles), or 2.2 x 106 (open circles) FHVH33-CD8BBZ-expressing T cells on the indicated days after CAR T-cell infusion. Untreated mice are represented by open triangles. Figure 9C is a graph showing survival of mice shown in Figure 9B after treatment with 0.2 x 106 (filled triangles), 0.7 x 106 (filled circles), or 2.2 x 106 (open circles) FHVH33-CD8BBZ-expressing T cells on the indicated days after CAR T-cell infusion. Untreated mice are represented by open triangles. [Figure 10-1] Figure 10A is a graph showing tumor volume (mm3) measured in mice treated with T cells expressing SP6-CD828Z (triangles), 11D5-3-CD8BBZ (squares), FHVH33-CD8BBZ (open circles), or FHVH33-CD828Z (filled circles) CARs at the indicated days after CAR T cell infusion. Untreated mice are represented by diamonds. [Figure 10-2] Figure 10B is a graph showing survival of mice treated with T cells expressing SP6-CD828Z (triangles), 11D5-3-CD8BBZ (squares), FHVH33-CD8BBZ (open circles), or FHVH33-CD828Z (filled circles) CARs at the indicated days after CAR T cell infusion. Untreated mice are represented by diamonds. DETAILED DESCRIPTION OF THE INVENTION

[0009] An embodiment of the present invention provides a CAR comprising an antigen recognition domain, a TM domain, and a T cell activation domain, with antigen specificity for BCMA. A CAR is an artificially constructed hybrid protein or polypeptide containing the antigen recognition domain of an antibody linked to a T cell signaling domain or a T cell activation domain. CARs have the ability to redirect T cell specificity and reactivity to selected targets in a non-MHC-restricted manner, utilizing the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition confers on CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thereby bypassing a major mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

[0010] The CARs of the present invention have antigen specificity for B-cell maturation antigen (BCMA, also known as CD269). BCMA is a member of the tumor necrosis factor receptor superfamily (see, e.g., Thompson et al., J. Exp. Medicine, 192(1):129-135 (2000) and Mackay et al., Annu. Rev. Immunol., 21:231-264 (2003)). BCMA binds to B-cell 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 benign cells, BCMA has been reported to be expressed predominantly in 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 RNA has been ubiquitously detected in multiple myeloma cells, and BCMA protein has been detected on the surface of plasma cells from multiple myeloma patients by several investigators (see, e.g., Novak et al., Blood, 103(2):689-694 (2004); Neri et al., Clinical Cancer Research, 13(19):5903-5909 (2007); Bellucci et al., Blood, 105(10):3945-3950 (2005); and Moreaux et al., Blood, 103(8):3148-3157 (2004)). BCMA expression has also been detected on the surface of Hodgkin's lymphoma cells (see, e.g., Chiu et al., Blood, 109(2):729-739 (2007)). Human BCMA has the amino acid sequence of SEQ ID NO:42.

[0011] The phrases "having antigen specificity" and "eliciting an antigen-specific response," as used herein, mean that the CAR is capable of specifically binding to and immunologically recognizing an antigen, such that binding of the CAR to the antigen elicits an immune response.

[0012] The CARs of the present invention can provide any one or more of a variety of advantages. For example, the CARs of the present invention can reduce anti-CAR immunogenicity. CARs comprising one or both of a non-human domain (e.g., a mouse domain) and an artificial linker peptide can elicit an anti-CAR immune response when administered to a patient. Such an anti-CAR immune response can be This may reduce the persistence of CAR-expressing cells and reduce or eliminate the efficacy of CAR therapy. Without being bound by any particular theory or mechanism, it is believed that any one or more of the following features of the CARs of the present invention can reduce or eliminate potential sources of anti-CAR immunogenicity: (i) all domains of the CAR are human; (ii) the CAR does not contain an artificial linker peptide, e.g., a linker peptide having a length of about 10 to about 25 amino acid residues and consisting of any one or more of glycine, serine, and threonine; (iii) the CAR does not contain an antibody light chain variable region; and (iv) the antigen recognition domain contains only a single antibody heavy chain variable region. Reducing or eliminating potential sources of anti-CAR immunogenicity is believed to improve the persistence of CAR-expressing cells and the efficacy of CAR therapy. Furthermore, any one or more of the aforementioned features (ii) to (iv) can facilitate the preparation of CARs that target one or more different antigens (other than BCMA) in addition to BCMA.

[0013] The CARs of the present invention may have reduced anti-CAR immunogenicity compared to conventional CARs, which may have any one or more of the following characteristics: (i) not all of the domains of the conventional CAR are human; (ii) the conventional CAR includes an artificial linker peptide, e.g., a linker peptide having a length of about 10 to about 25 amino acid residues and consisting of any one or more of glycine, serine, and threonine; and (iii) the conventional CAR includes an antibody light chain variable region (hereinafter referred to as a "conventional CAR").

[0014] Anti-CAR immunogenicity is reduced by the present invention when the immune response to the CAR of the present invention is quantitatively or qualitatively attenuated compared to the immune response to a conventional CAR. A quantitative reduction in anti-CAR immunogenicity encompasses a reduction in the magnitude or extent of the anti-CAR immune response. The magnitude or extent of anti-CAR immunogenicity can be measured based on any number of known parameters, such as a reduction in the level of cytokine (e.g., CAR-specific cytokine) production (cytokine concentration), a reduction in activation (e.g., lymphocyte (e.g., CAR-specific lymphocyte) proliferation) or the number of recruited lymphocytes, and / or a reduction in antibody (CAR-specific antibody) production (antibody concentration), a reduction in the ability of host (recipient) T cells to kill CAR-expressing T cells, etc. A qualitative reduction in anti-CAR immunogenicity encompasses any change in the nature of the anti-CAR immune response that reduces the effectiveness of the anti-CAR immune response in mediating a reduction in the cytotoxic activity of the CAR. Methods for measuring anti-CAR immunogenicity are known in the art. For example, anti-CAR immunogenicity can be measured by measuring the type and level of cytokines produced. Reduced anti-CAR immunogenicity may be characterized by reduced production of any one or more of cytokines, such as IFN-γ, TNF-α, and granzyme B, and / or reduced stimulation of a cell-mediated anti-CAR immune response, e.g., reduced proliferation and activation of T cells and / or macrophages specific for the CAR of the present invention compared to that obtained with a conventional CAR. Reduced anti-CAR immunogenicity may be characterized by any one or more of reduced stimulation of anti-CAR T cells, reduced proliferation of anti-CAR T cells, reduced secretion of IFNγ and / or granzyme B by anti-CAR T cells, and reduced ability of host T cells to kill CAR-expressing T cells. Qualitative and quantitative attenuation of anti-CAR immunogenicity may occur simultaneously and are not mutually exclusive. The phrase "anti-CAR immunogenicity," as used herein, refers to the immune response against the CAR itself, and not to any aspect of the immune response against the target antigen BCMA that the CAR may provide.

[0015] The CAR comprises an antigen recognition domain. The antigen recognition domain recognizes and binds to BCMA. In an embodiment of the invention, the antigen recognition domain comprises the heavy chain variable region of a human anti-BCMA antibody. A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain comprises an N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain comprises an N-terminal variable (VL) region and a C-terminal constant (CL) region. The VH and VL regions have the same general structure, and each region contains four framework regions, the sequences of which are relatively conserved. The framework regions are connected by three complementarity-determining regions (CDRs), i.e., CDR1, CDR2, and CDR3. However, as explained above, in an embodiment of the present invention, the CAR does not contain an antibody light chain variable region. Therefore, in an embodiment of the present invention, the antigen recognition domain contains only a single antibody heavy chain variable region.

[0016] In embodiments, the antigen recognition domain comprises the CDR1, CDR2, and CDR3 regions of the heavy chain variable region of a human anti-BCMA antibody. In this regard, in embodiments of the invention, the antigen recognition domain comprises: (a) one or more of a heavy chain CDR1 region comprising SEQ ID NO: 1, a heavy chain CDR2 region comprising SEQ ID NO: 2, and a heavy chain CDR3 region comprising SEQ ID NO: 3 (CDR regions of the FHVH74 heavy chain variable region); (b) one or more of a heavy chain CDR1 region comprising SEQ ID NO: 4, a heavy chain CDR2 region comprising SEQ ID NO: 5, and a heavy chain CDR3 region comprising SEQ ID NO: 6 (CDR regions of the FHVH32 heavy chain variable region); (c) one or more of a heavy chain CDR1 region comprising SEQ ID NO: 7, a heavy chain CDR2 region comprising SEQ ID NO: 8, and a heavy chain CDR3 region comprising SEQ ID NO: 9 (CDR regions of the FHVH33 heavy chain variable region); or (d) one or more of a heavy chain CDR1 region comprising SEQ ID NO: 10, a heavy chain CDR2 region comprising SEQ ID NO: 11, and a heavy chain CDR3 region comprising SEQ ID NO: 12 (CDR regions of the FHVH93 heavy chain variable region) may include: Preferably, the antigen recognition domain comprises the amino acid sequences of (a) all of SEQ ID NOs: 1 to 3, (b) all of SEQ ID NOs: 4 to 6, (c) all of SEQ ID NOs: 7 to 9, or (d) all of SEQ ID NOs: 10 to 12.

