Chimeric antigen receptors with BCMA specificity and uses thereof

Abstract

To provide B cell maturation antigen (BCMA)-specific chimeric antigen receptors, and to provide cells expressing such chimeric antigen receptors.SOLUTION: Provided is a B-cell maturation antigen (BCMA)-specific chimeric antigen receptor comprising, from N-terminus to C-terminus: (a) an extracellular ligand-binding domain comprising an anti-BCMA antigen-binding domain; (b) a hinge derived from CD8; (c) a transmembrane domain derived from CD8; and (d) a cytoplasmic domain comprising a costimulatory domain derived from 4-1BB and a signaling domain derived from CD3 zeta. In certain embodiments, engineered cells expressing the chimeric antigen receptors of the present invention are capable of inhibiting the growth of tumors expressing BCMA. The engineered cells of the invention are useful for the treatment of diseases and disorders in which an upregulated or induced BCMA-targeted immune response is desired and / or therapeutically beneficial.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Sequence Listing Reference This application incorporates by reference the Sequence Listing, which was filed in computer readable form as file 10455WO01-Sequence.txt, created on July 17, 2019, and contains 66,907 bytes.

[0002] The present invention relates to chimeric antigen receptors (CARs) and engineered cells containing such CARs that are specific for B-cell maturation antigen (BCMA), and methods of their use. [Background technology]

[0003] B-cell maturation antigen (BCMA), also known as TNFRSF17, or CD269, is a type III transmembrane protein lacking a signal peptide and containing a cysteine-rich extracellular domain. BCMA, along with closely related proteins, promotes the survival of B cells at different stages of development. BCMA is expressed exclusively in B-cell lineage cells, particularly in the interfollicular regions of germinal centers, as well as plasmablasts and differentiated plasma cells. BCMA is selectively induced during plasma cell differentiation and is required for optimal survival of long-lived plasma cells in the bone marrow. In multiple myeloma, BCMA is widely expressed at high levels on malignant plasma cells, and BCMA expression increases with progression from normal cells to active multiple myeloma. BCMA is also expressed in other B-cell malignancies, including Waldenstrom's macroglobulinemia, Burkitt's lymphoma, and diffuse large B-cell lymphoma. Non-patent literature 1.

[0004] Adoptive immunotherapy, which involves the transfer of ex vivo generated autoantigen-specific T cells, is a promising strategy for treating viral infections and cancer. The T cells used in adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or by redirection of T cells by genetic engineering.

[0005] New specificities of T cells have been successfully generated by gene transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Non-Patent Document 2). CARs are synthetic receptors consisting of a targeting moiety associated with one or more signaling domains in a single fusion molecule. In general, the binding portion of a CAR consists of the antigen-binding domain of a single-chain antibody (scFv), which contains the variable fragments of the light and heavy chains of a monoclonal antibody linked by a flexible linker. The signaling domain of first-generation CARs is derived from the cytoplasmic region of CD3 zeta or the gamma chain of the Fc receptor. First-generation CARs have been shown to successfully redirect the cytotoxicity of T cells. However, they have failed to provide long-term expansion and antitumor activity in vivo. Signaling domains from costimulatory molecules, as well as transmembrane and hinge domains, have been added to form second- and third-generation CARs, leading to some successful clinical trials in humans. For example, CAR-redirected T cells specific for the B cell differentiation antigen CD19 have shown dramatic efficacy in the treatment of B cell malignancies, and TCR-redirected T cells have shown benefit in patients suffering from solid tumors. Stauss et al. describe a strategy to modify therapeutic CARs and TCRs for use in the treatment of cancer, for example, to enhance antigen-specific effector function and limit the toxicity of engineered T cells (Non-Patent Document 3).

[0006] Engineered cells expressing chimeric antigen receptors that target BCMA are useful in therapeutic settings where specific targeting and T cell-mediated killing of cells expressing BCMA is desired. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Tai et al.,Immunotherapy,7(11):1187-1199,2015 [Non-Patent Document 2] Jena, Dotti et al. 2010 [Non-Patent Document 3] Current Opinion in Pharmacology 2015,24:113-118 Summary of the Invention

[0008] In one aspect, the invention provides a B-cell maturation antigen (BCMA)-specific chimeric antigen receptor comprising, from N-terminus to C-terminus, (a) an extracellular ligand binding domain comprising an anti-BCMA antigen binding domain, (b) a hinge, (c) a transmembrane domain, and (d) a cytoplasmic domain comprising a costimulatory domain and a signaling domain.

[0009] In some embodiments, the extracellular ligand binding domain comprises an anti-BCMA single chain variable fragment (scFv) domain comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR). In some embodiments, the anti-BCMA scFv domain comprises a linker between the LCVR and the HCVR. In some embodiments, the chimeric antigen receptor further comprises a linker between the extracellular ligand binding domain (e.g., scFv domain) and the hinge. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 93-96. In some embodiments, the linker comprises a linker selected from the group consisting of (G4S) n is a linker, n is 1 to 10 be.

[0010] In some embodiments, the hinge, the transmembrane domain, or both are derived from a CD8α polypeptide. In some embodiments, the costimulatory domain comprises a 4-1BB costimulatory domain. In some embodiments, the signaling domain comprises a CD3 zeta signaling domain. In some embodiments, the hinge comprises the amino acid sequence of SEQ ID NO: 97. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 98. In some embodiments, the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 100.

[0011] In some cases, the LCVR comprises a complementarity determining region (CDR) of the LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58 and 74. In some cases, the LCVR comprises an LCDR1-LCDR2-LCDR3 domain comprising an amino acid sequence of SEQ ID NOs: 12-14-16, 28-30-32, 44-46-48, 60-62-64, or 76-78-80, respectively. In some cases, the HCVR comprises a CDR of the HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50 and 66. In some cases, the HCVR comprises an HCDR1-HCDR2-HCDR3 domain comprising an amino acid sequence of SEQ ID NOs: 4-6-8, 20-22-24, 36-38-40, 52-54-56, or 68-70-72, respectively.

[0012] In some embodiments, the LCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, and 74, or an amino acid sequence having 95%-99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, and 74, and the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, and 66, or an amino acid sequence having 95%-99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, and 66. In some embodiments, the LCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, and 74, and the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, and 66. The amino acid sequence is selected from the group consisting of:

[0013] In some embodiments, the scFv domain comprises an LCVR / HCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO: 10 / 2, 26 / 18, 42 / 34, 58 / 50, or 74 / 66. Optionally, the LCVR and HCVR are linked by a linker, optionally with n=1-3 (G4S) n are joined by a linker.

[0014] In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, or SEQ ID NO: 90. In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 90.

[0015] In another aspect, the present invention provides an isolated nucleic acid molecule encoding a chimeric antigen receptor as described above or herein. Optionally, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 81, 83, 85, 87 and 89.

