Human domain-containing anti-B cell maturation antigen chimeric antigen receptor

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 of 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] Description of federally supported research or development This invention was made with government support from the National Institutes of Health and the National Cancer Institute under project number ZIABC01143905. The government reserves certain rights in this invention.

[0003] References to electronically submitted literature The computer-readable nucleotide / amino acid sequence listing submitted concurrently with this specification and identified below is incorporated herein by reference in its entirety: a 67,061-byte ASCII (text) file named "739534_ST25.TXT" dated June 25, 2018. [Background technology]

[0004] Cancer is a public health concern. Despite advances in treatments such as chemotherapy, many cancers have poor prognoses. For example, while multiple myeloma (MM) can go into remission with treatment, many patients eventually relapse and die. Therefore, there is an unmet need for further cancer treatments. [Overview of the project]

[0005] Embodiments of the present invention provide 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 sequences 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 present invention provide related nucleic acids, recombinant expression vectors, host cells, cell populations, and pharmaceutical compositions related to the CARs of the present invention.

[0007] Additional embodiments of the present invention provide related methods for treating or preventing cancer in mammals. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] [Figure 1-1] Figures 1A - 1D are diagrams showing CARs containing only the antigen recognition domain of the complete human heavy chain 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 co-stimulatory molecule, and the cytoplasmic portion of the CD3ζ T cell activation domain. [Figure 1-2] Figures 1E - 1H are diagrams showing CARs containing only the antigen recognition domain of the complete human heavy chain 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 co-stimulatory molecule, and the cytoplasmic portion of the CD3ζ T cell activation domain. [Figure 1-3] Figures 1I - 1L are diagrams showing CARs containing only the antigen recognition domain of the complete human heavy chain 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 co-stimulatory protein (ICOS), and the cytoplasmic portion of the CD3ζ T cell activation domain. [Figure 2]Figure 2 is a series of graphs showing experimental data demonstrating that the four FHVH CARs shown were expressed by primary human T cells, as described in Example 2. Non-transduced (UT) T cells were included as a negative control, and 11D5-3-CD828Z was provided as the positive control CAR. T cells were transduced on day 2 of culture, and the cells were stained with BCMA-Fc protein reagent on day 7 of culture. The plots represent gated live lymphocytes. The numbers on the plots represent the percentage of CD3+ cells expressing the CAR (top) or not expressing the CAR (bottom). [Figure 3] Figure 3 shows a series of graphs representing experimental data demonstrating BCMA-specific degranulation by T cells expressing FHVH CARs. These graphs show the results for primary human T cells transduced with one of four FHVH CARs in a CD107a degranulation assay to evaluate antigen-specific function. As described in Example 3, T cells expressing each CAR degranulated more 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 was provided as a positive control CAR. The plots represent gated CD3+ lymphocytes. The numbers on the plots represent the percentage of CD3+ cells upregulating CD107a (top) or not upregulating CD107a (bottom). [Figure 4-1] Figure 4A is a graph showing experimental data demonstrating that the 11D5-3-CD828Z CAR proliferated specifically to BCMA. The plots represent gated live CD3+ lymphocytes. The white histograms represent CAR+ T cells stimulated with BCMA-K562 (BCMA-expressing) target cells, and the black histograms represent 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 the FHVH74-CD828Z(B) or FHVH32-CD828Z(C) CAR proliferated specifically to BCMA, as described in Example 5. [Figure 4-2] Figures 4D-4E are graphs showing experimental data demonstrating that FHVH33-CD828Z(D) or FHVH93-CD828Z(E)CAR proliferated specifically in BCMA, as described in Example 5. Figure 4F is a graph showing experimental data demonstrating that the absolute number of CAR+ T cells increased when T cells transduced with the displayed CAR were cultured together with BCMA+ target cells. When CAR T cells were cultured together with BCMA-K562 cells, the number of CAR+ T cells increased for all T cells expressing CAR. The Y-axis represents the number of CAR+ T cells (×10⁶). The X-axis represents the number of days the T cells were cultured together with BCMA+ target cells. [Figure 5] Figure 5A is a graph showing experimental data illustrating the ability of the FHVH33-CD828Z CAR to kill BCMA+ target cells compared to the ability of UT cells to kill BCMA+ target cells. T cells expressing the FHVH33-CD828Z CAR were cultured in vitro with RPMI8226 target cells for 4 hours at the effector-to-target ratio shown. Cytotoxicity was determined by duplication. Results are presented as the mean + / - standard error. 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 showing experimental data illustrating the ability of the FHVH33-CD8BBZ CAR to kill BCMA+ target cells compared to the ability of UT cells to kill BCMA+ target cells. T cells expressing the FHVH33-CD8BBZ CAR were cultured in vitro with RPMI8226 target cells for 4 hours at the effector-to-target ratio shown. Cytotoxicity was determined by duplication. Results are presented as the mean + / - standard error. The Y-axis represents the cytotoxicity % of 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 represent gated live lymphocytes. The numbers on the plots represent the percentage of cells stained with BCMA-Fc (top number) or unstained (bottom number). [Figure 7-1] Figures 7A-7B show the expression of this CAR in UT cells (A) compared to T cells (B) expressing FHVH33-CD8BBZ. The plots represent gated live CD3+ lymphocytes. The numbers in the plots are the percentages for BCMA-PE+ (top) and BCMA-PE- (bottom). [Figure 7-2] Figures 7C-7F show experimental data demonstrating that T cells transduced with FHVH33-CD8BBZ underwent BCMA-specific degranulation when evaluated by CD107a staining. This data compares the CD107a upregulation in UT+BCMA-K562(C) and UT+NGFR-K562 cells(D) with that in FHVH33+BCMA-K562(E) and FHVH33+NGFR-K562 cells(F). The same T cell cultures as those presented in Figures 7A-7B were used. The plots represent gated live CD3+ lymphocytes. The numbers in the plots represent the percentages of CD107a+ (top) and CD107a- (bottom). [Figure 7-3]Figures 7C-7F show experimental data demonstrating that T cells transduced with FHVH33-CD8BBZ underwent BCMA-specific degranulation when evaluated by CD107a staining. This data compares the CD107a upregulation in UT+BCMA-K562(C) and UT+NGFR-K562 cells(D) with that in FHVH33+BCMA-K562(E) and FHVH33+NGFR-K562 cells(F). The same T cell cultures as those presented in Figures 7A-7B were used. The plots represent gated live CD3+ lymphocytes. The numbers in the plots represent the percentages of CD107a+ (top) and CD107a- (bottom). [Figure 7-4] Figure 7G shows experimental data demonstrating that CAR-expressing T cells specifically produced IFNγ in a BCMA-specific manner. When T cells were cultured with 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 (pg / mL) of IFNγ secreted by non-transduced (UT) or transduced T cells with FHVH33-CD828Z or FHVH33-CD8BBZ CAR 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 dose titer measurement of FHVH33-CD8BBZ T cells in mice. Female (F) 7-8 week (wk) aged NSG mice were intradermally (id) injected with 8 million (M) RPMI8226 cells, and tumors were allowed to grow for 10 days. On day 0, varying numbers of FHVH33-CD8BBZ-expressing T cells were intravenously (IV) injected into the mice. Tumors were measured every 3 days (d). [Figure 9-2]Figure 9B is a graph showing the tumor volume (mm3) measured in mice treated with 0.2 × 10⁶ (black triangles), 0.7 × 10⁶ (black circles), or 2.2 × 10⁶ (white circles) FHVH33-CD8BBZ-expressing T cells as shown in Figure 9A, at the indicated number of days after CAR T cell injection. Untreated mice are represented by white triangles. Figure 9C is a graph showing the survival rate of mice shown in Figure 9B after treatment with 0.2 × 10⁶ (black triangles), 0.7 × 10⁶ (black circles), or 2.2 × 10⁶ (white circles) FHVH33-CD8BBZ-expressing T cells, at the indicated number of days after CAR T cell injection. Untreated mice are represented by white triangles. [Figure 10-1] Figure 10A is a graph showing tumor volume (mm3) measured in mice treated with T cells expressing SP6-CD828Z (triangle), 11D5-3-CD8BBZ (square), FHVH33-CD8BBZ (white circle), or FHVH33-CD828Z (black circle) CARs, at the indicated number of days after CAR T cell injection. Untreated mice are represented by diamonds. [Figure 10-2] Figure 10B is a graph showing the survival rates of mice treated with T cells expressing SP6-CD828Z (triangle), 11D5-3-CD8BBZ (square), FHVH33-CD8BBZ (white circle), or FHVH33-CD828Z (black circle) CARs, at the indicated number of days after CAR T cell injection. Untreated mice are represented by diamonds. [Modes for carrying out the invention]