[0017] In embodiments of the invention, the antigen recognition domain comprises a heavy chain variable region of a human anti-BCMA antibody. In this regard, the antigen recognition domain may comprise the amino acid sequence of (a) SEQ ID NO: 13 (FHVH74 heavy chain variable region), (b) SEQ ID NO: 14 (FHVH32 heavy chain variable region), (c) SEQ ID NO: 15 (FHVH33 heavy chain variable region), or (d) SEQ ID NO: 16 (FHVH93 heavy chain variable region).

[0018] In embodiments of the present invention, the antigen-recognition domain does not include a linker peptide. The antigen-recognition domain of a conventional CAR may be composed of a single-chain variable fragment (scFv). An scFv is a monovalent molecule comprising two domains (i.e., VL and VH) of an Fv fragment linked by an artificial linker peptide, which allows the two domains to be synthesized as a single polypeptide chain. Any one or more of the following features of the CARs of the present invention can advantageously reduce or eliminate potentially immunogenic linkages connecting different components of the CAR, such as two potentially immunogenic linkages connecting the VL and VH of an scFv used in conventional CARs: (i) the absence of a linker peptide, such as those typically found in scFvs used in conventional CARs; (ii) the absence of an antibody light chain variable region (also used in conventional CARs); and (iii) the presence of only a single antibody heavy chain variable region. The linkages and linker peptide(s) are artificial sequences not normally found in humans and therefore may be immunogenic. Alternatively or additionally, any one or more of the foregoing features (i)-(iii) can eliminate any potentially immunogenic regions in one or both of the peptide linker and the antibody light chain variable region. The purpose of the linker peptide is generally to form a flexible link between two other peptides or proteins (e.g., between an antibody heavy chain and an antibody light chain). Linker peptides can be of any length, and many are of any length. In an embodiment of the present invention, the linker peptide may have a length of about 5 to about 100 amino acid residues, about 8 to about 75 amino acid residues, about 8 to about 50 amino acid residues, about 10 to about 25 amino acid residues, about 8 to about 30 amino acid residues, about 8 to about 40 amino acid residues, or about 8 to about 50 amino acid residues. In an embodiment of the present invention, the antigen-recognition domain does not include a linker peptide having a length of about 8 to about 40 amino acid residues. For example, the linker peptide may contain or consist of one or more of glycine, serine, and threonine, with or without other amino acid residues. In an embodiment of the present invention, the antigen-recognition domain does not include a linker peptide having a length of about 8 to about 40 amino acid residues and consisting of one or more of glycine, serine, and threonine.

[0019] In another embodiment, the CAR of the present invention comprises a leader domain. The leader domain may be located amino-terminal to the antigen recognition domain (e.g., the heavy chain variable region of an anti-BCMA antibody). The leader domain may comprise any suitable leader sequence. Preferably, the leader domain is a human leader domain. In one embodiment, the leader domain is a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor sequence or a human CD8α leader sequence.

[0020] In another embodiment, the CAR comprises a hinge domain. Those skilled in the art will appreciate that a hinge domain is a short amino acid sequence that promotes antibody flexibility (see, for example, Woof (See, e.g., [Delta] et al., Nat. Rev. Immunol., 4(2):89-99 (2004)). The hinge domain may be located between the antigen recognition domain and the T cell activation domain. The hinge domain may comprise any suitable sequence derived from or obtained from any suitable molecule. Preferably, the hinge domain comprises a human sequence. In one embodiment, for example, the hinge domain is a portion of a human CD8α molecule or a human CD28 molecule.

[0021] The CAR may comprise a TM domain. The TM domain may be any TM domain derived from or obtained from any molecule known in the art. Preferably, the TM domain is a human TM domain. For example, the TM domain may comprise the TM domain of a human CD8α molecule or a human CD28 molecule. CD8 is a TM glycoprotein that functions as a co-receptor for the T cell receptor (TCR) and is primarily expressed on the surface of cytotoxic T cells. The most common form of CD8 exists as a dimer composed of CD8α and CD8β chains. CD28 is expressed on T cells and provides a costimulatory signal necessary for T cell activation. CD28 is a receptor for CD80 (B7.1) and CD86 (B7.2).

[0022] The CAR may comprise a T cell activation domain. The T cell activation domain may comprise an intracellular (i.e., cytoplasmic) T cell signaling domain. The intracellular T cell signaling domain may be obtained or derived from a CD28 molecule, a CD3 zeta (ζ) molecule, an Fc receptor gamma (FcRγ) chain, a CD27 molecule, an OX40 molecule, a 4-1BB molecule, an inducible T cell costimulatory protein (ICOS), or other intracellular signaling molecules known in the art, or modified versions of any of the foregoing. As discussed above, CD28 is a T cell marker important for T cell costimulation. CD3ζ associates with the TCR to generate a signal and contains an immunoreceptor tyrosine-based activation motif (ITAM). 4-1BB, also known as CD137, delivers a potent costimulatory signal to T cells, promoting T lymphocyte differentiation and enhancing long-term survival. ICOS is a CD28 superfamily costimulatory molecule expressed on activated T cells. In a preferred embodiment, CD28, CD3 zeta, FcRγ, ICOS, 4-1BB, OX40, and CD27 are human.

[0023] The CARs of the present invention may comprise any one of the aforementioned TM domains and any one or more of the aforementioned intracellular T cell signaling domains in any combination. For example, the CARs of the present invention may comprise the TM domain of CD8α and the intracellular T cell signaling domains of CD28 and CD3 zeta. Alternatively, for example, the CARs of the present invention may comprise the TM domain of CD8α and the intracellular T cell signaling domains of CD3 zeta and 4-1BB. In yet another example, the CARs of the present invention may comprise the TM domain of CD8α and the intracellular T cell signaling domains of ICOS and CD3 zeta.

[0024] In one embodiment, a CAR of the invention comprises, from amino to carboxyl terminus, a human CD8α leader domain, a human anti-BCMA antibody heavy chain variable region, a hinge and transmembrane region of a human CD8α molecule, a cytoplasmic T cell signaling domain of a human CD28 molecule, and a cytoplasmic T cell signaling domain of a human CD3ζ molecule. In another embodiment, a CAR of the invention comprises, from amino to carboxyl terminus, a human CD8α leader domain, a human anti-BCMA antibody heavy chain variable region, a hinge and transmembrane region of a human CD8α molecule, a cytoplasmic T cell signaling domain of a human 4-1BB molecule, and a cytoplasmic T cell signaling domain of a human CD3ζ molecule. In yet another embodiment, a CAR of the invention comprises, from amino to carboxyl terminus, a human CD8α leader domain, a human anti-BCMA antibody heavy chain variable region, a hinge and transmembrane region of a human CD8α molecule, a cytoplasmic T cell signaling domain of a human ICOS molecule, and a cytoplasmic T cell signaling domain of a human CD3ζ molecule. A further embodiment of the invention provides a CAR comprising, consisting of, or consisting essentially of the amino acid sequence of any one of SEQ ID NOs: 17 to 28. The components of the CARs of SEQ ID NOs: 17 to 28 are set forth in Table A below.

[0025] [Table A]

[0026] In embodiments of the present invention, all domains of the CAR are human. In this regard, the leader domain, hinge domain, antigen recognition domain, TM domain, and T cell activation domain are all human. Thus, the CAR of the present invention may advantageously have reduced anti-CAR immunogenicity, as described herein with respect to other aspects of the present invention, compared to a CAR comprising any one or more of a non-human leader domain, a non-human hinge domain, a non-human antigen recognition domain, a non-human TM domain, and a non-human T cell activation domain.