[0016] In another aspect, the present invention provides a vector that comprises the nucleic acid molecule described above or herein.In some cases, the vector is a DNA vector, an RNA vector, a plasmid, a lentivirus vector, an adenovirus vector, or a retrovirus vector.In some embodiments, the vector is a lentivirus vector.

[0017] In another aspect, the invention provides a cell comprising a nucleic acid molecule or a vector as described above or herein. Optionally, the cell is a human T cell.

[0018] In another aspect, the present invention provides an engineered cell comprising a chimeric antigen receptor as described above or herein. Optionally, the engineered cell is an immune cell. Optionally, the immune cell is an immune effector cell. Optionally, the immune effector cell is a T lymphocyte. In some embodiments, the T lymphocyte is an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte. In some embodiments, the engineered cell is a CD8+ cytotoxic T lymphocyte.

[0019] In some cases, the engineered cells of the invention are for use in treating a BCMA-expressing cancer. In some cases, the BCMA-expressing cancer is multiple myeloma.

[0020] In another aspect, the invention provides an engineered human T cell comprising a chimeric antigen receptor comprising, from N-terminus to C-terminus, (a) an extracellular ligand binding domain comprising an anti-BCMA single chain variable fragment (scFv) domain comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR); (b) a hinge; (c) a transmembrane domain; and (d) a cytoplasmic domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain.

[0021] In some cases, the scFv domain comprises an LCVR / HCVR amino acid sequence pair comprising the amino acid sequences of SEQ ID NO: 10 / 2, 26 / 18, 42 / 34, 58 / 50, or 74 / 66. In some cases, the hinge comprises the amino acid sequence of SEQ ID NO: 97. In some cases, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 98. In some cases, the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 99. In some cases, the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 100.

[0022] In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO: 82. In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO: 84. In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO: 86. In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO: 88. In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO: 90.

[0023] In another aspect, the present invention provides a pharmaceutical composition comprising a genetically modified human T cell and a pharma- ceutically acceptable carrier, wherein the genetically modified human T cell comprises a chimeric antigen receptor as described above or herein. In some cases, the pharmaceutical composition is for use in treating a cancer that expresses BCMA. In some embodiments, the cancer that expresses BCMA is multiple myeloma.

[0024] In another aspect, the present invention provides an engineered cell as described above or herein. In some cases, the pharmaceutical composition is for use in treating a BCMA-expressing cancer. In some embodiments, the BCMA-expressing cancer is multiple myeloma.

[0025] In another aspect, the present invention provides the use of a chimeric antigen receptor, a nucleic acid molecule, a vector, a cell, or an engineered cell as described above or herein in the manufacture of a medicament for the treatment of a cancer that expresses BCMA.In some cases, the cancer that expresses BCMA is multiple myeloma.In various embodiments, the chimeric antigen receptor, a nucleic acid molecule, a vector, a cell, or an engineered cell as described above or herein is intended for use in any of the methods described above or herein.For example, in some embodiments, the CAR or engineered cell as described herein is for use in medicine or for use in the treatment of cancer, as described above or herein.

[0026] In another aspect, the present invention provides a method of enhancing T lymphocyte activity in a subject comprising introducing into the subject T lymphocytes that comprise a chimeric antigen receptor as described above or herein.

[0027] In another aspect, the present invention provides a method for treating a subject suffering from cancer comprising introducing into the subject a therapeutically effective amount of T lymphocytes comprising a chimeric antigen receptor as described above or herein.

[0028] In another aspect, the invention provides a method for stimulating a T cell-mediated immune response against a target cell population or tissue in a subject comprising administering to the subject an effective amount of cells genetically modified to express a chimeric antigen receptor as described above or herein.

[0029] In another aspect, the present invention provides a method of providing anti-tumor immunity to a subject, the method comprising administering to the subject an effective amount of cells genetically modified to express a chimeric antigen receptor as described above or herein.

[0030] In some embodiments of the method discussed above, the subject is human.In some cases, the subject suffers from multiple myeloma, B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin's lymphoma, leukemia and lymphoma, acute lymphoblastic leukemia, Hodgkin's lymphoma, or childhood acute lymphoblastic leukemia.In some embodiments, the subject suffers from multiple myeloma.

[0031] In another aspect, the invention provides a method of engineering a population of cells to express a chimeric antigen receptor, the method comprising: (a) providing a population of immune cells, (b) introducing into the immune cells a nucleic acid molecule encoding a chimeric antigen receptor as discussed above or herein, (c) culturing the immune cells under conditions to express the nucleic acid molecule, and (d) isolating the immune cells that express the chimeric antigen receptor on their cell surface. Optionally, the method further comprises obtaining the population of immune cells from a subject prior to introducing the nucleic acid molecule.

[0032] In another aspect, the invention provides a method of treating a BCMA-expressing cancer in a subject, the method comprising (a) manipulating a population of cells according to the methods discussed above, and (b) reintroducing into the subject a population of immune cells that express a chimeric antigen receptor. In some embodiments, the BCMA-expressing cancer is multiple myeloma.

[0033] Other embodiments will become apparent from review of the following detailed description. [Brief description of the drawings]

[0034] [Figure 1] 1 shows an exemplary nucleotide construct for expressing a chimeric antigen receptor (CAR) construct. The exemplary nucleotide construct includes an anti-BCMA VL-linker-VH scFv, a human CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, a CD3 zeta signaling domain, and an IRES:eGFP sequence for tracking CAR-transduced cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Before describing the present invention, it should be understood that the present invention is not limited to the specific method and experimental conditions described, since such methods and conditions may vary. It should also be understood that the terms used in this specification are only for describing specific embodiments, and are not intended to be limiting, since the scope of the present invention is limited only by the scope of the appended claims. Any embodiment or embodiment features can be combined with each other, and such combinations are expressly included within the scope of the present invention. Any specific value discussed above or herein can be combined with another related value discussed above or herein to recite a range with values ​​representing the upper and lower limits of the range, and such ranges are included within the scope of the present disclosure.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs.As used in the present invention, the term "about" when used in relation to a specific recited numerical value means that the value can vary by 1% or less from the recited value.For example, as used in the present invention, the expression "about 100" includes 99 and 101, and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0037] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0038] definition As used herein, the term "BCMA" refers to B cell maturation antigen. BCMA (also known as TNFRSF17 and CD269) is a cell surface protein expressed on malignant plasma cells and plays a central role in regulating B cell maturation and differentiation into immunoglobulin-producing plasma cells. As used herein, "BCMA" refers to B cell maturation antigen expressed in non-human species ( Unless otherwise specified as being derived from a human BCMA protein (e.g., "mouse BCMA", "monkey BCMA", etc.), the term refers to a human BCMA protein. The human BCMA protein has the amino acid sequence shown in SEQ ID NO: 101.

[0039] As used herein, an "antibody that binds BCMA" or an "anti-BCMA antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize BCMA.