[0009] Embodiments of the present invention provide a CAR comprising an antigen recognition domain, a TM domain, and a T cell activation domain, wherein the CAR has antigen specificity to BCMA. The CAR is an artificially constructed hybrid protein or polypeptide containing an antigen recognition domain of an antibody linked to a T cell signaling domain or a T cell activation domain. The CAR has the ability to redirect the specificity and responsiveness of T cells to a selected target in a non-MHC-restricted manner by utilizing the antigen-binding properties of a monoclonal antibody. Non-MHC-restricted antigen recognition recognizes antigens independently of antigen treatment, thereby conferring the ability of T cells expressing the CAR to bypass the main mechanism of tumor escape. Furthermore, when expressed in T cells, the CAR advantageously does not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

[0010] The CAR of the present invention has antigen specificity for the 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 activator (BAFF) and proliferation-inducing ligand (APRIL) (see, e.g., Mackay et al., above, and Kalled et al., Immunological Reviews, 204:43-54 (2005)). Among benign cells, BCMA has been reported to be expressed mostly in a subset of plasma cells and mature B cells (see, for example, Laabi et al., EMBO J., 11(11):3897-3904 (1992); Laabi et al., Nucleic Acids Res., 22(7):1147-1154 (1994); Kalled et al., see above; 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 is universally detected in multiple myeloma cells, and BCMA protein has been detected on the surface of plasma cells from multiple myeloma patients by several researchers (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 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] When used herein, the phrases "having antigen specificity" and "eliciting an antigen-specific response" mean that the CAR specifically binds to the antigen and can immunologically recognize the antigen in such a way that the binding of the CAR to the antigen elicits an immune response.

[0012] The CAR of the present invention can provide one or more of the following advantages. For example, the CAR of the present invention can reduce anti-CAR immunogenicity. A CAR containing one or both of a non-human domain (e.g., a mouse domain) and / or an artificial linker peptide may elicit an anti-CAR immune response when administered to a patient. Such an anti-CAR immune response is This may reduce the persistence of CAR-expressing cells and reduce or eliminate the effectiveness of CAR therapy. While not bound by any specific theory or mechanism, it is believed that one or more of the following characteristics of the CAR 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 artificial linker peptides, such as linker peptides having a length of about 10 to about 25 amino acid residues and consisting of one or more of glycine, serine, and threonine; (iii) the CAR does not contain antibody light chain variable regions; and (iv) the antigen recognition domain contains only a single antibody heavy chain variable region. It is believed that reducing or eliminating potential sources of anti-CAR immunogenicity improves the persistence of CAR-expressing cells and the effectiveness of CAR therapy. Furthermore, one or more of the aforementioned characteristics (ii) to (iv) can facilitate the preparation of CARs targeting one or more different antigens (other than BCMA) in addition to BCMA.

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

[0014] Anti-CAR immunogenicity is reduced by the present invention if the immune response to CARs according to the present invention is quantitatively or qualitatively attenuated compared to the immune response to conventional CARs. A quantitative reduction in anti-CAR immunogenicity includes a reduction in the magnitude or degree of the anti-CAR immune response. The magnitude or degree of anti-CAR immunogenicity can be measured based on any number of known parameters, such as a decrease in cytokine (e.g., CAR-specific cytokine) production levels (cytokine concentration), a decrease in the number of activated (e.g., lymphocyte (e.g., CAR-specific lymphocyte)) or mobilized lymphocytes, and / or a decrease in antibody (CAR-specific antibody) production (antibody concentration), a decrease in the ability of host (recipient) T cells to kill CAR-expressing T cells, etc. A qualitative reduction in anti-CAR immunogenicity includes any change in the nature of the anti-CAR immune response that reduces the effectiveness of the anti-CAR immune response in mediating the reduction of the cytotoxic activity of CARs. 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 a decrease in the production of one or more cytokines, such as IFN-γ, TNF-α, and granzyme B, and / or a decrease in the stimulation of cell-mediated anti-CAR immune responses, such as a decrease in the proliferation and activation of CAR-specific T cells and / or macrophages compared to those obtained with conventional CARs. Reduced anti-CAR immunogenicity may be characterized by one or more of the following: decreased stimulation of anti-CAR T cells, decreased proliferation of anti-CAR T cells, decreased secretion of IFNγ and / or granzyme B by anti-CAR T cells, and a decrease in the 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. As used herein, the term "anti-CAR immunogenicity" refers to the immune response to the CAR itself and not to any aspect of the immune response to the target antigen BCMA that the CAR may provide.

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

[0016] In the embodiment, the antigen recognition domain includes the CDR1, CDR2, and CDR3 regions of the heavy chain variable region of the human anti-BCMA antibody. In relation thereto, in the embodiment of the present invention, the antigen recognition domain is (a) One or more of the following: the heavy chain CDR1 region containing SEQ ID NO: 1, the heavy chain CDR2 region containing SEQ ID NO: 2, and the heavy chain CDR3 region containing SEQ ID NO: 3 (the CDR region of the FHVH74 heavy chain variable region); (b) One or more of the following: the heavy chain CDR1 region containing SEQ ID NO: 4, the heavy chain CDR2 region containing SEQ ID NO: 5, and the heavy chain CDR3 region containing SEQ ID NO: 6 (the CDR region of the FHVH32 heavy chain variable region); (c) One or more of the heavy chain CDR1 region containing SEQ ID NO: 7, the heavy chain CDR2 region containing SEQ ID NO: 8, and the heavy chain CDR3 region containing SEQ ID NO: 9 (CDR region of the FHVH33 heavy chain variable region); or (d) One or more of the following: the heavy chain CDR1 region containing SEQ ID NO: 10, the heavy chain CDR2 region containing SEQ ID NO: 11, and the heavy chain CDR3 region containing SEQ ID NO: 12 (CDR region of the FHVH93 heavy chain variable region). It may include. Preferably, the antigen recognition domain includes (a) all of SEQ ID NOs: 1-3, (b) all of SEQ ID NOs: 4-6, (c) all of SEQ ID NOs: 7-9, or (d) all of SEQ ID NOs: 10-12.

[0017] In embodiments of the present invention, the antigen recognition domain includes the heavy chain variable region of a human anti-BCMA antibody. In this regard, the antigen recognition domain may include 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 contain a linker peptide. The antigen recognition domain of a conventional CAR may consist of a single-stranded variable fragment (scFv). The scFv is a monovalent molecule containing 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. One or more of the following features of the CAR of the present invention can advantageously reduce or eliminate potentially immunogenic linkers that link different components of the CAR, for example, two potentially immunogenic linkers that link the linker peptide to the VL and VH of the scFv used in conventional CARs: (i) no linker peptide, such as those typically found in scFvs used in conventional CARs; (ii) no antibody light chain variable region (also used in conventional CARs); and (iii) only a single antibody heavy chain variable region. The linkers and linker peptides are artificial sequences not normally found in humans and therefore may be immunogenic. Alternatively, one or more of the aforementioned features (i) to (iii) can eliminate any potentially immunogenic regions in either or both of the peptide linker and the antibody light chain variable region. The purpose of a 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 arbitrary. The amino acid sequence includes the following. In embodiments 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 embodiments of the present invention, the antigen recognition domain does not contain a linker peptide having a length of about 8 to about 40 amino acid residues. For example, the linker peptide may contain one or more of glycine, serine, and threonine, with or without other amino acid residues. In embodiments of the present invention, the antigen recognition domain has a length of about 8 to about 40 amino acid residues and does not contain a linker peptide consisting of one or more of glycine, serine, and threonine.