[0027] Functional portions of the CARs of the invention described herein are included 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 (the parent CAR). A functional portion includes, for example, a portion of a CAR that retains the ability to recognize target cells or detect, treat, or prevent disease to a similar extent, the same extent, or a greater extent than the parent CAR. With respect to the parent CAR, a functional portion can comprise, for example, about 10%, about 25%, about 30%, about 50%, about 68%, about 80%, about 90%, about 95%, or more of the parent CAR.

[0028] A functional portion can include additional amino acids at the amino or carboxy terminus, or at both termini, of the portion, which additional amino acids are not found in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, e.g., recognizing target cells, detecting cancer, treating or preventing cancer, etc. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent CAR.

[0029] Functional variants of the CARs of the present invention described herein are included within the scope of the present invention. The term "functional variant" as used herein refers to a CAR, polypeptide, or protein that has substantial or significant sequence identity or similarity to a parent CAR, and the functional variant retains the biological activity of the CAR from which it is a variant. Functional variants include, for example, variants of the CARs described herein (parent CARs) that retain the ability to recognize target cells to a similar extent, the same extent, or a greater extent than the parent CAR. With respect to the parent CAR, the functional variant may, for example, have an amino acid sequence that is at least about 30%, at least about 50%, at least about 75%, at least about 80%, at least about 90%, at least about 98%, or more identical to the parent CAR.

[0030] A functional variant can, for example, comprise the amino acid sequence of a parent CAR with at least one conservative amino acid substitution. Alternatively, or in addition, a functional variant can comprise the amino acid sequence of a parent CAR with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent CAR.

[0031] The amino acid substitutions in the CAR of the present invention are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having particular physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be substitutions of an acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), substitutions of amino acids with nonpolar side chains for amino acids with other nonpolar side chains (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), substitutions of a basic / positively charged polar amino acid for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), substitutions of uncharged amino acids with polar side chains for uncharged amino acids with other polar side chains (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), substitutions of amino acids with beta-branched side chains for amino acids with beta-branched side chains (e.g., Ile, Thr, and Val), substitutions of amino acids with aromatic side chains for amino acids with other aromatic side chains (e.g., His, Phe, Trp, and Tyr), etc.

[0032] A CAR may consist essentially of the specific amino acid sequence(s) described herein, such that other components, e.g., other amino acids, do not substantially alter the biological activity of the functional variant.

[0033] CARs (including functional portions and functional variants) of embodiments of the present invention can be of any length, i.e., contain any number of amino acids, so long as the CAR (or functional portion or functional variant thereof) retains its biological activity, such as the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent a disease in a mammal. For example, a CAR can be about 50 to about 1000 amino acids in length, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more amino acids in length.

[0034] CARs of embodiments of the present invention (including functional portions and functional variants of the present invention) may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl- Examples include cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-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.

[0035] The CARs of the present embodiments (including functional portions and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via a disulfide bond, or converted into an acid addition salt, and / or optionally dimerized or multimerized, or conjugated.

[0036] CARs (including functional portions and functional variants thereof) of the present embodiments can be obtained by methods known in the art. CARs can be produced by any suitable method for producing polypeptides or proteins. For example, CARs can be produced recombinantly using standard recombinant methods and the nucleic acids described herein. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4 th ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2012. Alternatively, the CARs described herein (including functional portions and functional variants thereof) can be commercially synthesized by companies such as, for example, Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA). In this regard, the CARs of the invention can be synthetic, recombinant, isolated, and / or purified.

[0037] Further provided by embodiments of the present invention are nucleic acids comprising a nucleotide sequence encoding any of the CARs described herein (including functional portions and functional variants thereof). The nucleic acids of the present invention may comprise a nucleotide sequence encoding any of the leader domain, hinge domain, antigen recognition domain, TM domain, and T cell activation domain described herein. In embodiments of the invention, the nucleic acid may comprise, consist of, or consist essentially of the nucleotide sequence of any one of SEQ ID NO:29 (FHVH74-CD828Z), SEQ ID NO:30 (FHVH32-CD828Z), SEQ ID NO:31 (FHVH33-CD828Z), SEQ ID NO:32 (FHVH93-CD828Z), SEQ ID NO:33 (FHVH74-CD8BBZ), SEQ ID NO:34 (FHVH32-CD8BBZ), SEQ ID NO:35 (FHVH33-CD8BBZ), SEQ ID NO:36 (FHVH93-CD8BBZ), SEQ ID NO:37 (FHVH74-CD8ICOSZ), SEQ ID NO:38 (FHVH32-CD8ICOSZ), SEQ ID NO:39 (FHVH33-CD8ICOSZ), and SEQ ID NO:40 (FHVH93-CD8ICOSZ).

[0038] "Nucleic acid," as used herein, includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally refers to a polymer of DNA or RNA, which may be single- or double-stranded, which may be synthetic or obtained (e.g., isolated and / or purified) from natural sources, and which may contain natural, non-natural, or modified nucleotides, including the nucleotides of unmodified oligonucleotides. Instead of the phosphodiester bond found between nucleotides, the nucleic acid may contain natural, non-natural, or modified internucleotide bonds, such as phosphoramidate or phosphorothioate bonds. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, in some instances, as discussed herein, it may be preferable for the nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0039] Nucleic acids of embodiments of the present invention may be recombinant. As used herein, the term "recombinant" refers to (i) molecules constructed outside a living cell by joining natural or synthetic nucleic acid segments into a nucleic acid molecule capable of replication in the living cell, or (ii) molecules resulting from replication as described in (i) above. For purposes herein, replication may be in vitro or in vivo.

[0040] Recombinant nucleic acids may have sequences that do not occur in nature or that are created by the artificial combination of two otherwise separate segments of sequence. This artificial combination is often achieved by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acid, by genetic engineering techniques such as those described in Green and Sambrook, supra. Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, e.g., Green and Sambrook, supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N-acetyl-3-methyl-4-methyl-5-methyl-1-methyl-2 ... 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 Examples of nucleic acids include, but are not limited to, -isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queusine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0041] The nucleic acid may comprise any isolated or purified nucleotide sequence encoding a CAR or any functional portion or variant thereof. Alternatively, the nucleotide sequence may comprise a nucleotide sequence that is degenerate to any of the sequences or a combination of degenerate sequences.

[0042] Also, embodiments of the present invention include nucleotide sequences that are complementary to, or that hybridize under stringent conditions to, the nucleotide sequences of any of the nucleic acids described herein. Isolated or purified nucleic acids are provided.

[0043] Nucleotide sequences that hybridize under stringent conditions can also hybridize under highly stringent conditions. "Highly stringent conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of a nucleic acid described herein) in an amount detectably greater than nonspecific hybridization. Highly stringent conditions include conditions that distinguish polynucleotides with exact complementary sequences, or those containing only a few scattered mismatches, from random sequences that happen to have a few small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity melt more easily than full-length complements of 14-17 or more bases, making them readily distinguishable by highly stringent hybridization. Relatively highly stringent conditions include, for example, low-salt and / or high-temperature conditions, such as those provided by about 0.02-0.1 M NaCl or its equivalent at a temperature of about 50-70°C. Such highly stringent conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting expression of any of the CARs of the present invention. It is generally understood that conditions can be made more stringent by adding increasing amounts of formamide.

[0044] The present invention also provides nucleic acids comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of the nucleic acids described herein.

[0045] In embodiments, the nucleic acids of the present invention can be incorporated into a recombinant expression vector. In this regard, embodiments of the present invention provide recombinant expression vectors comprising any of the nucleic acids of the present invention. For purposes herein, the term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and causes a host cell to express the mRNA, protein, polypeptide, or peptide when the vector is contacted with the cell under conditions sufficient to cause expression of the mRNA, protein, polypeptide, or peptide in the cell. The vectors of the present invention are not naturally occurring as a whole. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the present invention may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or modified nucleotides or internucleotide bonds do not interfere with the transcription or replication of the vector.

[0046] In embodiments, the recombinant expression vector of the present invention may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and propagation, or for expression, or both, such as plasmids and viruses. The vector may be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 may also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector may be a viral vector, such as a retroviral vector (e.g., a gamma retroviral vector) or a lentiviral vector.

[0047] In embodiments, the recombinant expression vectors of the invention are those described, for example, in Sambrook and Green, supra, can be prepared using standard recombinant DNA techniques. Circular or linear expression vector constructs can be prepared containing replication systems that function in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from ColEl, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0048] The recombinant expression vector may include regulatory sequences, such as transcriptional and translational initiation and termination codons, specific to the type of host cell into which the vector will be introduced (e.g., bacterial, fungal, plant, or animal), as needed, and considering whether the vector is DNA- or RNA-based. The recombinant expression vector may include restriction enzyme sites to facilitate cloning. In addition to the nucleic acid sequence of the invention encoding a CAR, the recombinant expression vector preferably includes expression control sequences, such as a promoter, enhancer, polyadenylation signal, transcription terminator, internal ribosome entry site (IRES), etc., that direct the expression of the nucleic acid sequence in the host cell.