[0040] The terms "ligand binding domain" and "antigen binding domain" are used interchangeably herein and refer to the portion of a chimeric antigen receptor or corresponding antibody that specifically binds to a given antigen (e.g., BCMA). Reference to a "corresponding antibody" refers to the antibody from which the CDRs or variable regions (HCVR and LCVR) used in the chimeric antigen receptor are derived. For example, the chimeric antigen receptor construct discussed in Example 2 comprises an scFv with variable regions derived from a specific anti-BCMA antibody. These anti-BCMA antibodies are the "corresponding antibodies" to the respective chimeric antigen receptor.

[0041] The term "antibody" as used herein refers to any antigen-binding molecule or molecular complex that contains at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., BCMA). The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). The term "antibody" also includes immunoglobulin molecules that consist of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (HCVR or VVR, as used herein). H and heavy chain constant The heavy chain constant region contains three domains, C H 1. C H 2, and C H Includes 3. Each light chain comprises a light chain variable region (referred to herein as LCVR or V L (abbreviated as ) and light chain constant The light chain constant region comprises one domain (C L 1) is included. H Area and V L region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-BCMA antibody (or antigen-binding portion thereof) may be identical to the human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on the parallel analysis of two or more CDRs.

[0042] The term "antibody" as used herein also includes antigen-binding fragments of complete antibody molecules. An "antigen-binding portion" of an antibody, an "antigen-binding fragment" of an antibody, and similar terms as used herein include naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from complete antibody molecules using any suitable standard techniques, such as, for example, proteolytic or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated, for example, chemically or by using molecular biology techniques, to place one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0043] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) Fc fragments, (v) Fd fragments, (v) Fc ... v) single chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs), such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. Domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0044] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and generally contains at least one CDR that is adjacent to or in frame with one or more framework sequences. L V bound to the domain H In an antigen-binding fragment having a domain, H Domains and V L The domains can be arranged relative to each other in any suitable configuration. For example, the variable region is a dimer and the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of the antibody may comprise a monomeric V H D Main or V L It may include a domain.

[0045] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) a V H -C H 1, (ii) V H -C H 2. (iii) V H -C H 3. (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2 -C H 3. (vii) V H -C L , (viii) V L -C H 1, (ix) VL -C H 2, (x) V L -C H 3. (xi) V L -C H 1-C H 2. (xii) V L -C H 1-C H 2-C H 3. (x iii) V L -C H 2-C H 3, and (xiv) V L -C L The above examples are listed below. In any configuration of the variable and constant domains, including any of the configurations shown, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible link between adjacent variable and / or constant domains in a single polypeptide molecule. Moreover, antigen-binding fragments of the antibodies of the present invention may be linked to each other and / or to one or more monomeric V H D Main or V L Non-covalently linked to the domain (e.g., by disulfide bond(s)) The combination may include homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above.

[0046] In certain embodiments, the anti-BCMA antibody is a human antibody. The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibody of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation), for example in the CDRs, particularly CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.

[0047] The antibody may in some embodiments be a recombinant human antibody. As used herein, the term "recombinant human antibody" refers to an antibody that has been prepared, expressed, created or isolated by recombinant means, such as an antibody expressed using a recombinant expression vector transfected into a host cell. It is intended to include all human antibodies (described below), antibodies isolated from recombinant combinatorial human antibody libraries (described below), antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created or isolated by any other means including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, in the case of animals transgenic for human Ig sequences, in vivo somatic mutagenesis) to thereby modify the V and V regions of the recombinant antibodies. H and V L The amino acid sequence of the region is H and V LThese are sequences that, although derived and related to the sequences, may not naturally occur within the human antibody germline repertoire in vivo.

[0048] Human antibodies can exist in two forms related to hinge heterogeneity. In the first form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked via interchain disulfide bonds, and a molecule of approximately 75-80 kDa consisting of covalently linked light and heavy chains is formed (half antibody). These forms have proven extremely difficult to separate, even after affinity purification.

[0049] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed using a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present invention provides a method for determining whether the second form occurs in the hinge, C H 2 or C H Antibodies with one or more mutations in three regions are included, which may be desirable, for example, in production to improve the yield of the desired antibody form.

[0050] The antibody may be an isolated antibody. As used herein, "isolated antibody" refers to an identified antibody, as well as an antibody that has been separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which it naturally occurs or is naturally produced, is an "isolated antibody" for the purposes of the present invention. An isolated antibody also includes an antibody in situ in a recombinant cell. An isolated antibody is an antibody that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0051] The anti-BCMA antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the antibody was derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily produce many antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain In the embodiment, V H and / or V LAll framework and / or CDR residues within the domain are mutated back to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., the mutated residue is found within the first 8 amino acids of FR1, or the mutated residue is found within the last 8 amino acids of FR4, or the mutated residue is found only in CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations in the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the scope of the present invention.

[0052] Anti-BCMA antibodies may include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, anti-BCMA antibodies may have HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or less, 8 or less, 6 or less, or 4 or less conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences described herein.

[0053] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes may be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are those generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include portions of sugars, phosphoryl groups, or sulfonyl groups on an antigen.

[0054] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or a fragment thereof, indicates that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% nucleotide sequence identity of the nucleotide bases as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0055] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT using predefined gap weights. Preferably, residue positions that are not identical differ only by conservative amino acid substitutions. "Conservative amino acid substitutions" refer to amino acid residues that have side chains (R groups) with similar chemical properties (e.g., charge or hydrophobicity). ) is replaced by another amino acid residue having a different structure. In general, conservative amino acid substitutions are not expected to substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, (2) aliphatic-hydroxyl side chains: serine and threonine, (3) amide-containing side chains: asparagine and glutamine, (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, (5) basic side chains: lysine, arginine, and histidine, (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0056] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software uses similarity measurements assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions, to match similar sequences. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the region of best overlap between the query sequence and the search sequence (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0057] As used herein, the term "nucleic acid" or "polynucleotide" refers to nucleotides and / or polynucleotides (such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)), oligonucleotides, fragments generated by polymerase chain reaction (PCR), and fragments generated by either ligation, cleavage, endonuclease action, or exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have changes in the sugar moiety and / or in the pyrimidine or purine base moiety. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar can be functionalized as an ether or ester. In addition, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as azasugars and carbocyclic sugar analogs. Examples of modifications of the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutions. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Nucleic acids can be either single-stranded or double-stranded.

[0058] The term "chimeric antigen receptor" (CAR) refers to a molecule that combines a T cell receptor activating intracellular domain with a binding domain for a component present on a target cell, such as specificity for a desired antigen (e.g., a tumor antigen such as BCMA), to generate a chimeric protein that exhibits specific anti-target cell immune activity. Generally, CARs consist of an extracellular single-chain antibody binding domain (scFv) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain, and when expressed in a T cell, have the ability to redirect antigen recognition based on the specificity of a monoclonal antibody.