[0019] In another embodiment, the CAR of the present invention includes a leader domain. The leader domain may be located at the amino terminus of an antigen recognition domain (e.g., the heavy chain variable region of an anti-BCMA antibody). The leader domain may include 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 includes a hinge domain. Those skilled in the art will understand that the hinge domain is a short amino acid sequence that promotes the flexibility of the antibody (e.g., Woof See 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 be derived from or contain any suitable sequence obtained from any suitable molecule. Preferably, the hinge domain contains a human sequence. In one embodiment, for example, the hinge domain is part of a human CD8α molecule or a human CD28 molecule.

[0021] The CAR may contain a TM domain. The TM domain may be 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 contain a TM domain from a human CD8α molecule or a human CD28 molecule. CD8 is a TM glycoprotein that functions as a co-receptor to the T cell receptor (TCR) and is mainly expressed on the surface of cytotoxic T cells. The most common form of CD8 is a dimer composed of CD8α and CD8β chains. CD28 is expressed on T cells and provides the co-stimulatory signals necessary for T cell activation. CD28 is a receptor for CD80(B7.1) and CD86(B7.2).

[0022] CAR may contain a T cell activation domain. The T cell activation domain may contain an intracellular (i.e., cytoplasmic) T cell signaling domain. The intracellular T cell signaling domain can be obtained from or derived from the CD28 molecule, the CD3 zeta (ζ) molecule, the Fc receptor gamma (FcRγ) chain, the CD27 molecule, the OX40 molecule, the 4-1BB molecule, the inducible T cell costimulatory protein (ICOS), or other intracellular signaling molecules known in the art, or modified versions of any of the above. As discussed above, CD28 is an important T cell marker in T cell costimulatory stimulation. CD3ζ associates with the TCR to generate a signal and contains an immune receptor tyrosine activation motif (ITAM). 4-1BB, also known as CD137, transmits 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 preferred embodiments, CD28, CD3 zeta, FcRγ, ICOS, 4-1BB, OX40, and CD27 are human.

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

[0024] In one embodiment, the CAR of the present invention comprises, from the amino terminus to the 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, the CAR of the present invention comprises, from the amino terminus to the 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, the CAR of the present invention comprises, from the amino terminus to the 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. Further embodiments of the present invention provide CARs comprising, or essentially comprising, any one of the amino acid sequences of SEQ ID NOs. 17 to 28. The components of the CARs of SEQ ID NOs. 17 to 28 are listed 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. Therefore, the CAR of the present invention may have lower anti-CAR immunogenicity compared to a CAR containing one or more of the non-human leader domain, non-human hinge domain, non-human antigen recognition domain, non-human TM domain, and non-human T cell activation domain, as described herein in relation to other embodiments of the present invention.

[0027] The functional portions of the CARs of the present invention as described herein are within the scope of the invention. When used in relation to a CAR, the term “functional portion” refers to any portion or fragment of the CAR of the present invention that retains the biological activity of the CAR from which the portion or fragment is part (parent CAR). A functional portion includes, for example, a portion of the CAR that retains the ability to recognize target cells or to detect, treat, or prevent disease to the same degree, the same degree, or a higher degree as the parent CAR. Based on the parent CAR, a functional portion may include, 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] The functional portion may contain additional amino acids at its amino or carboxyl terminus or at both ends, and these additional amino acids are not found in the amino acid sequence of the parent CAR. Preferably, the additional amino acids do not interfere with the biological function of the functional portion, such as recognizing target cells, detecting cancer, or treating or preventing cancer. More preferably, the additional amino acids enhance the biological activity compared to that of the parent CAR.

[0029] Functional variants of the CARs of the present invention described herein are within the scope of the invention. The term “functional variant,” as used herein, means a CAR, polypeptide, or protein having substantial or significant sequence identity or similarity to the parent CAR, wherein 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 the same degree, the same degree, or a higher degree as the parent CAR. With respect to the parent CAR, the functional variant may, for example, have amino acid sequence identity with the parent CAR by 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.

[0030] A functional mutant may, for example, include the amino acid sequence of the parent CAR having at least one conserved amino acid substitution. Alternatively, the functional mutant may further include the amino acid sequence of the parent CAR having at least one non-conserved amino acid substitution. In this case, it is preferable that the non-conserved amino acid substitution neither interferes with nor inhibits the biological activity of the functional mutant. The non-conserved amino acid substitution may enhance the biological activity of the functional mutant, resulting in increased biological activity compared to the parent CAR.

[0031] The amino acid substitutions of CARs in 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 an amino acid having certain 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 may include the substitution of an acidic / negatively charged polar amino acid with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), the substitution of an amino acid with a nonpolar side chain with another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), the substitution of a basic / positively charged polar amino acid with another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), the substitution of an uncharged amino acid with a polar side chain with another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), the substitution of an amino acid with a beta-branched side chain with another amino acid with a beta-branched side chain (e.g., Ile, Thr, and Val), and the substitution of an amino acid with an aromatic side chain with another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr).

[0032] CAR may essentially consist of specific amino acid sequences described herein, such that other components, for example, other amino acids, do not substantially alter the biological activity of the functional mutant.

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

[0034] The CARs of embodiments of the present invention (including the functional parts and functional variants of the present invention) may contain synthetic amino acids instead 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'-methyllysine, 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 (including functional moieties and functional variants) of embodiments of the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via disulfide bonds, etc., or converted to acid addition salts, and / or optionally dimerized, polymerized, or conjugated.

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

[0037] Furthermore, embodiments of the present invention provide nucleic acids comprising nucleotide sequences encoding any of the CARs (including their functional parts and functional variants) described herein. The nucleic acids of the present invention may comprise nucleotide sequences encoding any of the leader domain, hinge domain, antigen recognition domain, TM domain, and T cell activation domain described herein. In embodiments of the present invention, the nucleic acid may contain, consist of, or essentially consist of any one of the nucleotide sequences 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] As used herein, "nucleic acid" includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally means polymers of DNA or RNA, which may be single-stranded or double-stranded, may be synthesized or obtained from natural sources (e.g., isolated and / or purified), may contain natural, non-natural, or modified nucleotides, and may also include unmodified oligonucleotides. Instead of the phosphodiesters found between the nucleotides, natural, unnatural, or modified internucleotide bonds, such as phosphoramidate or phosphorothioate bonds, may be present. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, in some examples, as discussed herein, it may be preferable for the nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0039] The nucleic acids of embodiments of the present invention may be recombinant. As used herein, the term “recombinant” means (i) a molecule constructed outside of a living cell by linking a natural or synthetic nucleic acid segment to a nucleic acid molecule that can be replicated in a living cell, or (ii) a molecule obtained from replication of the one described in (i) above. For the purposes of this specification, replication may be in vitro or in vivo.

[0040] Recombinant nucleic acids may have sequences that do not exist in nature, or sequences that are created by artificial combinations of two sequences that would otherwise be separated. These artificial combinations are often achieved by chemical synthesis, or more generally, by artificially manipulating isolated segments of nucleic acids by genetic engineering techniques, such as those described above, for example, Green and Sambrook. Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Green and Sambrook, above. 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 the physical stability of the double strands formed during hybridization. Examples of modified nucleotides that can be used to produce 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-galactosylqueusin, inosine, and N2. 6 -Isopentenyl adenine, 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-mannosylquosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 Examples of nucleic acids of the present invention include, but are not limited to, isopentenyl adenine, uracil-5-oxyacetic acid (v), weybutoxosin, pseudouracil, cuosin, 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 contain any isolated or purified nucleotide sequence encoding either a CAR or a functional part or functional variant thereof. Alternatively, the nucleotide sequence may contain a nucleotide sequence or combination of degenerate sequences that are degenerate to any of the sequences.