[0049] The recombinant expression vector may contain one or more marker genes to allow for the selection of transformed or transfected host cells. Marker genes include biocide resistance, resistance to, e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to confer prototrophy, etc. Suitable marker genes for the expression vectors of the present invention include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0050] The recombinant expression vector may comprise a native or non-native promoter operably linked to a nucleotide sequence encoding a CAR (including functional portions and functional variants thereof) or to a nucleotide sequence complementary to or hybridizing to the nucleotide sequence encoding a CAR. For example, the selection of strong, weak, inducible, tissue-specific, and developmental stage-specific promoters is within the ordinary skill of one of ordinary skill in the art. Similarly, the combination of a nucleotide sequence with a promoter is also within the skill of one of ordinary skill in the art. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long terminal repeat of murine stem cell virus.

[0051] The recombinant expression vectors of the invention can be designed for either transient expression, stable expression, or both, and can be constructed for constitutive or inducible expression.

[0052] Additionally, the recombinant expression vector may be engineered to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of a cell in which it is expressed. A suicide gene may be a gene that confers sensitivity to an agent, e.g., a drug, on the cell in which it is expressed, causing the cell to die when contacted or exposed to the agent. Suicide genes are known in the art and can be used in a variety of applications, including, for example, Examples include the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleotide phosphorylase, and nitroreductase.

[0053] Embodiments of the present invention further provide host cells containing any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain a recombinant expression vector of the present invention. Host cells can be eukaryotic cells, such as plants, animals, fungi, or algae, or prokaryotic cells, such as bacteria or protozoa. Host cells can be cultured or primary cells, i.e., cells isolated directly from an organism, such as a human. Host cells can be adherent or suspension cells, i.e., cells that grow in suspension. Suitable host cells are known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For purposes of amplifying or replicating a recombinant expression vector, the host cell can be a prokaryotic cell, such as a DH5α cell. For purposes of producing a recombinant CAR, the host cell can be a mammalian cell. The host cell can be a human cell. The host cells may be of any cell type, may be derived from any type of tissue, and may be at any stage of development. The host cells may be peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs).

[0054] In an embodiment of the present invention, the host cell is a T cell. For purposes herein, a T cell may be any T cell, for example, a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell 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 T cell may be enriched or purified. The T cell may be a human T cell. The T cell may be a T cell isolated from a human. The T cell may be any type of T cell, at any stage of development, and may be CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD8 +These include, but are not limited to, T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, etc. T cells include CD8 + T cells or CD4 + It may be a T cell.

[0055] In an embodiment of the present invention, the host cells are natural killer (NK) cells. NK cells are a type of cytotoxic lymphocyte that plays a role in the innate immune system. NK cells are defined as large granular lymphocytes and constitute the third type of cell that differentiates from a common lymphoid progenitor cell that also gives rise to B and T lymphocytes (see, e.g., Immunobiology, 99(1):101-104). th ed., Janeway et al., eds., Garland Publishing, New York, NY (2016)). 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 some abnormal cells, such as some tumor cells and virus-infected cells, and are thought to be important in innate immune defense against intracellular pathogens. As described above with respect to T cells, NK cells can be any NK cell, for example, cultured NK cells, e.g., primary NK cells, or NK cells from a cultured NK cell line, or NK cells obtained from a mammal. If obtained from a mammal, NK cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. NK cells may be enriched or purified. 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 derivatives thereof.

[0056] Also provided by embodiments of the present invention are populations of cells comprising at least one host cell described herein. The population of cells may be a heterogeneous population comprising host cells comprising any of the described recombinant expression vectors in addition to at least one other cell, e.g., a host cell (e.g., a T cell) that does not comprise any of the recombinant expression vectors, or a cell other than a T cell, e.g., a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc. Alternatively, the population of cells may be a substantially homogeneous population (the population primarily comprises (e.g., consists essentially of) host cells comprising the recombinant expression vector). The population may also be a clonal population of cells, in which all cells in the population are clones of a single host cell comprising the recombinant expression vector, such that all cells in the population comprise the recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.

[0057] The recombinant expression vector of the invention encoding a CAR can be introduced into cells by "transfection," "transformation," or "transduction." As used herein, "transfection," "transformation," or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Many transfection techniques are known in the art, including, for example, calcium phosphate DNA co-precipitation; DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-promoted microprojectile bombardment; and strontium phosphate DNA co-precipitation. Phage or viral vectors may be introduced into host cells after propagation of infectious particles in suitable packaging cells, many of which are commercially available.

[0058] Conjugates, e.g., bioconjugates, comprising any of the CARs of the invention (including any functional portion or variant thereof), nucleic acids, recombinant expression vectors, host cells, or populations of host cells are within the scope of the invention. Conjugates, and generally methods for synthesizing conjugates, are known in the art.

[0059] The CAR (including functional portions and variants thereof), nucleic acids, recombinant expression vectors, and host cells (including populations thereof) (all of which are hereinafter collectively referred to as the "CAR material of the invention") may be isolated and / or purified. The term "isolated," as used herein, means removed from its natural environment. The terms "purified" or "isolated" do not require absolute purity or isolation, but are intended to be relative terms. Thus, for example, a purified (or isolated) host cell preparation is one in which the host cells are more pure than the cells in their natural environment in the body. Such host cells can be produced, for example, by standard purification techniques. In some embodiments, a preparation of host cells is purified such that the host cells represent at least about 50%, e.g., at least about 70%, of the total cell content of the preparation. For example, the purity can be at least about 50%, greater than about 60%, greater than about 70%, or greater than about 80%, or even about 100%.

[0060] The CAR materials of the present invention may be formulated into compositions, e.g., pharmaceutical compositions. In this regard, embodiments of the present invention provide pharmaceutical compositions comprising any of a CAR, functional portion, functional variant, nucleic acid, expression vector, or host cell (including populations thereof) and a pharmaceutically acceptable carrier. Pharmaceutical compositions of the present invention containing any of the CAR materials of the present invention may contain more than one CAR material of the present invention, e.g., a CAR and a nucleic acid, or two or more different CARs. Alternatively, pharmaceutical compositions may contain other pharmaceutically active agents or drugs, e.g., chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methionine, ribonucleotides ... It may also comprise the CAR material of the present invention in combination with trexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In a preferred embodiment, the pharmaceutical composition comprises the host cell of the present invention or a population thereof.

[0061] Preferably, the carrier is a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier may be any of those conventionally used for the particular CAR material of the present invention under consideration. Such pharmaceutically acceptable carriers are well known to those skilled in the art and are readily available to the public. Preferably, the pharmaceutically acceptable carrier has no harmful side effects or toxicity under the conditions of use.

[0062] The choice of carrier is determined in part by the specific CAR material of the present invention and the specific method used to administer the CAR material of the present invention. In a preferred embodiment, the CAR is expressed by host cells, preferably T cells or NK cells, and the CAR-expressing host cells are administered to a patient. These cells may be autologous or allogeneic to the recipient of the cells. A nucleic acid encoding a CAR may be introduced into the cells by any of a variety of genetic modification methods, including, but not limited to, gamma-retroviral, lentiviral, or transposon-based transduction. Various suitable formulations of the pharmaceutical composition of the present invention exist. Suitable formulations may include parenteral, subcutaneous, intravenous, intramuscular, intratumoral, intraarterial, intrathecal, or intraperitoneal administration. The CAR material of the present invention may be administered using more than one route, and in certain instances, certain routes may provide a more immediate and effective response than others.

[0063] Preferably, the CAR material of the present invention is administered, for example, by intravenous injection. When the CAR material of the present invention is a host cell expressing the CAR of the present invention (or a functional variant thereof), a pharmaceutically acceptable carrier for the cells for injection can be any isotonic carrier, such as normal saline (about 0.90% w / v NaCl in water, about 300 mOsm / L in water). NaCl, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or lactated Ringer's solution, etc. In embodiments, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0064] The compositions may use sustained-release, delayed-release, and sustained-release delivery systems so that the compositions of the present invention are delivered before and for a time sufficient to sensitize the treated area. Many types of release delivery systems are available and known to those skilled in the art. Such systems can avoid repeated administration of the composition, thereby increasing convenience for the subject and the physician, and may be particularly suitable for certain embodiments of the compositions of the present invention.