[0059] The term "vector" as used herein includes, but is not limited to, viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules that may consist of chromosomal, non-chromosomal, semisynthetic or synthetic nucleic acids. In some cases, vectors are capable of autonomous replication (episomal vectors) and / or expression of nucleic acids to which they are linked (expression vectors). Many suitable vectors are known to those of skill in the art and are commercially available. Viral vectors include negative-stranded RNA viruses such as retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, orthomyxoviruses (e.g., influenza viruses), positive-stranded RNA viruses such as rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), picornaviruses and alphaviruses, as well as double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox and canarypox).Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus and hepatitis virus.Examples of retroviruses include avian leukemia-sarcoma, mammalian C, B, D viruses, HTLV-BLV group, and lentivirus.

[0060] "Costimulatory domain" or "costimulatory molecule" refers to the cognate binding partner on T cells that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors. Examples of costimulatory molecules include ligands that specifically bind to CD27, CD28, CD8, 4-1BB (CD137) (SEQ ID NO: 99), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and CD83, etc. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for an effective immune response.

[0061] "Costimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal in addition to the primary signal provided, for example, by the binding of a peptide-loaded MHC molecule to the TCR / CD3 complex, to mediate T cell responses, including, but not limited to, proliferation, activation, differentiation, etc. Costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1 These include, but are not limited to, agonists or antibodies that bind CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, Toll ligand receptor, and ligands that specifically bind B7-H3.

[0062] A "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or up- or down-regulation of key molecules.

[0063] The term "extracellular ligand-binding domain" as used herein refers to an oligo- or polypeptide capable of binding to a ligand, e.g., a cell surface molecule. For example, the extracellular ligand-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state (e.g., cancer). Examples of cell surface markers that act as ligands include those associated with viral, bacterial and parasitic infections, autoimmune diseases and cancer cells.

[0064] As used herein, the term "subject" or "patient" includes all members of the animal kingdom, including non-human primates and humans. In one embodiment, the patient is a human with cancer (e.g., multiple myeloma).

[0065] As used herein, the "signaling domain" or "signaling domain" of a CAR is responsible for intracellular signal transduction following binding of the extracellular ligand-binding domain to a target, resulting in immune cell activation and immune response. In other words, the signaling domain is responsible for activation of at least one normal effector function of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be cytotoxic activity or helper activity, including secretion of cytokines. Thus, the term "signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Examples of signaling domains for use in CARs can be the cytoplasmic sequences of T cell receptors and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences, as well as any synthetic sequence with the same functional capabilities. In some cases, the signaling domain includes two different classes of cytoplasmic signaling sequences, those that initiate antigen-dependent primary activation and those that act antigen-independently to provide secondary or costimulatory signals. The primary cytoplasmic signaling sequence may comprise a signaling motif known as the immunoreceptor tyrosine-based activation motif of ITAM. ITAM is a well-defined signaling motif found in the cytoplasmic tail of various receptors that serves as a binding site for syk / zap70 class tyrosine kinases. Exemplary ITAMs include those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d. In some embodiments, the signaling domain of the CAR can comprise the CD3 zeta signaling domain (SEQ ID NO: 100).

[0066] Chimeric antigen receptors (CARs) Chimeric antigen receptors (CARs) redirect T cell specificity to antibody-recognized antigens expressed on the cell surface (e.g., cancer cells), while T cell receptors (TCRs) expand the range of targets to include intracellular antigens (e.g., tumor antigens).

[0067] One aspect of the present invention comprises a chimeric antigen receptor (CAR) specific for B cell maturation antigen (BCMA) expressed on the surface of malignant plasma cells. In one embodiment of the present invention, a CAR as described herein comprises an extracellular target specific binding domain, a transmembrane domain, an intracellular signaling domain (such as a signaling domain derived from CD3 zeta or FcR gamma), and a CAR-specific binding domain (such as a CAR-specific binding domain derived from CD3 zeta or FcR gamma). ), and / or one or more costimulatory signaling domains derived from costimulatory molecules such as, but not limited to, 4-1BB. In one embodiment, the CAR comprises a hinge or spacer region between the extracellular binding domain and the transmembrane domain, such as a CD8 alpha hinge.

[0068] The binding domain or extracellular domain of the CAR provides the CAR with the ability to bind to a target antigen of interest. The binding domain (e.g., ligand binding domain or antigen binding domain) can be any protein, polypeptide, oligopeptide, or peptide that has the ability to specifically recognize and bind to a biomolecule (e.g., a cell surface receptor or tumor protein, or a component thereof). The binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for the biomolecule of interest. For example, as further described herein, the binding domain can be an antibody light chain and heavy chain variable region, or the light chain and heavy chain variable region can be bound to each other in a single chain and in any orientation (e.g., VL-VH or VH-VL). Various assays are known to identify the binding domain of the present disclosure that specifically binds to a particular target, including Western blot, ELISA, flow cytometry, or surface plasmon resonance analysis (e.g., using BIACORE analysis). The antigen can be an antigen of clinical interest where it is desired to induce an effector immune response that results in tumor killing. In one embodiment, the target antigen of the binding domain of the chimeric antigen receptor is the BCMA protein on the surface of a tumor cell, particularly a tumor cell of the B-cell lineage, such as a multiple myeloma cell.

[0069] Exemplary ligand binding domains include antigen binding proteins such as antigen-binding fragments of antibodies, such as scFvs, scTCRs, extracellular domains of receptors, ligands for cell surface molecules / receptors, or receptor binding domains thereof, and tumor binding proteins. In certain embodiments, the antigen binding domains included in the CARs of the present invention can be variable regions (Fvs), CDRs, Fabs, scFvs, VHs, VLs, domain antibody variants (dAbs), camelid antibodies (VHHs), fibronectin 3 domain variants, ankyrin repeat variants, and other antigen-specific binding domains derived from other protein scaffolds.

[0070] In one embodiment, the binding domain of the CAR is an anti-BCMA single chain antibody (scFv), which can be a mouse, human, or humanized scFv. Single chain antibodies can be cloned from the V region genes of hybridomas specific for the desired target. Techniques that can be used to clone the variable heavy chain (VH) and variable light chain (VL) are described, for example, in Orlandi et al., PNAS, 1989;86:3833-3837. Thus, in certain embodiments, the binding domain comprises an antibody-derived binding domain, but can also be a non-antibody-derived binding domain. An antibody-derived binding domain can be a fragment of an antibody, or a genetically engineered product of one or more fragments of an antibody, which fragment is involved in binding to an antigen.