[0042] Furthermore, embodiments of the present invention include a nucleotide sequence complementary to any of the nucleotide sequences of the nucleic acids described herein, or a nucleotide sequence that hybridizes to any of the nucleotide sequences of the nucleic acids described herein under stringent conditions. We provide isolated or purified nucleic acids.

[0043] Nucleotide sequences that hybridize under stringent conditions can also hybridize under highly stringent conditions. “Highly stringent conditions” means that the nucleotide sequence hybridizes specifically to a target sequence (any nucleotide sequence of the nucleic acids described herein) in a detectably greater amount than in nonspecific hybridization. Highly stringent conditions include conditions for distinguishing polynucleotides having precisely complementary sequences, or those containing only a few scattered mismatches, from random sequences that coincidentally have several small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such complementary small regions melt more readily than full-length complements of 14-17 or more bases, and they become readily identifiable by highly stringent hybridization. Relatively highly stringent conditions include low-salt and / or high-temperature conditions, such as those provided by approximately 0.02-0.1 M NaCl or its equivalent at a temperature of approximately 50-70°C. Such highly stringent conditions tolerate only slight mismatches (if any) between the nucleotide sequence and the template or target chain, making them particularly suitable for detecting the expression of any of the CARs of the present invention. Generally, it is understood that conditions can be made more stringent by adding gradually increasing amounts of formamide.

[0044] Furthermore, the present invention provides nucleic acids comprising nucleotide sequences that are at least about 70% identical, for example, 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 the purposes of this specification, the term “recombinant expression vector” means a genetically modified oligonucleotide or polynucleotide construct comprising a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, which causes the cell to express the mRNA, protein, polypeptide, or peptide when the vector is brought into contact with the cell under conditions sufficient to cause the cell to express the mRNA, protein, polypeptide, or peptide. The vectors of the present invention do not exist in nature as a whole. However, parts of the vectors may exist in nature. 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, may be synthesized or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vectors may contain nucleotide-nucleotide bonds that are present in nature or not present in nature, or both types of bonds. Preferably, nucleotides or internucleotide bonds that are not naturally present or have been modified 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 that can be used to transform or transfect any suitable host cell. Suitable vectors include plasmids and viruses, etc., designed for propagation and proliferation, or for expression, or both. 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 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). Recombinant expression vectors may be viral vectors, such as retroviral vectors (e.g., gamma retroviral vectors) or lentiviral vectors.

[0047] In the embodiments, the recombinant expression vector of the present invention is, for example, Sambrook and Green can be prepared using the standard recombinant DNA techniques described above. The expression vector construct, which may be round or linear, can be prepared to include a replication system that functions in prokaryotic or eukaryotic host cells. The replication system may be derived from, for example, ColEl, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0048] The recombinant expression vector may include, for example, regulatory sequences such as transcriptional and translational start and stop codons, which are specific to the type of host cell into which the vector is introduced (e.g., bacteria, fungi, plants, or animals), as needed and taking into consideration whether the vector is DNA-based or RNA-based. The recombinant expression vector may include restriction enzyme sites to facilitate cloning. In addition to the nucleic acid sequence of the present invention encoding the CAR, the recombinant expression vector preferably includes expression regulatory sequences that cause the nucleic acid sequence to be expressed in the host cell, such as promoters, enhancers, polyadenylation signals, transcriptional terminators, intrasequence ribosome entry sites (IRESs), etc.

[0049] Recombinant expression vectors may contain one or more marker genes that enable the selection of transformed or transfected host cells. Marker genes include those for biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., and nutritional complementarity in the host to provide protrophotrophy. Suitable marker genes for the expression vectors of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0050] Recombinant expression vectors may include native or non-native promoters operably ligated to the CAR-coding nucleotide sequence (including its functional portion and functional variants), or to nucleotide sequences complementary to or hybridizing with the CAR-coding nucleotide sequence. For example, the selection of strong, weak, inducible, tissue-specific, and developmental stage-specific promoters is within the ordinary skill of those skilled in the art. Similarly, the combination of nucleotide sequences and promoters is also within the skill of those skilled in the art. Promoters may be non-viral promoters, or they may be viral promoters, such as cytomegalovirus (CMV) promoters, SV40 promoters, RSV promoters, or promoters found in long-chain terminal repeat sequences of mouse stem cell viruses.

[0051] The recombinant expression vector of the present invention can be designed for transient expression, stable expression, or both. Furthermore, the recombinant expression vector can be prepared for constitutive or inducible expression.

[0052] Furthermore, recombinant expression vectors may be constructed to include suicide genes. As used herein, the term “suicide gene” means a gene that causes cells expressing a suicide gene to die. A suicide gene may be a gene that confers sensitivity to an agent, such as a drug, to cells expressing the gene, and causes the cells to die when they come into contact with or are exposed to the agent. Suicide genes are known in the art, 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” means any type of cell that may contain the recombinant expression vector of the present invention. The host cell may be a eukaryotic cell, e.g., a plant, animal, fungus, or algae, or a prokaryotic cell, e.g., a bacterium or protist. The host cell may be a cultured cell or a primary cell, i.e., a cell directly isolated from an organism, e.g., a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell that grows in a suspension. Suitable host cells are known in the art and include, for example, DH5α Escherichia coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating the recombinant expression vector, the host cell may be a prokaryotic cell, e.g., a DH5α cell. For the purpose of generating recombinant CARs, the host cell may be a mammalian cell. The host cell may be a human cell. The host cells may be any cell type, may originate from any type of tissue, and may be at any developmental stage. The host cells may be peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs).

[0054] In embodiments of the present invention, the host cell is a T cell. For the purposes of this specification, the T cell may be any T cell, e.g., cultured T cell, e.g., primary T cell, or T cell derived from a cultured T cell line, e.g., Jurkat, SupT1, etc., or T cell obtained from a mammal. When 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 concentrated 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, may be at any developmental stage, and may be CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD8 +Examples 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 can be CD8 + T cells or CD4 + T cells.

[0055] In an embodiment of the present invention, the host cell is a natural killer (NK) cell. 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 a third type of cell that differentiates from lymphoid common progenitor cells that also give rise to B lymphocytes and T lymphocytes (see, for example, Immunobiology, 9 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 circulating blood as large lymphocytes with characteristic cytotoxic granules. NK cells can recognize and kill several abnormal cells, such as some tumor cells and virus-infected cells, and are thought to be important in the innate immune defense against intracellular pathogens. As described above for T cells, NK cells can be any NK cells, such as cultured NK cells, for example primary NK cells, or NK cells derived from a cultured NK cell line, or NK cells obtained from a mammal. When 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 concentrated 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] Furthermore, embodiments of the present invention provide a population of cells comprising at least one host cell as described herein. The population of cells may be a heterogeneous population comprising at least one other cell that does not contain any of the recombinant expression vectors, for example, a host cell (e.g., a T cell), or a cell other than a T cell, for example, a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc., in addition to a host cell containing any of the recombinant expression vectors described herein. Alternatively, the population of cells may be a substantially homogeneous population (the population mainly comprising host cells containing recombinant expression vectors (e.g., essentially consisting of)). The population may also be a clonal population of cells in which all cells are clones of one host cell containing a recombinant expression vector, and as a result all cells in the population contain a recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells containing a recombinant expression vector as described herein.

[0057] The recombinant expression vectors of the present invention encoding CARs can be introduced into cells by “transfection,” “transformation,” or “transduction.” “Transfection,” “transformation,” or “transduction,” as used herein, refer to the introduction of one or more exogenous polynucleotides into a host cell by physical or chemical methods. Many transfection techniques are known in the art, including, for example, calcium phosphate DNA coprecipitation; DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-enhanced particulate guns; and strontium phosphate DNA coprecipitation. In many cases, infected particles may be grown in suitable commercially available packaging cells, after which the phage or viral vector may be introduced into the host cell.