[0065] Without being bound by any particular theory or mechanism, it is believed that by eliciting an antigen-specific response against BCMA, the CARs of the present invention provide one or more of the following: targeting and destroying BCMA-expressing cancer cells, reducing or eliminating cancer cells, promoting the infiltration of immune cells into the tumor site(s), and enhancing / prolonging the anti-cancer response.

[0066] It is contemplated that the CAR materials of the present invention can be used in methods for treating or preventing diseases, such as cancer, in mammals. Without being bound by a particular theory or mechanism, the CARs of the present invention have biological activity, e.g., the ability to recognize an antigen, such as BCMA, such that when expressed by a cell, the CAR can mediate an immune response against cells expressing the antigen for which the CAR is specific, e.g., BCMA. In this regard, an embodiment of the present invention is a method for treating or preventing cancer in a mammal, comprising: Methods are provided that comprise administering to the mammal a therapeutically or prophylactically effective amount of any of the CARs, nucleic acids, recombinant expression vectors, host cells, populations of cells, and / or pharmaceutical compositions of the invention. In a preferred embodiment, the methods comprise injecting host cells transduced with the CARs of the invention into the mammal.

[0067] One or more isolated host cells expressing the BCMA CAR of the invention described herein may be contacted with a population of BCMA-expressing cancer cells ex vivo, in vivo, or in vitro. "Ex vivo" refers to a method performed in or on cells or tissues in an artificial environment outside of an organism with minimal alteration from their natural conditions. In contrast, the term "in vivo" refers to a method performed within a living organism in its normal, intact state, while "in vitro" methods are performed using components of an organism that have been isolated from its usual biological context. The methods of the invention preferably include ex vivo and in vivo components. In this context, for example, the isolated host cells may be contacted with an anti-BCMA CAR of the invention. The cells may be cultured ex vivo under conditions that express the CAR, and then directly transferred into a mammal (preferably a human) suffering from a BCMA-positive cancer, such as multiple myeloma. This cell transfer method is referred to in the art as "adoptive cell transfer (ACT)," in which immune-derived cells are transferred into a recipient to transfer the function of the immune-derived cells to the host. The immune-derived cells may be derived from the recipient or another individual. Adoptive cell transfer methods are used to treat various types of cancer, including hematological cancers such as myeloma.

[0068] After administering a composition comprising a host cell expressing a nucleic acid sequence encoding a CAR of the present invention or a vector comprising a nucleic acid sequence encoding a CAR of the present invention 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 methods of the present invention, the CAR binds to BCMA in cancer cells and destroys the cancer cells. Binding of the CAR to BCMA on the surface of cancer cells can be assayed using any suitable method known in the art, including, for example, ELISA (enzyme-linked immunosorbent assay) and flow cytometry. The ability of the CAR to destroy cells can be measured using any suitable method known in the art, such as the cytotoxicity assay 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 assaying the expression of certain cytokines, such as CD107a, IFNγ, IL-2, and TNF.

[0069] Embodiments of the present invention further include lymphodepleting the mammal prior to administering the CAR material of the present invention. Examples of lymphodepletion may include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.

[0070] For purposes of the methods of the present invention in which a host cell or population of cells is administered, the cells may be allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0071] An "effective amount" or "therapeutically effective amount" refers to a dose appropriate for preventing or treating cancer in an individual. Amounts effective for therapeutic or prophylactic use will depend, for example, on the stage and severity of the disease or disorder being treated, the age, weight, and general health of the patient, and the judgment of the prescribing physician. The size of the dose will also be determined by the particular CAR material selected, the method of administration, the timing and frequency of administration, the existence, nature, and extent of any adverse side effects that may accompany the administration of the particular CAR material, and the desired physiological effect. Various diseases or disorders (e.g., Those skilled in the art will appreciate that certain conditions (e.g., cancer) may require long-term treatment, possibly involving multiple administrations using the CAR material of the invention at each or various administration rounds. By way of example, and not intended to limit the invention, the dose of the CAR material of the invention may be about 0.001 to about 1000 mg / kg (body weight of the subject being treated) / day, about 0.01 to about 10 mg / kg (body weight) / day, or about 0.01 to about 1 mg / kg (body weight) / day. In embodiments of the invention, the dose is about 1 x 10 cells expressing a CAR of the invention per kg body weight. 4 ~Approx. 1×10 10 When the CAR material of the invention is a host cell, an exemplary dose of host cells is at least 1 million cells (1 mg cells / dose), e.g., 1 x 10 cells per kg of body weight. 9 When the CAR material of the present invention is a nucleic acid packaged in a virus, an exemplary dose of the virus may be 1 ng / administration.

[0072] For purposes of the present invention, the amount or dose of the CAR material of the present invention administered should be sufficient to produce a therapeutic or prophylactic response in a subject or animal over an appropriate time frame. For example, a dose of the CAR material of the present invention should be sufficient to bind to an antigen or detect, treat, or prevent a disease, such as cancer, for a period of about 2 hours or more, e.g., about 12 to about 24 hours or more, from the time of administration. In certain embodiments, the period may be even longer. The dose will be determined by the efficacy of the specific CAR material of the present invention and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) being treated.

[0073] For purposes of the present invention, an assay comprising, for example, comparing the extent to which target cells are lysed and / or IFN-γ is secreted by T cells expressing a CAR of the invention upon administration of a particular dose of such T cells to a mammal, between a set of mammals each receiving different doses of T cells, can be used to determine a starting dose to administer to a mammal. The extent to which target cells are lysed and / or IFN-γ is secreted upon administration of a particular dose can be assayed by methods known in the art.

[0074] When the CAR material of the present invention is administered with one or more additional therapeutic agents, the one or more additional therapeutic agents may be co-administered to a mammal. "Co-administration" means that the administration of the one or more additional therapeutic agents and the CAR material of the present invention is sufficiently close in time that the CAR material of the present invention can enhance the effect of the one or more additional therapeutic agents, or vice versa. In this regard, the CAR material of the present invention may be administered first and the one or more additional therapeutic agents may be administered second, or vice versa. Alternatively, the CAR material of the present invention and the one or more additional therapeutic agents may be administered simultaneously. An exemplary therapeutic agent that may be co-administered with the CAR material is IL-2. IL-2 is believed to enhance the therapeutic effect of the CAR material of the present invention. Without being bound by a particular theory or mechanism, it is believed that IL-2 enhances therapy by enhancing the in vivo expansion of the number of cells expressing the CAR of the present invention.

[0075] A mammal referred to herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, rodent mammals, e.g., mice and hamsters, and lagomorph mammals, e.g., rabbits. A mammal may be a feline mammal, including felines (cats) and canines (dogs). A mammal may be an artiodactyla mammal, including cattle and pigs, or an odd-toed ungulate mammal, including horses. A mammal may be a primate, ceboid, or simian (monkey), or an anthropoid (human and ape) mammal. Preferably, the mammal is a human.

[0076] In the context of the methods of the present invention, the cancer may be any cancer. In an embodiment of the present invention, the cancer is a BCMA-expressing cancer. In an embodiment of the present invention, the cancer is multiple myeloma or Hodgkin's lymphoma.

[0077] As discussed herein, multiple myeloma, also known as plasma cell myeloma or Kahler's disease, is a cancer of plasma cells, a type of white blood cell that is normally involved in producing antibodies (Raab et al., Lancet, 374:324-329 (2009)). Multiple myeloma affects 1 to 4 people per 100,000 per year. The disease is more prevalent in men and, for reasons that remain unclear, is twice as prevalent in African Americans as in Caucasians. Multiple myeloma is the least common hematologic malignancy (14%) and constitutes 1% of all cancers (Raab et al., supra). Treatment for multiple myeloma typically involves high-dose chemotherapy followed by hematopoietic stem cell transplantation (allogeneic or autologous), but multiple myeloma patients undergoing such treatment usually have a high relapse rate. As discussed above, BCMA is highly expressed by multiple myeloma cells (eg, Novak et al., supra; Neri et al., supra; Bellucci et al., supra; and Moreaux et al., supra).

[0078] Hodgkin lymphoma (formerly known as Hodgkin's disease) is a cancer of the immune system characterized by the presence of a multinucleated cell type called Reed-Sternberg cells. The two main types of Hodgkin lymphoma are classical Hodgkin lymphoma and nodular lymphocyte-predominant Hodgkin lymphoma. Currently, Hodgkin lymphoma is treated with radiation therapy, chemotherapy, or hematopoietic stem cell transplantation, and the choice of treatment depends on the patient's age and sex, as well as the stage, volume, and histological subtype of the disease. BCMA expression has been detected on the surface of Hodgkin lymphoma cells (see, e.g., Chiu et al., Blood, 109(2):729-739 (2007)).