[0071] In certain embodiments, the CAR of the present invention may include linkers between various domains, added for proper spacing and conformation of the molecule. For example, in one embodiment, there may be a linker between the binding domains VH or VL, which may be 1-10 amino acids long. In other embodiments, the linker between any of the domains of the chimeric antigen receptor may be between 1-20 or 20 amino acids long. In this regard, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. In further embodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids long. Ranges including the numbers described herein are also included herein, for example, linkers of 10-30 amino acids long.

[0072] In certain embodiments, a linker suitable for use in the CAR described herein is a flexible linker. Suitable linkers can be easily selected and can be of any of different lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 3 to 12 amino acids, including 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0073] Exemplary flexible linkers include glycine polymers (G), glycine-serine polymers (n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can function as neutral tethers between domains of fusion proteins such as the CARs described herein. Glycine has access to much more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Those skilled in the art will recognize that the design of a CAR can include a linker that is fully or partially flexible, such that the linker can include a flexible linker and one or more moieties that confer a less flexible structure to provide the desired CAR structure. Specific linkers include (G4S) n Linkers (n=1 to 3, as shown in SEQ ID NOs: 95 to 97), and sequences An example of the linker is shown as number 96.

[0074] The binding domain of a CAR may be followed by a "spacer" or "hinge," which refers to a region that distances the antigen-binding domain from the effector cell surface to allow proper cell / cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). The hinge region of a CAR is generally between the transmembrane (TM) and the binding domain. In certain embodiments, the hinge region is an immunoglobulin hinge region, which may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. Other exemplary hinge regions used in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins, such as CD8 alpha, CD4, CD28 and CD7, which may be wild-type hinge regions from these molecules or may be modified. In one embodiment, the hinge region comprises a CD8 alpha hinge (SEQ ID NO: 97).

[0075] The "transmembrane" region or domain is the portion of the CAR that anchors the extracellular binding moiety to the plasma cell membrane of immune effector cells and facilitates the binding of the binding domain to the target antigen. The transmembrane domain can be the CD3 zeta transmembrane domain, but other transmembrane domains that can be used include those obtained from CD8 alpha, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain is the transmembrane domain of CD137. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 98. In certain embodiments, the transmembrane domain is synthetic, in which case it will comprise mainly hydrophobic residues such as leucine and valine.

[0076] "Intracellular signaling domain" or "signaling domain" refers to the portion of the chimeric antigen receptor protein that is involved in transmitting the message of effective CAR binding to a target antigen to the inside of immune effector cells and inducing effector cell functions, such as, for example, cytotoxic activity including activation, cytokine production, proliferation and release of cytotoxic factors against CAR-bound target cells, or other cellular responses elicited by antigen binding to the extracellular CAR domain. The term "effector function" refers to a specialized function of a cell. Effector functions of T cells include, for example, cytotoxic activity, or helper cell functions including secretion of cytokines. The term "intracellular signaling domain" or "signaling domain" as used interchangeably herein therefore refers to the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Usually, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the entire domain, so long as it transmits an effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal. The intracellular signaling domain is also known as the "signaling domain" and is typically derived from a portion of the human CD3 or FcRy chain.

[0077] It is known that signals generated solely through the T cell receptor are insufficient for the complete activation of T cells, and that secondary or costimulatory signals are also required. Thus, it can be said that T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation (primary cytoplasmic signaling sequences) and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). Cytoplasmic signaling sequences that act in a costimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0078] Examples of ITAMs comprising primary cytoplasmic signaling sequences that are particularly useful in the present invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d. In one particular embodiment, the intracellular signaling domain of the anti-BCMA CAR described herein is derived from CD3 zeta. In some embodiments, the signaling domain comprises the amino acid sequence of SEQ ID NO: 100.

[0079] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or an Fc receptor that, upon binding to an antigen, provides a second signal necessary for effective activation and function of T lymphocytes. Examples of such costimulatory molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2, and CD83. Thus, although the present disclosure provides exemplary costimulatory domains derived from CD3 zeta and 4-1BB, other costimulatory domains are contemplated for use with the CARs described herein. The inclusion of one or more costimulatory signaling domains can promote the efficacy and expansion of T cells expressing a CAR receptor. The intracellular signaling and costimulatory signaling domains can be tandemly linked in any order to the carboxyl terminus of the transmembrane domain. In some embodiments, the costimulatory domain comprises the amino acid sequence of SEQ ID NO:99.

[0080] Although scFv-based CARs engineered to contain signaling domains from CD3 or FcR gamma have been shown to deliver potent signals for T cell activation and effector function, they are not sufficient to induce signals that promote T cell survival and expansion in the absence of concomitant costimulatory signals. Other CARs containing signaling domains derived from CD3 zeta or FcR gamma with binding domains, hinges, transmembrane domains and one or more costimulatory signaling domains (e.g., intracellular costimulatory domains from CD28, CD137, CD134 and CD278) have demonstrated antitumor activity as well as in CAR expressing T cells in vitro and in animal models and In cancer patients, increased cytokine secretion, lytic activity, survival and T cell proliferation may be more effectively directed (Milone et al., Molecular Therapy, 2009; 17: 1453-1464; Zhong et al., Molecular Therapy, 2010; 18: 413-420; Carpenito et al., PNAS, 2009; 106: 3360-3365).

[0081] The BCMA CAR of the present invention comprises (a) an anti-BCMA scFv as a binding domain (a scFv having a binding region (e.g., CDR or variable domain) from one or more of the BCMA antibodies identified in Table 1), (b) a hinge region derived from human CD8 alpha, (c) a human CD8 alpha transmembrane domain, and (d) a human T cell receptor CD3 zeta chain (CD3) intracellular signaling domain, and optionally one or more costimulatory signaling domains (e.g., 4-1BB). In one embodiment, the different protein domains are arranged from amino-terminus to carboxyl-terminus in the following order: binding domain, hinge region, and transmembrane domain. The intracellular signaling domain and optional costimulatory signaling domain are linked in tandem in any order to the transmembrane carboxy terminus to form a single chain chimeric polypeptide. In one embodiment, the nucleic acid construct encoding a BCMA CAR is a chimeric nucleic acid molecule comprising different coding sequences, for example, a nucleic acid molecule comprising a (5' to 3') coding sequence for a human anti-BCMA scFv, a human CD8 alpha-hinge, a human CD8 alpha transmembrane domain, and a CD3 zeta intracellular signaling domain. In another embodiment, the nucleic acid construct encoding a BCMA CAR is a chimeric nucleic acid molecule comprising a nucleic acid molecule comprising a (5' to 3') coding sequence for a human anti-BCMA scFv, a human CD8 alpha-hinge, a human CD8 alpha transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta costimulatory domain.