[0058] The present invention includes conjugates, such as bioconjugates, that comprise any of the following: CARs (including either a functional portion or a variant thereof), nucleic acids, recombinant expression vectors, host cells, or populations of host cells. Conjugates and methods for synthesizing conjugates in general are known in the art.

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

[0060] The CAR material of the present invention may be formulated into a composition, for example, a pharmaceutical composition. In this regard, embodiments of the present invention provide a pharmaceutical composition comprising any of the following: a CAR, a functional portion, a functional variant, a nucleic acid, an expression vector, or a host cell (including a population thereof), and a pharmaceutically acceptable carrier. A pharmaceutical composition of the present invention containing any of the CAR materials of the present invention may contain one or more of the CAR materials of the present invention, for example, a CAR and a nucleic acid, or two or more different CARs. Alternatively, the pharmaceutical composition may contain other pharmaceutically active agents or drugs, for example, chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate. The CAR material of the present invention may be included in combination with trexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In a preferred embodiment, the pharmaceutical composition includes the host cells or population thereof of the present invention.

[0061] Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to the pharmaceutical composition, the carrier may be any of those conventionally used with the specific 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 does not have 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 preferred embodiments, the CAR is expressed by host cells, preferably T cells or NK cells, and the host cells expressing the CAR are administered to the patient. These cells may be autologous or allogeneic to the cell recipient. The nucleic acid encoding the CAR may be introduced into the cells by any of a variety of genetic modification methods, including but not limited to transduction by gamma-retroviruses, lentiviruses, or transposon systems. A variety of suitable formulations of the pharmaceutical composition of the present invention exist. Suitable formulations may include those administered parenterally, subcutaneously, intravenously, intramuscularly, intratumorally, intraarterially, subarachnoidally, or intraperitoneally. The CAR material of the present invention may be administered using more than one route, and in certain examples, a particular route may provide a more immediate and effective response than another route.

[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 (or a functional variant thereof) of the present invention, a pharmaceutically acceptable carrier for the cells for injection is any isotonic carrier, for example, normal physiological saline (about 0.90% w / v NaCl in water, about 300 mOsm / L in water). The solution may contain NaCl (or approximately 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), approximately 5% dextrose in water, or Ringer's lactate solution, etc. In the embodiment, human serum albumin is supplied to a pharmaceutically acceptable carrier.

[0064] The compositions may be delivered using sustained-release, delayed-release, and prolonged-release delivery systems, so that the compositions of the present invention are delivered before and for a sufficient amount of time to sensitize the site to be treated. Many types of delivery systems are available and known to those skilled in the art. Such systems can avoid repeated administration of the compositions, 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] Although not bound by any particular theory or mechanism, it is believed that the CAR of the present invention provides one or more of the following by eliciting an antigen-specific response to BCMA: targeting and destroying BCMA-expressing cancer cells, reducing or eliminating cancer cells, promoting the invasion of immune cells into the tumor site(s), and enhancing / extending the anti-cancer response.

[0066] It is intended that the CAR material of the present invention can be used in methods for treating or preventing diseases in mammals, such as cancer. Although not bound by any particular theory or mechanism, the CAR of the present invention has biological activity, such as the ability to recognize antigens such as BCMA, and as a result, when expressed by cells, the CAR can mediate an immune response against cells expressing a specific antigen, such as BCMA. In this regard, embodiments of the present invention are methods for treating or preventing cancer in mammals, wherein cancer in mammals The present invention provides a method comprising administering to a mammal an amount effective for treatment or prevention of any of the CARs, nucleic acids, recombinant expression vectors, host cells, cell populations, and / or pharmaceutical compositions of the present invention. In a preferred embodiment, the method comprises injecting a host cell transduced with the CAR of the present invention into a mammal.

[0067] One or more isolated host cells expressing the BCMA CAR of the present invention as described herein may be brought into contact with a population of cancer cells expressing BCMA ex vivo, in vivo, or in vitro. “Ex vivo” refers to a method performed inside or on cells or tissues in an artificial extra vivo environment with minimal alteration from 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 isolated from its normal biological context. The methods of the present invention preferably include ex vivo and in vivo components. In this regard, for example, the isolated host cells express the anti-BCMA of the present invention. Cells may be cultured ex vivo under conditions that express CAR, and then directly transferred to mammals (preferably humans) with BCMA-positive cancer, such as multiple myeloma. Such a cell transfer method is referred to in the art as "adoptive cell transfer (ACT)," in which immune-derived cells are transferred to a recipient, and the functions of the immune-derived cells are transferred to the host. The immune-derived cells may originate from the recipient or another individual. This is an adoptive cell transfer method for treating various types of cancer, including hematological cancers such as myeloma.

[0068] When a composition containing host cells expressing a nucleic acid sequence encoding the CAR of the present invention, or a vector containing a nucleic acid sequence encoding the CAR of the present invention, is administered to a mammal (e.g., human), the biological activity of the CAR can be measured by any suitable method known in the art. According to the method of the present invention, the CAR binds to BCMA in cancer cells, destroying them. The 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 assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009) and Herman et al., J. Immunological Methods, 285(1):25-40 (2004). The biological activity of the CAR can also be measured by assaying the expression of specific cytokines such as CD107a, IFNγ, IL-2, and TNF.

[0069] Embodiments of the present invention further include lymphocyte depletion in mammals before administration of the CAR material of the present invention. Examples of lymphocyte depletion include, but are not limited to, non-myeloablative lymphocyte depletion chemotherapy, myeloablative lymphocyte depletion chemotherapy, and total body irradiation.

[0070] For the purposes of the method of the present invention, in which host cells or a population of cells are administered, the cells may be homogeneous or self-cells to the mammal. Preferably, the cells are self-cells to the mammal.

[0071] "Effective dose" or "therapeutic dose" refers to the appropriate dose for preventing or treating cancer in an individual. The effective dose for therapeutic or prophylactic use depends, for example, on the stage and severity of the disease or disorder being treated, the patient's age, weight, and general health, as well as the judgment of the prescribing physician. The size of the dose is also determined by the specific CAR material selected, the method of administration, the timing and frequency of administration, the presence, nature, and extent of any adverse side effects that may accompany the administration of a particular CAR material, and the desired physiological effect. (Examples) Those skilled in the art will understand that, for example, cancer, long-term treatment may be required, possibly involving multiple doses using the CAR material of the present invention in each or various rounds of administration. For example, and not intended to limit the present invention, the dose of the CAR material of the present invention may be about 0.001 to about 1000 mg / kg (body weight of the treated subject) / 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 present invention, the dose is about 1 × 10 cells expressing the CAR of the present invention per kg of body weight. 4 ~Approx. 1×10 10 It may be a single cell. When the CAR material of the present invention is a host cell, an exemplary dose of host cells is a minimum of 1 million cells (1 mg cells / administration), for example, 1 × 10 per kg of body weight. 9 It may be a single cell. When the CAR material of the present invention is nucleic acid packaged with a virus, the exemplary dose of the virus may be 1 ng / administer.

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

[0073] For the purposes of the present invention, an assay may be used to determine the starting dose to administer to a mammal, for example, by comparing the extent to which target cells are lysed and / or IFN-γ is secreted by T cells expressing the CAR of the present invention upon administration of a particular dose to a mammal, between a set of mammals each given different doses of T cells. 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 together with one or more additional therapeutic agents, the one or more additional therapeutic agents may be co-administered to mammals. "Co-administration" means that the administration of the one or more additional therapeutic agents and the CAR material of the present invention occurs within a sufficiently close time interval so that the CAR material of the present invention can enhance the effects of the one or more additional therapeutic agents, and vice versa. In this regard, the CAR material of the present invention may be administered first, followed by the one or more additional therapeutic agents, and vice versa. Alternatively, the CAR material of the present invention and one or more additional therapeutic agents may be administered simultaneously. An exemplary therapeutic agent that can be co-administered with the CAR material is IL-2. IL-2 is thought to enhance the therapeutic effect of the CAR material of the present invention. Although not bound by any particular theory or mechanism, IL-2 is thought to enhance the treatment by enhancing the in vivo increase in the number of cells expressing the CAR of the present invention.