[0079] The terms "treat" and "prevent," and derivatives thereof, as used herein, do not necessarily mean 100%, i.e., complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art will recognize as having potential benefit or therapeutic effect. In this regard, the methods of the present invention can provide any amount or level of treatment or prevention of cancer in a mammal. Furthermore, the treatment or prevention provided by the methods of the present invention can include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented, e.g., cancer. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, e.g., cancer, or its symptoms or conditions.

[0080] Another embodiment of the invention provides any of the CARs, nucleic acids, recombinant expression vectors, host cells, populations of cells, and / or pharmaceutical compositions described herein in connection with other aspects of the invention for use in a method of treating or preventing cancer in a mammal. Yet another embodiment of the invention provides the use of any of the CARs, nucleic acids, recombinant expression vectors, host cells, populations of cells, and / or pharmaceutical compositions described herein in connection with other aspects of the invention in the manufacture of a medicament for treating or preventing cancer in a mammal. The cancer may be any of the cancers described herein.

[0081] The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope. [Example]

[0082] The materials and methods used in Examples 1-10 are provided below.

[0083] Cell lines and primary cells Multiple myeloma (BCMA) + Cell lines H929, U266, and RPMI8226 were obtained from ATCC. BCMA-negative lung cancer cell line A549 was obtained from ATCC. BCMA-negative sarcoma cell lines were obtained from ATCC.

[0084] Prior to the following experiments, BCMA-K562 and K562 cells obtained from ATCC were transduced in the laboratory with the gene for full-length BCMA. Prior to the following experiments, NGFR-K562 and K562 cells were transduced in the laboratory with the gene for low-affinity nerve growth factor. The same gammaretroviral vector and method were used to transduce BCMA-K562 and NGFR-K562 cells.

[0085] Tissue samples or peripheral blood mononuclear cells (PMBCs) from six patients with multiple myeloma were designated Myeloma Patients 1 to 6. PBMCs from three subjects with myeloma were used, and the donors were designated Donor A, Donor B, and Donor C. Primary CD34 cells from three healthy donors were used. + Hematopoietic cells were also obtained. All human samples used were obtained from patients enrolled in clinical trials approved by the Institutional Review Board at the National Cancer Institute.

[0086] Construction of fully human heavy chain only (FHVH) CAR A series of CARs containing fully human heavy chain-only antigen recognition (FHVH) domains were prepared. The sequence of each CAR followed this pattern from the 5' to 3' end: the CD8α leader sequence, one of four single heavy chain variable region domains, and the hinge and transmembrane regions of the human CD8α molecule. The cytoplasmic portion of either CD28, 4-1BB, or the inducible T cell costimulatory (ICOS) molecule was then added, followed by the cytoplasmic portion of the CD3ζ molecule. The complete amino acid sequences of these CARs are provided in SEQ ID NOS: 17-28.

[0087] As shown in Figures 1A-1L, the four fully human heavy chain-only CAR antigen recognition domains were designated FHVH74, 32, 33, and 93. The CAR designations also include the CD8α hinge and transmembrane domains, the included costimulatory domain, and the CD3ζ domain. For example, FHVH74-CD828Z contains the FHVH74 antigen recognition domain, the CD8α-derived hinge and transmembrane domains, the CD28 costimulatory domain, and the CD3ζ T cell activation domain. The 11D5-3-CD828Z anti-BCMA CAR was used as a positive control.

[0088] These CARs were constructed, and the CAR nucleotide sequences were ligated into the MSGV gammaretroviral vector backbone using standard methods. The complete nucleotide sequences of the CARs are provided in SEQ ID NOS: 29-40. BCMA-specific variable heavy chain sequences were synthesized as GBLOCK fragments (Integrated DNA Technologies (IDT), Skokie, IL). Each synthesized fragment contained a GTC trinucleotide, an Ncol site, a CD8α leader sequence, an FHVH sequence, a portion of the CD8α hinge and transmembrane domain, a Blpl site, and a TATCGT hexanucleotide (provided as SEQ ID NOS: 41). GTC and TATCGT (SEQ ID NOS: 41) nucleotides were added to ensure complete terminal cleavage by Ncol and Blpl. The fragments were cleaved with Blpl and NCOI-HF (New England Biolabs, Ipswich, MA) at 37°C for 2 hours. The cleaved fragments were then purified using a QIAQUICK PCR purification kit (Qiagen). The fragment was ligated into Blpl / Ncol-HF digested and gel-purified MSGV vector backbone, which also contains other components of the CAR not included in the GBLOCK (Integrated DNA Technologies (IDT), Skokie, IL) fragment.

[0089] The CAR components included in the MSGV vector backbone were the remainder of the CD8α domain that was not included in the GBLOCK (Integrated DNA Technologies (IDT), Skokie, IL) fragment, sequences encoding the costimulatory domains of either CD28, 4-1BB, or ICOS, and the CD3ζ domain. By using the DNA Ligation Kit (Roche Applied Sciences), each GBLOCK (Integrated DNA Tech Ligation of the CAR fragment and the MSGV vector backbone fragment was performed using IDT (Institute of Technology, Skokie, IL).

[0090] CAR detection in T cells T cells transduced with one of the CAR vectors and untransduced T cells were washed and stained with phycoerythrin-labeled BCMA-Fc protein to detect cell surface CAR molecules. 0.5 million T cells were suspended in 50 mL of staining buffer, and a titrated amount of BCMA-Fc-PE reagent was added. CD3, CD4, and CD8 staining was also performed using standard methods. Dead cells were removed using 7-AAD (7-amino-acinomycin dye, BD Biosciences).

[0091] T cell culture PBMCs were thawed and washed in T cell medium containing AIM V medium (Invitrogen, Waltham, MA) + 5% AB serum (Valley Biomedical, Winchester, VA), 100 U / mL penicillin, and 100 μg / mL streptomycin. Prior to transduction, T cell medium + 50 ng / mL anti-CD3 monoclonal antibody OKT3 (Ortho, Bridgewater, NJ) and 300 IU / mL 1 × 10 in IL-2 6 PBMCs were suspended at a concentration of 1000 cells / mL. After transduction, T cells were maintained in T cell medium + IL-2.

[0092] Gammaretroviral transduction To generate replication-incompetent gammaretrovirus, the CAR-encoding plasmid was transfected into packaging cells together with a plasmid encoding the RD114 envelope protein. Gammaretroviral transduction of T cells was performed 2 days after initiation of T cell culture.

[0093] Interferon-γ and tumor necrosis factor-alpha ELISA 100,000 BCMAs +BCMA-negative target cells were mixed with 100,000 CAR-transduced T cells in 200 μL of AIM-V medium + 5% human serum in duplicate wells of a 96-well round-bottom plate. The plates were incubated at 37°C for 18-20 hours. After incubation, ELISA for interferon gamma (INFγ) was performed using standard methods (Pierce). ELISA for tumor necrosis factor alpha (TNF) was performed using standard methods (R&D).

[0094] CD107a assay Two tubes were prepared for each T cell culture tested. One tube contained BCMA-K562 cells, and the other tube contained NGFR-K562 cells. Both tubes contained CAR-transduced T cells, 1 mL of AIM-V medium + 5% human AB serum, a titrated concentration of anti-CD107a antibody (eBioscience, clone eBioH4A3), and 1 μL of GOLGI STOP (monesin, BD Biosciences, San Jose, CA). All tubes were incubated at 37°C for 4 hours and then stained for CD3, CD4, and CD8.

[0095] Flow cytometry For anti-BCMA staining, cells were stained with a polyclonal biotin-conjugated goat anti-human BCMA antibody (R&D Systems, catalog number BAF 193) followed by streptavidin (BD). Bone marrow cells were also stained with anti-CD38 (eBioscience). Bone marrow cells were also stained with anti-CD38 (eBioscience) and anti-CD56 (BD). FLOWJO software (Tree Star, Flow cytometry analysis of all experiments was performed using a cytometer (Cell Signaling Technology, Inc. Oregon, US).