[0082] In certain embodiments, the polynucleotide encoding the CAR described herein is inserted into a vector. A vector is a vehicle into which a polynucleotide encoding a protein can be covalently inserted to result in the expression of the protein and / or cloning of the polynucleotide. Such vectors are sometimes referred to as "expression vectors". An isolated polynucleotide can be inserted into a vector using methods known in the art, such as, but not limited to, digesting the vector with an appropriate restriction enzyme and then ligating with the isolated polynucleotide with the corresponding restriction end. An expression vector has the ability to incorporate and express heterologous or modified nucleic acid sequences that code for at least a portion of a gene product that can be transcribed in a cell. In most cases, the RNA molecule is then translated into a protein. An expression vector can contain various control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to the control sequences that govern transcription and translation, vectors and expression vectors can contain nucleic acid sequences that also serve other functions, which are discussed below. An expression vector may contain additional elements, for example, an expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example, a human cell for expression and a prokaryotic host for cloning and amplification.

[0083] The expression vector may have necessary 5' upstream and 3' downstream regulatory elements such as promoter sequences such as CMV, PGK and EF1 alpha promoters, ribosome recognition and binding TATA boxes, and 3'UTR AAUAAA transcription termination sequence for effective gene transcription and translation in the respective host cells. Other suitable promoters include the constitutive promoter of Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukosis virus promoter, EBV immediate early promoter and Rous sarcoma virus promoter. Actin promoter, myosin promoter, hemoglob ... Human gene promoters can also be used, including but not limited to Robin promoter and creatine kinase promoter.In certain embodiments, inducible promoters are also contemplated as part of the vector that expresses chimeric antigen receptor.This provides a molecular switch that can turn on or off the expression of the polynucleotide sequence of interest.Examples of inducible promoters include but are not limited to metallothionein promoter, glucocorticoid promoter, progesterone promoter or tetracycline promoter.

[0084] The expression vector may have additional sequences such as 6x-histidine, c-Myc, and FLAG tags that are incorporated into the expressed CAR. Thus, the expression vector may be engineered to include 5' and 3' untranslated regulatory sequences that can sometimes act as enhancer sequences, promoter regions and / or terminator sequences that can facilitate or enhance the effective transcription of the nucleic acid(s) of interest carried in the expression vector. The expression vector may also be engineered for replication and / or expression function (e.g., transcription and translation) in a specific cell type, cell location, or tissue type. The expression vector may include a selectable marker for the maintenance of the vector in the host or recipient cell.

[0085] In various embodiments, vector is a plasmid, an autonomously replicating sequence, and a transposable element.Additional exemplary vectors include, but are not limited to, plasmid, phagemid, cosmid, yeast artificial chromosome (YAC), artificial chromosome such as bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC), bacteriophage such as lambda phage or M13 phage, and animal virus.Examples of animal virus categories useful as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), pox virus, baculovirus, papilloma virus, and papova virus (e.g., SV40). Examples of expression vectors are the Lenti-X™ bicistronic expression system (Neo) vector (Clontrch), pClneo vectors (Promega) for expression in mammalian cells, such as pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequence of the CAR disclosed herein can be linked to such expression vectors for expression of chimeric proteins in mammalian cells.

[0086] In certain embodiments, the nucleic acid encoding the CAR of the present invention is provided in a viral vector. The viral vector can be derived from a retrovirus, lentivirus, or foamy virus. As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into a viral vector particle. The viral vector can contain the coding sequence of various chimeric proteins described herein in place of non-essential viral genes. The vector and / or particle can be utilized to transfer DNA, RNA, or other nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0087] In certain embodiments, the viral vector comprising the coding sequence of CAR described herein is a retroviral vector or lentiviral vector.The term "retroviral vector" refers to the vector that comprises the structural and functional genetic elements that are mainly derived from retrovirus.The term "lentiviral vector" refers to the vector that comprises the structural and functional genetic elements outside LTR that are mainly derived from lentivirus.

[0088] Retroviral vectors as used herein can be derived from any known retrovirus (e.g., c-type retroviruses such as Moloney Murine Sarcoma Virus (MoMSV), Harvey Murine Sarcoma Virus (HaMuSV), Murine Mammary Tumor Virus (MuMTV), Gibbon Ape Leukemia Virus (GaLV), Feline Leukemia Virus (FLV), Spumavirus, Friend, Murine Stem Cell Virus (MSCV) and Avian Sarcoma Virus (RSV)). "Retroviruses" of the present invention also include the lentivirus family of retroviruses, such as human T-cell leukemia viruses, HTLV-1 and HTLV-2, as well as human immunodeficiency viruses, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV) and other classes of retroviruses.

[0089] Lentiviral vector for use herein refers to vector derived from lentivirus, which is a group (or genus) of retroviruses that cause slowly progressing diseases. Viruses included in this group include HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2), Visna-Maedi, Caprine Arthritis-Encephalitis Virus, Equine Infectious Anemia Virus, Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV). Preparation of recombinant lentivirus can be achieved using the method by Dull et al. and Zufferey et al. (Dull et al., J.Virol., 1998; 72: 8463-8471 and Zufferey et al., J.Virol. 1998; 72: 9873-9880).

[0090] Retroviral vectors (i.e., both lentiviral and non-lentiviral) for use in the present invention can be formed using standard cloning techniques by combining the desired DNA sequences in the order and orientation described herein (Current Protocols in Molecular Biology, Ausubel, FM et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals; Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1989 ... al.(1990)Proc.Natl.Acad.Sci.USA87:6141-6145;Huber et al.(1991)Proc.Natl.Acad.Sci.USA88:8039-8043;Ferry et al.(1991)Proc.Natl.Acad.Sci.USA88:8377-8381;Chowdhury et al. al.(1991)Science254:1802-1805;van Beusechem et al.(1992)Proc.Natl.Acad.Sci.USA89:7640-7644;Kay et al.(1992)Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application No. WO 89 / 07136; PCT Application No. WO 89 / 02468; PCT Application No. WO 89 / 05345; and PCT Application No. WO 92 / 07573).

[0091] Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) sequences for use in forming vectors include, for example, Type 2 retroviral vectors. The sequence includes genomic RNA and cDNA available from commercial sources, including the American College of Culture Collection (ATCC), Rockville, Md. It can also be chemically synthesized.

[0092] For the expression of BCMA CAR, the vector can be introduced into a host cell to allow expression of the polypeptide in the host cell. The expression vector can include various elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements can be appropriately selected by those skilled in the art, as described above. For example, the promoter sequence can be selected to promote the transcription of the polynucleotide in the vector. Suitable promoter sequences include but are not limited to T7 promoter, T3 promoter, SP6 promoter, beta-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter. Enhancer sequences can be selected to enhance the transcription of the polynucleotide. The selectable marker can be selected to allow the host cell inserted with the vector to be selected from those that are not, for example, the selectable marker can be a gene that confers antibiotic resistance. The signal sequence can be selected to allow the expressed polypeptide to be transported out of the host cell.