[0075] The mammals referred to herein may be any mammal. As used herein, the term “mammal” means any mammal, including but not limited to rodents such as mice and hamsters, and mammals of the order Lagomorpha such as rabbits. Mammals may also be mammals of the order Carnivora, including felines (cats) and canines (dogs). Mammals may also be mammals of the order Artiodactyla, including cattle and pigs, or mammals of the order Persodactyla, including horses. Mammals may also be mammals of the order Primates, Ceboids or Simoids (monkeys), or Haplorhini (humans and apes). Preferably, the mammal is human.

[0076] With respect to the method of the present invention, the cancer may be any type of cancer. In embodiments of the present invention, the cancer is a BCMA-expressing cancer. In embodiments of the present invention, the cancer is multiple myeloma or Hodgkin 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 normally involved in antibody production (Raab et al., Lancet, 374:324-329 (2009)). Multiple myeloma affects 1 to 4 people per 100,000 annually. The disease is more common in men and, for reasons still unknown, is twice as common in African Americans as in Caucasian Americans. Multiple myeloma is the least common hematological malignancy (14%) and accounts for 1% of all cancers (Raab et al., ibid.). Treatment for multiple myeloma typically involves high-dose chemotherapy followed by hematopoietic stem cell transplantation (allogeneic or autologous), but patients receiving such treatment usually have a high recurrence rate. As discussed above, BCMA is highly expressed by multiple myeloma cells (e.g., Novak et al., as mentioned above; Neri et al., as mentioned above; Bellucci et al., as mentioned above; and Moreaux et al., as mentioned above).

[0078] Hodgkin lymphoma (formerly known as Hodgkin's disease) is a cancer of the immune system characterized by the presence of multinucleated cells called Reed-Sternberg cells. The two main types of Hodgkin lymphoma include classical Hodgkin lymphoma and nodular lymphocyte-predominant Hodgkin lymphoma. Currently, Hodgkin lymphoma is treated with radiotherapy, 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 any terms derived therefrom, as used herein, do not necessarily imply 100%, i.e., complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that those skilled in the art would recognize as having potential benefits or therapeutic effects. In this regard, the methods of the present invention can provide treatment or prevention of any amount or level of cancer in mammals. Furthermore, the treatment or prevention provided by the methods of the present invention may include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented, e.g., cancer. Also, for the purposes of this specification, “prevention” may include delaying the onset of the disease, e.g., cancer or its symptoms or conditions.

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

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

[0082] The materials and methods used in Examples 1 to 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 with the full-length BCMA gene in the laboratory. Prior to the following experiments, NGFR-K562 and K562 cells were transduced with the low-affinity nerve growth factor gene in the laboratory. BCMA-K562 and NGFR-K562 cells were transduced using the same gamma retroviral vector and method.

[0085] Tissue samples or peripheral blood mononuclear cells (PMBCs) from six patients with multiple myeloma were named Myeloma Patients 1-6. PBMCs from three subjects with myeloma were used, and the donors were named Donor A, Donor B, and Donor C. Primary CD34 cells were obtained from three healthy donors. + 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 in the United States.

[0086] Construction of a fully human heavy chain (FHVH) CAR A series of CARs containing only the full human heavy chain antigen recognition (FHVH) domain were prepared. The sequence of each CAR followed this pattern from the 5' end to the 3' end: CD8α leader sequence, one of four single heavy chain variable region domains, and the hinge and transmembrane region of the human CD8α molecule. The cytoplasmic portion of either CD28, 4-1BB, or an inducible T cell costimulatory system (ICOS) molecule was then added, followed by the addition of 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 CAR antigen recognition domains of only four complete human heavy chains were named FHVH74, 32, 33, and 93. Furthermore, the CAR names include the CD8α hinge and transmembrane domains, the included costimulatory domains, and the CD3ζ domain. For example, FHVH74-CD828Z possesses the FHVH74 antigen recognition domain, the CD8α-derived hinge and transmembrane domains, the CD28 costimulatory domain, and the CD3ζ T cell activation domain. 11D5-3-CD828Z anti-BCMA CAR was used as a positive control.

[0088] These CARs were constructed, and the CAR nucleotide sequences were ligated to the MSGV gamma retroviral vector backbone using standard methods. The complete CAR nucleotide sequences are provided as 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 reader sequence, an FHVH sequence, a portion of the CD8α hinge and transmembrane domain, a Blpl site, and a TATCGT hexanucleotide (provided as SEQ ID NO: 41). To ensure perfect end cleavage by Ncol and Blpl, GTC and TATCGT (SEQ ID NO: 41) nucleotides were added. The fragments were cleaved at 37°C for 2 hours using Blpl and NCOI-HF (New England Biolabs, Ipswich, MA). The cleaved fragments were then purified using the QIAQUICK PCR purification kit (Qiagen). The GBLOCK (Integrated DNA Technologies (IDT), Skokie, IL) fragment was ligated to a Blpl / Ncol-HF digested and gel-purified MSGV vector backbone, which also contained other components of the CAR not present in the GBLOCK fragment.

[0089] The CAR components included in the MSGV vector skeleton were the remainder of the CD8α domain that was not present in the GBLOCK (Integrated DNA Technologies (IDT), Skokie, IL) fragment, a sequence encoding one of the CD28, 4-1BB, or ICOS co-stimulatory domains, and the CD3ζ domain. By using the DNA Ligation Kit (Roche Applied Sciences), each GBLOCK (Integrated DNA Tech) We performed ligation of nologies(IDT), Skokie, IL)CAR fragments and MSGV vector skeleton fragments.

[0090] CAR detection in T cells Transduced and untransduced T cells were washed and stained with phycoerythrin-labeled BCMA-Fc protein to detect cell surface CAR molecules. 500,000 T cells were suspended in 50 mL of staining buffer and titrated amounts of BCMA-Fc-PE reagent were added. CD3, CD4, and CD8 staining were also performed using standard methods. Dead cells were removed using 7-AAD (7-amino-asinomycin 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. Before transduction, T cell medium + 50 ng / mL anti-CD3 monoclonal antibody OKT3 (Ortho, Bridgewater, NJ) and 300 IU / mL IL-2 medium 1 x 10 6 PBMCs were suspended at a concentration of cells / mL. After transduction, T cells were maintained in T cell medium + IL-2.

[0092] Gamma retrovirus transduction To generate non-replicating gamma retroviruses, packaging cells were transfected with a plasmid encoding the RD114 envelope protein along with a plasmid encoding the CAR. Two days after the start of T cell culture, gamma retrovirus transduction of the T cells was performed.

[0093] ELISA for interferon-γ and tumor necrosis factor alpha 100,000 BCMA +Alternatively, 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 plate was incubated at 37°C for 18–20 hours. After incubation, ELISA for interferon-gamma (INFγ) was performed using the standard method (Pierce). ELISA for tumor necrosis factor alpha (TNF) was performed using the standard method (R&D).

[0094] CD107a assay Two tubes were prepared for each T cell culture tested. One tube contained BCMA-K562 cells, and the other contained NGFR-K562 cells. Both tubes contained CAR transduced T cells, 1 mL of AIM-V medium + 5% human AB serum, titrated 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 polyclonal biotin-labeled 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). Furthermore, bone marrow cells were stained with anti-CD38 (eBioscience) and anti-CD56 (BD). FLOWJO software (Tree Star, Flow cytometry analysis was performed for all experiments using Inc. Oregon, US.