[0096] Proliferation assay Co-cultures were established in 24-well plates. The target cells included in the co-cultures were 0.5 × 10 6irradiated BCMA-K562 cells or 0.5 × 10 6 Co-cultures also contained 1 x 10 T cells from cultures transduced with either anti-bcma2 or SP6. 6 Each co-culture contained 100 T cells. T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE, Invitrogen) as previously described. The medium used for co-culture was AIM V + 5% human AB serum. IL-2 was not added to the medium. After 4 days of initiation, viable cells in each co-culture were counted using trypan blue to exclude dead cells, and flow cytometry was performed.

[0097] Cytotoxicity assay BCMA on the survival of CCRF-CEM cells as a negative control + Cytotoxicity was measured by comparing target cell survival. Both of these cell types were mixed in the same tube with CAR-transduced T cells. CCRF-CEM negative control cells were labeled with the fluorescent dye 5-(and 6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR) (Invitrogen) and BCMA. + Target cells were labeled with CFSE. Co-cultures were set up in duplicate in 5 mL sterile tubes (BD) at multiple T cell to target cell ratios. The target cells contained 50,000 BCMA along with 50,000 CCRF-CEM negative control cells. + The cultures were incubated at 37°C for 4 hours. Immediately after incubation, 7AAD (7-amino-actinomycin D) (BD) was added and flow cytometry was performed. For each T cell + target cell culture, viable BCMA + BCMA by dividing the percentage of cells by the percentage of viable CCRF-CEM negative control cells. + The viability of target cells was determined. BCMA in each T cell + target cell culture + Target cell viability was assessed by BCMA immunoreactivity, without effector T cells. +Viable BCMA in tubes containing only target cells and CCRF-CEM cells + BCMA corrected by dividing the ratio of the percentage of target cells to the percentage of viable CCRF-CEM negative control cells + The viability of target cells was calculated. This correction was necessary to account for variability in starting cell numbers and natural death of target cells. Cytotoxicity was calculated as follows: BCMA + Target cell cytotoxicity rate = 100 - corrected BCMA + Target cell viability.

[0098] In vivo mouse model treatment experiments NSG mice (NOD.Cg-Prkdc) obtained from The Jackson Laboratory scid Il2rg tm1Wjl / SzJ) were used. Mice were intradermally injected with RPMI8226 cells. Tumors were allowed to grow for 10 days. Mice were then intravenously injected with human T cells transduced or not transduced with the CARs indicated in Example 9 (Figures 9B-9C) or Example 10 at the doses described in Example 9 (Figures 9B-9C) or Example 10. Tumors were measured every 3 days with a caliper. The tumor size (area) (mm ) was calculated by multiplying the longest length by the length perpendicular to the longest length. 2 ) were obtained. Mice were sacrificed when the longest length reached 15 mm. Animal experiments were approved by the Animal Care and Use Committee of the National Cancer Institute.

[0099] Example 1 This example demonstrates the design of a CAR with an antigen recognition domain only on the heavy chain.

[0100] As shown in Figures 1A-1L, we designed 12 CARs with fully human heavy chain-only antigen recognition domains. The general design of these CARs includes, from N- to C-terminus, a CD8a leader sequence, a fully human heavy chain variable region, the hinge and transmembrane domains of CD8a, the cytoplasmic portion of one of three costimulatory domains, and the cytoplasmic portion of the CD3ζ activation domain. The three costimulatory domains tested were CD28, 4-1BB, and inducible T cell costimulatory domain (ICOS). This example represents the first reported CAR with a heavy chain-only antigen recognition domain.

[0101] Example 2 This example demonstrates that a heavy chain-only CAR was expressed on the surface of T cells.

[0102] To conduct the experiment, primary human T cells from multiple myeloma (MM) patients were transduced with the heavy chain-only CARs shown in Figure 2. CAR surface expression was assessed by staining the cells with BCMA-Fc reagents followed by flow cytometry (Figure 2). As shown in Figure 2, all four FHVH CARs were constitutively expressed on the surface of T cells. For unknown reasons, the median fluorescence intensity of staining was slightly higher for the 11D5-3-CD828Z control CAR, which contains both the light and heavy chain variable regions.

[0103] Example 3 This example demonstrates that heavy chain-only CARs degranulated BCMA specifically.

[0104] We measured BCMA-specific degranulation of T cells expressing each of the four FHVH CARs shown in Figure 3 and the 11D5-3-CD828Z CAR. As shown in Figure 3, T cells transduced with each FHVH CAR specifically upregulated CD107a in response to stimulation with BCMA-expressing target cells, but not BCMA-negative target cells. T cells expressing 11D5-3 also upregulated CD107a (Figure 3). Upregulation of CD107a indicates BCMA-specific degranulation of T cells, which is part of the perforin-mediated cytotoxicity process.

[0105] Example 4 This example shows that T cells expressing a heavy chain-only CAR released cytokines in a BCMA-specific manner.

[0106] Primary human T cells from MM patients were evaluated for their ability to release interferon gamma (IFNγ) and tumor necrosis factor alpha (TNF) when cultured in vitro with various target cell lines. All FHVH CARs shown in Tables 1-3 were found to release these cytokines in a highly BCMA-specific manner, as listed in Tables 1-3, respectively.

[0107] [Table 1]

[0108] Referring to Table 1, cultured T cells were transduced with the indicated CARs and co-cultured with the indicated target cells (top row) overnight. After overnight incubation, standard ELISA assays were performed on the culture supernatants. BCMA-K562 and RPMI8226 expressed BCMA + NGFR-K562, CCRF-CEM, and 293GP are BCMA-negative. The percentage of T cells expressing the indicated CAR was determined by staining with BCMA-Fc-PE reagent followed by flow cytometry. %CAR + Figure 1 shows the CD3 cells transduced with each CAR stained with BCMA-Fc-PE reagent. + Percentage of cells stained with BCMA-Fc-PE reagent were non-transduced CD3 + Equal to the percentage of lymphocytes minus %CAR + All figures are in pg / mL of interferon gamma except for the column.

[0109] [Table 2]

[0110] Referring to Table 2, cultured T cells were transduced with the indicated CARs and cultured overnight with the indicated target cells (top row). After overnight incubation, standard ELISA assays were performed on the culture supernatants. BCMA-K562 inhibited the expression of BCMA. + All other targets are BCMA-negative. The percentage of T cells expressing the indicated CAR was determined by staining with BCMA-Fc-PE reagent followed by flow cytometry. %CAR + Figure 1 shows the CD3 cells transduced with each CAR stained with BCMA-Fc-PE reagent. + Percentage of cells stained with BCMA-Fc-PE reagent were non-transduced CD3 + Equal to the percentage of lymphocytes minus %CAR + All figures are in pg / mL of interferon gamma except for the column.

[0111] The results in Tables 1 and 2 show that the indicted CARs are BCMA + It shows that the target cells are specifically recognized.

[0112] [Table 3]

[0113] Referring to Table 3, cultured T cells were transduced with the indicated CARs and cultured overnight with the indicated target cells (top row). After overnight incubation, standard ELISA assays were performed on the culture supernatants. BCMA-K562 inhibited the expression of BCMA. + All other targets are BCMA negative. Staining with BCMA-Fc-PE reagent followed by flow cytometry The percentage of T cells expressing the indicated CAR was determined by cytometry. %CAR + Figure 1 shows the CD3 cells transduced with each CAR stained with BCMA-Fc-PE reagent. + Percentage of cells stained with BCMA-Fc-PE reagent were non-transduced CD3 + Equal to the percentage of lymphocytes minus %CAR+ All numbers are in pg / mL of interferon gamma except for the column.

[0114] The results in Table 3 show that FHVH-CD828Z or T cells expressing FHVH-CD828Z induce BCMA + It shows that the target cells are specifically recognized.

[0115] Example 5 This example demonstrates that T cells expressing a heavy chain-only CAR proliferated in a BCMA-specific manner in vitro.

[0116] Primary T cells expressing CFSE-tagged CARs from MM patients were transfected with irradiated BCMA + T cells expressing the 11D5-3-CD828Z CAR also proliferated in a BCMA-specific manner (Figure 4A). In addition to documenting BCMA-specific proliferation by CFSE dilution, BCMA-specific proliferation was also demonstrated by BCMA-negative target cells. + This was also demonstrated by the increase in the absolute number of CAR-expressing T cells cultured with target cells. As shown in Figure 4F, T cells were transfected with BCMA + CAR when cultured with target cells + The absolute number of T cells increased.

[0117] Example 6 This example demonstrates that T cells expressing only a heavy chain CAR can express BCMA + It is shown to kill target cells.

[0118] The ability of FHVH CAR T cells to kill BCMA+ target cells was evaluated. As shown in Figures 5A and 5B, FHVH33-CD828Z and FHVH33-CD8BBZ were shown to have the ability to kill BCMA+ RPMI226 cells compared to UT cells.