[0093] For cloning polynucleotides, vectors can be introduced into host cells (isolated host cells) to allow the vectors to replicate themselves, thereby amplifying the copies of the polynucleotides contained therein. Cloning vectors can generally contain sequence components, including but not limited to, replication origins, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements can be appropriately selected by those skilled in the art. For example, replication origins can be selected to promote the autonomous replication of vectors in host cells.

[0094] In certain embodiments, the present disclosure provides an isolated host cell comprising the vector provided herein. The host cell comprising the vector can be useful for expressing or cloning the polynucleotide contained in the vector. Suitable host cells can include, but are not limited to, higher eukaryotic cells, such as prokaryotic cells, fungal cells, yeast cells, or mammalian cells. Suitable prokaryotic cells for this purpose include, but are not limited to, eubacteria, such as gram-negative or gram-positive bacteria, for example, Enterobacteriaceae, such as Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., S. seratina), and Shigella, as well as bacilli, such as Bacillus subtilis and Bacillus licheniformis, Pseudomonas, such as Pseudomonas aeruginosa, and Streptomyces.

[0095] The CAR of the present invention is introduced into a host cell using transfection and / or transduction techniques known in the art. As used herein, the terms "transfection" and "transduction" refer to the process by which an exogenous nucleic acid sequence is introduced into a host cell. The nucleic acid may be integrated into the DNA of the host cell or may be maintained extrachromosomally. The nucleic acid may be maintained transiently or may be stably introduced. Transfection may be achieved by a variety of methods known in the art, including but not limited to calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics. Transduction refers to the delivery of a gene(s) with a viral or retroviral vector by viral infection rather than transfection. In certain embodiments, a retroviral vector is transduced by packaging the vector into virions prior to contacting a cell. For example, the nucleic acid encoding the BCMA CAR carried by the retroviral vector is expressed by the combination of infection and provirus. Cells can be transduced via integration.

[0096] As used herein, the terms "genetically engineered" or "genetically modified" refer to the addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms "genetically modified cell," "modified cell," and "redirected cell" are used interchangeably.

[0097] In particular, the CARs of the present invention are introduced into and expressed in immune effector cells to redirect their specificity to a target antigen of interest, e.g., malignant plasma cells, e.g., multiple myeloma.

[0098] The present invention provides a method for producing immune effector cells expressing a CAR as described herein. In one embodiment, the method includes transfecting or transducing immune effector cells isolated from a subject, such as a subject with BCMA-expressing tumor cells, such that the immune effector cells express one or more CARs as described herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly re-administered to the individual. In a further embodiment, the immune effector cells are first activated, stimulated, and expanded in vitro before being genetically modified to express a CAR. In this regard, the immune effector cells can be cultured before or after being genetically modified (i.e., transduced or transfected to express a CAR as described herein).

[0099] Prior to in vitro manipulation or genetic modification of immune effector cells as described herein, a source of cells can be obtained from a subject. In particular, immune effector cells for use with CARs as described herein include T cells. T cells can be obtained from multiple sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus matter, tissue at the site of infection, ascites, pleural effusion, splenic tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL separation. In one embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing. In one embodiment of the present invention, the cells are washed with PBS. In alternative embodiments, the washed solution may lack calcium, lack magnesium, or lack many, if not all, divalent cations. As will be appreciated by those skilled in the art, the washing step may be accomplished by methods known to those skilled in the art, such as using a semi-automated flow-through centrifuge. After washing, the cells may be resuspended in a variety of biocompatible buffers or other saline solutions with or without buffers. In certain embodiments, undesirable components of the apheresis sample may be removed in culture medium resuspended directly on the cells.

[0100] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient. Specific subpopulations of T cells, such as CD28+, CD4+, CD8+, CD45RA+ and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, enrichment of T cell populations by negative selection can be achieved with a combination of antibodies directed against surface markers unique to the negatively selected cells. One method used herein uses a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells, Cell sorting and / or selection via magnetic immunoadhesion or flow cytometry.For example, to enrich CD4+ cells by negative selection, monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8.Flow cytometry and cell sorting can also be used to isolate the cell population of interest for use in the present invention.

[0101] PBMCs can be used directly for genetic modification with CARs using methods as described herein. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes can be classified into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion. CD8+ cells can be obtained using standard methods. In some embodiments, CD8+ cells are further classified into naive, central memory, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In embodiments, memory T cells are present in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs are classified into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, expression of phenotypic markers of central memory TCMs includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and is negative for granzyme B. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and are positive for granzyme B and perforin. In some embodiments, naive CD8+ T lymphocytes are characterized by expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD3, CD 127, and CD45RA.

[0102] In certain embodiments, CD4+ T cells are further classified into subpopulations. For example, CD4+ helper T cells can be classified into naive, central memory, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+ CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L positive and CD45RO positive. In some embodiments, effector CD4+ cells are CD62L and CD45RO negative.

[0103] Immune effector cells such as T cells can be genetically modified after isolation using known methods, or immune effector cells can be activated and expanded (or differentiated in the case of precursor cells) in vitro before being genetically modified. In another embodiment, immune effector cells such as T cells are genetically modified with a chimeric antigen receptor as described herein (e.g., transduced with a viral vector containing a nucleic acid encoding a CAR) and then activated and expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Pat. No. 6,905,874; U.S. Pat. No. 6,867,041; U.S. Pat. No. 6,797,514; WO2012 / 079000. In general, such methods include contacting PBMCs or isolated T cells with stimulatory and costimulatory agents, such as anti-CD3 and anti-CD28 antibodies, attached to beads or other surfaces, usually in a medium containing an appropriate cytokine, such as IL-2. Anti-CD3 and anti-CD28 antibodies attached to the same beads serve as "surrogate" antigen-presenting cells (APCs). In other embodiments, the T cells are those described in U.S. Pat. No. 6,040,177; U.S. Pat. No. 5,827,642; and WO2012 / 129514. Using methods such as those described, cells can be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines.

[0104] The present invention provides a population of engineered immune effector cells for the treatment of patients having a BCMA-expressing tumor, e.g., a malignant tumor caused by multiple myeloma, wherein the engineered immune effector cells comprise a BCMA CAR as disclosed herein.

[0105] The immune effector cells expressing CAR prepared as described herein can be used in methods and compositions for adoptive immunotherapy according to known techniques or their modifications that will be apparent to those skilled in the art based on this disclosure.See, for example, U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.See also U.S. Patent No. 4,690,915 to Rosenberg.

[0106] In some embodiments, the cells are formulated by first harvesting them from their culture medium, then washing and concentrating the cells in a medium and container system suitable for administration in a therapeutically effective amount (a "pharmaceutical acceptable" carrier). A suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), although 5% dextrose in water or Ringer's lactate can also be utilized. The infusion medium can be supplemented with human serum albumin.