[0096] Growth assay A co-culture was prepared in a 24-well plate. The target cells included in the co-culture were 0.5 × 10⁶ 6Individual irradiated BCMA-K562 cells or 0.5 × 10⁶ cells 6 It was one of several irradiated NGFR-K562 cells. The co-culture also contained 1 × 10 T cells derived from cultures transduced with either anti-bcma2 or SP6. 6 It contained [number] cells. As previously described, T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE, Invitrogen). The culture medium used for co-culture was AIM V + 5% human AB serum. IL-2 was not added to the medium. Four days after the start, viable cells in each co-culture were counted with trypan blue to exclude dead cells, and flow cytometry was performed.

[0097] Cytotoxic assay BCMA in relation to the survival of CCRF-CEM cells, which are a negative control. + Cytotoxicity was measured by comparing the survival of target cells. Both of these cell types were mixed in the same tube as 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 duplicated in 5 mL sterile test tubes (BDs) in multiple T cell-to-target cell ratios. The target cells contained in the tubes, along with 50,000 CCRF-CEM negative control cells, were labeled with 50,000 BCMA cells. + These were target 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 was obtained. + By dividing the percentage of cells by the percentage of surviving CCRF-CEM negative control cells, BCMA + The viability of target cells was determined. BCMA in each T cell + target cell culture. + Target cell survival rate, excluding effector T cells, BCMA +Viable BCMA cells in a tube containing only target cells and CCRF-CEM cells + Corrected BCMA by dividing the percentage of target cells by the ratio of the percentage of surviving CCRF-CEM negative control cells. + The viability of target cells was calculated. This correction was necessary to account for variability in the starting cell number and natural death of target cells. Cytotoxicity was calculated as follows: BCMA + Target cell cytotoxicity rate = 100 - corrected BCMA + The survival rate of target cells.

[0098] In vivo mouse model processing experiments NSG mice obtained from The Jackson Laboratory (NOD.Cg-Prkdc scid Il2rg tm1Wjl RPMI8226 cells were used. Mice were intradermally injected with RPMI8226 cells. The tumors were allowed to grow for 10 days. Then, mice were intravenously injected with human T cells transduced or not transduced with the CARs shown in Example 9 (Figures 9B-9C) or Example 10 at the doses described in Example 9 (Figures 9B-9C) or Example 10. The tumors were measured every 3 days using a calipas. The tumor size (area) (mm²) was calculated by multiplying the longest length by the length perpendicular to the longest length. 2 The longest length obtained was 15 mm. The mice were sacrificed when the longest length reached 15 mm. The animal experiment was approved by the Animal Experiments Committee of the National Cancer Institute.

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

[0100] As shown in Figures 1A–1L, twelve CARs were designed that possess an antigen recognition domain solely from the full human heavy chain. The general design of these CARs includes, from N-terminus to C-terminus, a CD8a reader sequence, a full 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 stimuli (ICOS). This example presents the first reported CARs to possess an antigen recognition domain solely from the heavy chain.

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

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

[0103] Example 3 This example demonstrates that only the heavy chain of CAR underwent BCMA-specific degranulation.

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

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

[0106] Primary human T cells derived 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 described in Tables 1-3.

[0107] [Table 1]

[0108] Referring to Table 1, cultured T cells were transduced with the indicated CARs and cultured overnight with the indicated target cells (top row). After overnight incubation, a standard ELISA assay was performed on the culture supernatant. BCMA-K562 and RPMI8226 were identified as 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 + Each CAR stained with BCMA-Fc-PE reagent is transduced into CD3 + From the percentage of cells, untransduced CD3 stained with BCMA-Fc-PE reagent + This is equivalent to the percentage of lymphocytes minus %CAR. + Except for the column marked with an asterisk, all numbers represent interferon-gamma in pg / mL.

[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, a standard ELISA assay was performed on the culture supernatant. BCMA-K562 is 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 + Each CAR stained with BCMA-Fc-PE reagent is transduced into CD3 + From the percentage of cells, untransduced CD3 stained with BCMA-Fc-PE reagent + This is equivalent to the percentage of lymphocytes minus %CAR. + Except for the column marked with an asterisk, all numbers represent interferon-gamma in pg / mL.

[0111] The results in Tables 1 and 2 show that the displayed (indicted) CAR is BCMA + This demonstrates the specific recognition of target cells.

[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, a standard ELISA assay was performed on the culture supernatant. BCMA-K562 is BCMA + All other targets are BCMA-negative. They are stained with BCMA-Fc-PE reagent, followed by flow cytosis. The percentage of T cells expressing the displayed CAR was determined by tometry. %CAR + Each CAR stained with BCMA-Fc-PE reagent is transduced into CD3 + From the percentage of cells, untransduced CD3 stained with BCMA-Fc-PE reagent + This is equivalent to the percentage of lymphocytes minus %CAR.+ Except for the column marked with an asterisk, all numbers represent interferon-gamma in pg / mL.

[0114] The results in Table 3 show that T cells expressing FHVH-CD828Z or FHVH-CD828Z are BCMA + This demonstrates the specific recognition of target cells.

[0115] Example 5 This example demonstrates that T cells expressing only the heavy chain CAR proliferated specifically in BCMA in vitro.

[0116] Primary T cells expressing CAR labeled with CFSE derived from MM patients were irradiated with BCMA. + Alternatively, they were cultured with BCMA-negative target cells. As shown in Figures 4B-4E, all four FHVH CARs shown in Figures 4B-4E proliferated specifically to BCMA. T cells expressing the 11D5-3-CD828Z CAR also proliferated specifically to BCMA (Figure 4A). In addition to confirming BCMA-specific proliferation by diluting CFSE, BCMA-specific proliferation is linked to BCMA + This was also demonstrated by an increase in the absolute number of CAR-expressing T cells cultured with target cells, as shown in Figure 4F. + When cultured with target cells, CAR + The absolute number of T cells increased.

[0117] Example 6 In this example, T cells expressing CAR only in the heavy chain were found to be BCMA + This indicates that it kills 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 CARs are expressed and function only in heavy chains containing the 4-1BB costimulatory domain.

[0120] Only four complete human heavy chains containing the 4-1BB costimulatory domain, as shown in Figure 6, were constructed and evaluated as CARs. All four of these CARs were expressed on the surface of primary human T cells, but FHVH33-CD8BBZ consistently showed the highest expression (Figure 6), and was therefore selected for further testing. The results obtained from the functional evaluation of FHVH33-CD8BBZ are shown in Figures 7A-7F. These CAR-expressing T cells produced IFNγ specifically in BCMA and degranulated.

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

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

[0123] [Table 4]

[0124] T cells that were either untransduced (UT) or expressing either FHVH33-CD828Z or FHVH33-CD8BBZ CAR were incubated overnight with autologous myeloma cells (90% purity) 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 FHVH33-CD828Z or FHVH33-CD8BBZ CAR secreted IFNγ after co-culture with target myeloma cells.

[0125] Example 9 This example demonstrates dose-response titer measurement of FHVH33-CD8BBZ-expressing T cells in mice.

[0126] As shown in Figure 9A, RPMI8226 cells were intradermally injected into NSG mice. Tumors were allowed to grow for 10 days. On day 0, the indicated number of FHVH33-CD8BBZ-expressing T cells were intravenously injected into the mice. One of three different doses of FHVH33-CD8BBZ CAR T cells was administered to the mice, 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 One FHVH33-CD8BBZ T cell was able to eradicate tumors from all mice, and the efficacy of FHVH33-CD8BBZ CAR T cells decreased in a dose-dependent manner. Tumors progressively enlarged in all untreated mice (n=5 mice / group).

[0128] The survival rate of mice is shown in Figure 9C. 2.2 × 10 6 All mice administered with FHVH33-CD8BBZ-expressing T cells survived and remained healthy throughout the experiment.

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

[0130] Mice were intradermally injected with RPMI8226 cells. Tumors were allowed to grow for 10 days. On day 0, 1 × 10 T cells expressing SP6-CD828Z, 11D5-3-CD8BBZ, FHVH33-CD8BBZ, or FHVH33-CD828Z CAR were introduced. 6 Either the individual units were intravenously injected into mice, or the mice were left untreated.