[0119] Example 7 This example demonstrates that only heavy chains with a 4-1BB costimulatory domain result in expressed and functional CARs.

[0120] Four fully human heavy chain-only CARs, shown in Figure 6, containing the 4-1BB costimulatory domain were constructed and evaluated. All four of these CARs were expressed on the surface of primary human T cells, but FHVH33-CD8BBZ consistently had the highest expression (Figure 6) and was therefore selected for further testing. Results from functional evaluation of FHVH33-CD8BBZ are shown in Figures 7A-7F. These CAR-expressing T cells produced IFNγ and degranulated in a BCMA-specific manner.

[0121] Example 8 This example demonstrates that anti-BCMA CAR-transduced T cells recognize primary multiple myeloma cells.

[0122] T cells either untransduced or expressing the FHVH33-CD828Z or FHVH33-CD8BBZ CAR were incubated with autologous bone marrow myeloma cells (90% pure) for 4 hours. Upregulation of CD107a was measured as a marker of T cell degranulation, and co-expression of CD8 or CD4 was also measured. The percentage of cells with the indicated phenotype is shown in Table 4. As shown in Table 4, FHVH33-CD T cells transduced with 828Z or FHVH33-CD8BBZ CAR upregulated CD107a expression after co-culture with target bone marrow myeloma cells.

[0123] [Table 4]

[0124] T cells, either untransduced (UT) or expressing the FHVH33-CD828Z or FHVH33-CD8BBZ CAR, were incubated overnight with autologous bone marrow myeloma cells (90% pure) or control PBMCs. Interferon-gamma release was measured using a standard ELISA assay. The results are shown in Figure 8. As shown in Figure 8, cells transduced with the FHVH33-CD828Z or FHVH33-CD8BBZ CAR secreted IFNγ after being co-cultured with target bone marrow myeloma cells.

[0125] Example 9 This example shows the titration of doses of FHVH33-CD8BBZ-expressing T cells in mice.

[0126] As shown in Figure 9A, NSG mice were intradermally injected with RPMI8226 cells. Tumors were allowed to grow for 10 days. On day 0, the mice were intravenously injected with the indicated number of FHVH33-CD8BBZ-expressing T cells. Mice were administered one of three different doses of FHVH33-CD8BBZ CAR T cells, while another group of mice was left untreated. All mice had established tumors at the time of T cell injection.

[0127] As shown in Figure 9B, 2.2 × 10 6 FHVH33-CD8BBZ T cells were able to eradicate tumors from all mice, and the efficacy of FHVH33-CD8BBZ CAR T cells decreased in a dose-dependent manner. Tumors progressed in all untreated mice (n=5 mice / group).

[0128] The survival rate of mice is shown in Figure 9C. 6 All mice that received FHVH33-CD8BBZ-expressing T cells survived and remained healthy throughout the experiment.

[0129] Example 10 This example demonstrates eradication of BCMA+ tumors in NSG mice.

[0130] Mice were injected intradermally with RPMI8226 cells. Tumors were allowed to grow for 10 days. On day 0, 1 x 10 T cells expressing SP6-CD828Z, 11D5-3-CD8BBZ, FHVH33-CD8BBZ, or FHVH33-CD828Z CARs were injected intradermally. 6 Mice were either intravenously injected with the compound or left untreated.

[0131] As shown in Figure 10A, mice injected with T cells expressing the negative control SP6-CD828Z CAR and untreated mice developed progressive tumor growth. Tumors were eradicated in mice receiving T cells expressing either the 11D5-3-CD8BBZ, FHVH33-CD8BBZ, or FHVH33-CD828Z CAR.

[0132] The survival rates of the mice are shown in Figure 10B. Mice that received T cells expressing either the 11D5-3-CD8BBZ, FHVH33-CD8BBZ, or FHVH33-CD828Z CAR survived.

[0133] Example 11 This example shows that T cells expressing a heavy chain-only CAR release IFN-gamma in a BCMA-specific manner.

[0134] Effector T cells were cultured overnight with the target cells listed in Table 5. Effector T cells were either untransduced T cells, T cells transduced with a nucleotide sequence encoding FHVH33-CD828Z, or T cells transduced with a nucleotide sequence encoding FHVH33-CD8BBZ. All T cells were derived from the same human donor. FHVH33-CD828Z-transduced T cells resulted in 71% CAR expression. FHVH33-CD8BBZ-transduced T cells resulted in 80% CAR expression.

[0135] BCMA+ target cells were BCMA-K562 and RPMI8226. BCMA-negative target cells were Panc10.05, U251, 293GP, primary normal human bronchial epithelial cells (NHBE), primary human capillary endothelial cells (HMVEC), and primary human intestinal epithelial cells (InEpC).

[0136] An ELISA for interferon (IFN) gamma was performed, and the results are shown in Table 5. Interferon gamma production by effector T cells alone is also shown in Table 5. All values ​​in Table 5 are in pg / mL of IFN gamma.

[0137] [Table 5]

[0138] As shown in Table 5, CAR T cells produced more interferon gamma in the presence of BCMA+ targets.

[0139] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference.

[0140] In the context of describing the present invention (particularly in the context of the claims which follow), use of the terms "a," "an," "the," and "at least one," and similar referents should be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item (A or B) selected from the listed items or any combination of two or more of the listed items (A and B), unless otherwise specified herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are used in conjunction with "comprising," "having," "including," and "containing," among others. Unless otherwise expressly stated, terms should be construed as open-ended (i.e., meaning "including, but not limited to"). The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method for individually referencing each separate value within the range, unless otherwise specified herein, and each separate value is incorporated into the specification as if it were individually listed herein. All methods described herein can be performed in any suitable order unless otherwise specified herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to further elucidate the invention and does not pose a limitation on the scope of the invention, unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0141] Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of the preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors anticipate that such variations will be employed by those skilled in the art, and the inventors intend 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 as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. A chimeric antigen receptor (CAR) comprising an antigen recognition domain, a transmembrane (TM) domain, and a T cell activation domain, the chimeric antigen receptor having antigen specificity for B cell maturation antigen (BCMA), wherein the antigen recognition domain is (a) SEQ ID NOs: 1 to 3; (b) SEQ ID NOs: 4 to 6; (c) SEQ ID NOs: 7 to 9, or (d) SEQ ID NOs: 10 to 12 A CAR comprising the amino acid sequence:

2. The CAR of claim 1, wherein all domains of the CAR are human.

3. The CAR according to claim 1 or 2, wherein the antigen recognition domain does not include a linker peptide having a length of about 8 to about 40 amino acid residues.

4. The CAR according to any one of claims 1 to 3, wherein the CAR does not comprise an antibody light chain variable region.

5. The antigen recognition domain is (a) SEQ ID NO: 13; (b) SEQ ID NO: 14; (c) SEQ ID NO: 15, or (d) SEQ ID NO: 16 The CAR according to any one of claims 1 to 4, comprising an amino acid sequence as follows:

6. The CAR of any one of claims 1 to 5, wherein the T cell activation domain comprises a T cell signaling domain of any one of the following proteins: human CD28 protein, human CD3-zeta protein, human FcRγ protein, CD27 protein, OX40 protein, human 4-1BB protein, human inducible T cell costimulatory protein (ICOS), a modified version of any of the foregoing, or any combination of the foregoing.

7. The CAR according to any one of claims 1 to 6, comprising an amino acid sequence of any one of SEQ ID NOs: 17 to 28.

8. A nucleic acid comprising a nucleotide sequence encoding the CAR according to any one of claims 1 to 7.

9. The nucleic acid according to claim 8, comprising the nucleotide sequence of any one of SEQ ID NOs: 29 to 40.

10. A vector comprising the nucleic acid of claim 8 or 9.

11. An isolated host cell comprising the vector of claim 10.

12. The isolated host cell of claim 11 , wherein the host cell is a T cell.

13. The isolated host cell of claim 11 , wherein the host cell is a natural killer (NK) cell.

14. A population of cells comprising at least one host cell according to any one of claims 11 to 13.

15. 15. The CAR of any one of claims 1 to 7, the nucleic acid of claim 8 or 9, the vector of claim 10, the host cell of any one of claims 11 to 13, or the population of claim 14, for use in a method for treating or preventing cancer in a mammal.

16. 16. The CAR, nucleic acid, vector, or host cell for use according to claim 15, wherein the cancer is multiple myeloma or Hodgkin's lymphoma.

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