[0131] As shown in Figure 10A, tumors progressively grew in mice injected with T cells expressing the negative control SP6-CD828Z CAR, as well as in untreated mice. In mice treated with T cells expressing either 11D5-3-CD8BBZ, FHVH33-CD8BBZ, or FHVH33-CD828Z CAR, tumors were eradicated.

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

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

[0134] Effector T cells were cultured overnight with target cells shown in Table 5. Effector T cells were either untransduced T cells, T cells transduced with the nucleotide sequence encoding FHVH33-CD828Z, or T cells transduced with the nucleotide sequence encoding FHVH33-CD8BBZ. All T cells originated from the same human donor. T cells transduced with FHVH33-CD828Z showed 71% CAR expression. T cells transduced with FHVH33-CD8BBZ showed 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. 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 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 herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as they are incorporated herein by reference, each reference being indicated to be incorporated individually and specifically as such.

[0140] In connection with the description of the present invention (particularly in connection with the following claims), the terms “a,” “an,” “the,” and “at least one,” as well as similar references, should be interpreted as encompassing both singular and plural forms unless otherwise specified herein or unless clearly contradicted by the 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 interpreted as meaning one item (A or B) selected from the enumerated items or any combination of two or more enumerated items (A and B), unless otherwise specified herein or unless clearly contradicted by the context. The terms “comprising,” “having,” “including,” and “containing” should be interpreted as encompassing both singular and plural forms. Unless otherwise specified, terms are open-ended (i.e., meaning "including but not limited to"). Enumerations of value ranges herein are intended, unless otherwise specified herein, simply to function as abbreviations referring individually to each distinct value within the range, and each distinct value is incorporated into the specification as if it were individually enumerated herein. All methods described herein may be performed in any preferred order unless otherwise specified herein or unless particularly evident from the context. The use of any and all examples or exemplary expressions provided herein (e.g., "etc.") is intended merely to further elucidate the invention and not to impose any limitation on the scope of the invention, unless specifically claimed. No expression in the specification should be construed as indicating that any unclaimed element is essential for the practice of the invention.

[0141] Preferred embodiments of the present invention, including the best modes known to the inventors for carrying out the invention, are described herein. Variations of the preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that those skilled in the art will use such variations as appropriate, and they intend that the invention may be carried out in ways other than those specifically described herein. Accordingly, the invention includes all variations and equivalents of the subject matter of the invention enumerated in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements in all possible variations is encompassed by the invention unless otherwise specified herein or is particularly evident from the context.

Claims

1. A pharmaceutical composition formulated for parenteral administration, comprising a pharmaceutically acceptable carrier and a population of host cells comprising a vector containing a nucleic acid having a nucleotide sequence encoding a chimeric antigen receptor (CAR) having antigen specificity for a B cell maturation antigen (BCMA), wherein the CAR is: (1) (a) Antigen recognition domain comprising an antibody heavy chain variable region including the heavy chain complementarity determining region (CDR) 1 amino acid sequence of SEQ ID NO: 1, the heavy chain CDR 2 amino acid sequence of SEQ ID NO: 2, and the heavy chain CDR 3 amino acid sequence of SEQ ID NO: 3, (b) Human CD8α transmembrane (TM) domain, and (c) Human CD28 T cell activation domain and human CD3 zeta T cell activation domain; (2) (a) Antigen recognition domain comprising an antibody heavy chain variable region including the heavy chain CDR1 amino acid sequence of SEQ ID NO: 4, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 5, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 6, (b) Human CD8α™ domain, and (c) Human CD28 T cell activating domain and human CD3 zeta T cell activating domain; (3) (a) Antigen recognition domain comprising an antibody heavy chain variable region including the heavy chain CDR1 amino acid sequence of SEQ ID NO: 7, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 8, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 9, (b) Human CD8α™ domain, and (c) Human CD28 T cell activation domain and human CD3 zeta T cell activation domain; (4) (a) Antigen recognition domain comprising an antibody heavy chain variable region including the heavy chain CDR1 amino acid sequence of SEQ ID NO: 10, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 11, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 12, (b) Human CD8α™ domain, and (c) Human CD28 T cell activation domain and human CD3 zeta T cell activation domain; (5) (a) Antigen recognition domain comprising an antibody heavy chain variable region including the heavy chain CDR1 amino acid sequence of SEQ ID NO: 1, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 2, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 3, (b) Human CD8α™ domain, and (c) Human 4-1BB T cell activation domain and human CD3 zeta T cell activation domain; (6) (a) Antigen recognition domain comprising an antibody heavy chain variable region including the heavy chain CDR1 amino acid sequence of SEQ ID NO: 4, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 5, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 6, (b) Human CD8α™ domain, and (c) Human 4-1BB T cell activation domain and human CD3 zeta T cell activation domain; (7) (a) Antigen recognition domain comprising an antibody heavy chain variable region including the heavy chain CDR1 amino acid sequence of SEQ ID NO: 7, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 8, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 9, (b) Human CD8α™ domain, and (c) Human 4-1BB T cell activation domain and human CD3 zeta T cell activation domain; (8) (a) Antigen recognition domain comprising an antibody heavy chain variable region including the heavy chain CDR1 amino acid sequence of SEQ ID NO: 10, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 11, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 12, (b) Human CD8α™ domain, and (c) Human 4-1BB T cell activation domain and human CD3 zeta T cell activation domain; (9) (a) Antigen recognition domain comprising an antibody heavy chain variable region including the heavy chain CDR1 amino acid sequence of SEQ ID NO: 1, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 2, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 3, (b) Human CD8α™ domain, and (c) Human ICOS T cell activation domain and human CD3 zeta T cell activation domain; (10) (a) Antigen recognition domain comprising an antibody heavy chain variable region including the heavy chain CDR1 amino acid sequence of SEQ ID NO: 4, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 5, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 6, (b) Human CD8α™ domain, and (c) Human ICOS T cell activation domain and human CD3 zeta T cell activation domain; (11) (a) Antigen recognition domain comprising an antibody heavy chain variable region including the heavy chain CDR1 amino acid sequence of SEQ ID NO: 7, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 8, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 9, (b) Human CD8α™ domain, and (c) Human ICOS T cell activation domain and human CD3 zeta T cell activation domain, (12) (a) Antigen recognition domain comprising an antibody heavy chain variable region including the heavy chain CDR1 amino acid sequence of SEQ ID NO: 10, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 11, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 12, (b) Human CD8α™ domain, and (c) Human ICOS T cell activation domain and human CD3 zeta T cell activation domain A pharmaceutical composition containing the following:

2. The pharmaceutical composition according to claim 1, wherein all domains of the CAR are human.

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

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

5. The aforementioned antigen recognition domain, (a) Sequence ID 13, (b) Sequence ID 14, (c) Sequence ID No. 15, or (d) Sequence ID 16 A pharmaceutical composition according to any one of claims 1 to 4, comprising the amino acid sequence.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the CAR comprises one amino acid sequence from sequence numbers 17 to 28.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the nucleic acid comprises one nucleotide sequence from sequence numbers 29 to 40.

8. A pharmaceutical composition according to any one of claims 1 to 7, formulated for injection.

9. A pharmaceutical composition according to any one of claims 1 to 8, formulated for intravenous administration.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutically acceptable carrier is supplemented with human serum albumin.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the pharmaceutically acceptable carrier is an isotonic carrier.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the pharmaceutically acceptable carrier comprises ordinary physiological saline, 5% dextrose in water, or Ringer's lactate solution.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the host cell population is a T cell population.

14. The pharmaceutical composition according to any one of claims 1 to 12, wherein the host cell population is a natural killer (NK) cell population.

15. A pharmaceutical composition according to any one of claims 1 to 14 for treating or preventing cancer in mammals.

16. The pharmaceutical composition according to claim 15, wherein the cancer is multiple myeloma.

17. The pharmaceutical composition according to claim 15, wherein the cancer is Hodgkin lymphoma.

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