[0107] At least 2 cells (e.g., at least one CD8+ central memory T cell and at least one CD4+ helper T cell subset) or, more commonly, 10 2 More than 10 cells 6 Up to 10 8 Or 10 9 Contains cells, 10 10 The number of cells will depend on the end use for which the composition is intended, as well as the type of cells contained therein.

[0108] The cells may be autologous or heterologous to the patient undergoing treatment. Optionally, the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-α, IL-18 and TNF-β, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein to enhance induction of an immune response.

[0109] The CAR-expressing immune effector cell population of the present invention can be administered as a pharmaceutical composition alone or in combination with other components such as diluents and / or IL-2 or other cytokines or cell populations. Briefly, the pharmaceutical composition of the present invention can include a CAR-expressing immune effector cell population, such as T cells, as described herein, in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents or excipients. Such compositions can include a buffer, such as neutral buffered saline, phosphate buffered saline; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants, such as aluminum hydroxide; and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0110] The anti-tumor immune response induced in a subject by administering the CAR-expressing T cells described herein using the methods described herein or other methods known in the art may include a cellular immune response mediated by cytotoxic T cells that can kill infected cells, regulatory T cells and helper T cell responses. A humoral immune response mediated primarily by helper T cells that can activate B cells to produce antibodies may also be induced. The anti-tumor immune response induced by the composition of the present invention, which has been fully described in the art, may also be induced. Various techniques can be used to analyze the type of immune response (e.g., Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, NY).

[0111] Thus, the invention provides a method of treating an individual diagnosed with, suspected of having, or at risk of developing a hematopoietic malignancy characterized in part by abnormal accumulation of immunoglobulin-producing plasma cells in the bone marrow, such as multiple myeloma, comprising administering to the individual a therapeutically effective amount of immune effector cells expressing a CAR as described herein.

[0112] In one embodiment, the invention provides a method of treating a subject diagnosed with a cancer that expresses BCMA comprising removing immune effector cells from a subject diagnosed with a cancer that expresses BCMA, genetically modifying said immune effector cells with a vector comprising a nucleic acid encoding a chimeric antigen receptor of the invention, thereby forming a population of modified immune effector cells, and administering the population of modified immune effector cells to the same subject. In one embodiment, the immune effector cells comprise T cells.

[0113] Methods for administering the cell compositions described herein include any method that is effective to result in the reintroduction of ex vivo genetically modified immune effector cells, either directly expressing a CAR of the invention in the subject, or reintroduction of genetically modified precursor cells of immune effector cells that differentiate into mature immune effector cells expressing a CAR when introduced into a subject. One method involves transducing peripheral blood T cells ex vivo with a nucleic acid construct according to the invention and returning the transduced cells to the subject.

[0114] Binding characteristics of chimeric antigen receptors and corresponding antibodies As used herein, the term "binding" in the context of binding of a chimeric antigen receptor or a corresponding antibody to either a given antigen, e.g., a cell surface protein or fragment thereof, refers to an interaction or association between at least two entities or molecular structures, such as an antigen-binding domain:antigen interaction.

[0115] For example, binding affinities, as determined by surface plasmon resonance (SPR) techniques, e.g., on a BIAcore3000 instrument, using an antigen as the ligand and an antibody or chimeric antigen receptor as the analyte (or antiligand), are typically around 10 -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 K below M D Corresponding values. Fluorescence-activated cell sorting ( Cell-based binding strategies such as FACS (FACS) binding assays are also routinely used, and FACS data correlate well with other methods such as radioligand competitive binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201(2):223-31; Geuijen, CA, et al. J Immunol Methods. 2005, 302(1-2):68-77).

[0116] Thus, the chimeric antigen receptor or corresponding antibody of the present invention has a K that is at least 10-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein). D The parent corresponding to the value According to the present invention, the antibody binds to a specific antigen or cell surface molecule with which it has affinity. D The affinity of the chimeric antigen receptor or the corresponding antibody having a value , which may be considered as undetectable binding.

[0117] The term “K D " (M) is the dissociation equilibrium constant of a particular antigen-binding domain:antigen interaction, K is the dissociation equilibrium constant of an antibody against an antigen. DThere is an inverse relationship between the binding affinity and Therefore, K D The smaller the value, the higher or stronger the affinity. The terms “higher affinity” or “stronger affinity” refer to a higher ability to form an interaction, i.e., a smaller K D Conversely, "lower affinity" or "weaker affinity" The term "ability" refers to a lower ability to form interactions, i.e., a larger K D Value In some situations, a higher binding affinity (or K) of a particular molecule (e.g., a chimeric antigen receptor or a corresponding antibody) for its interaction partner molecule (e.g., antigen X) compared to the binding affinity of the molecule (e.g., a chimeric antigen receptor or a corresponding antibody) for another interaction partner molecule (e.g., antigen Y) can be used. D ) is larger than K D The smaller the K value (lower, or weaker, affinity), the D (higher, or stronger, affinity) by dividing by The binding affinity is expressed as a binding ratio determined by, for example, a 5-fold or 10-fold higher binding affinity, as the case may be.

[0118] The term "k" d " (sec-1 or 1 / s) is a specific antigen-binding domain:antigen interaction or the dissociation rate constant of the chimeric antigen receptor or the corresponding antibody. Its value is k off Also called value.

[0119] The term "k" a " (M-1 × sec-1 or 1 / M) is the specific antigen-binding domain: antigen It refers to the association rate constant of the interaction, or the association rate constant of the chimeric antigen receptor or the corresponding antibody.

[0120] "K A The term "M-1 or 1 / M" refers to a specific antigen-binding domain:antigen interaction. The association equilibrium constant for the chimeric antigen receptor or the corresponding antibody is k a k d It is obtained by dividing by.

[0121] "EC50" or "EC 50 The term "half-maximal effective concentration" refers to the concentration of chimeric antigen receptor that induces a response halfway between the baseline and maximum after a particular exposure time. EC 50 essentially represents the concentration of chimeric antigen receptor at which 50% of its maximal effect is observed. In certain embodiments, the EC 50 The EC value is equal to the concentration of the chimeric antigen receptor of the present invention or the corresponding antibody that confers half-maximal binding to cells expressing an antigen (e.g., a tumor-associated antigen such as BCMA) as determined, for example, by a FACS binding assay. Thus, reduced or weak binding is an EC 50 An increase in the concentration of 100 mg / kg / day or half-maximal effective concentration values ​​is observed.

[0122] In one embodiment, the reduction in binding is an EC2 concentration that allows half-maximal binding to target cells. 50 This can be defined as an increase in chimeric antigen receptor or corresponding antibody concentration.

[0123] Chimeric antigen receptor sequence variants The chimeric antigen receptors herein may contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the individual antigen-binding domains of the corresponding antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The chimeric antigen receptors of the present invention may contain antigen-binding domains derived from any of the exemplary CDR or variable region amino acid sequences disclosed herein, where one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the corresponding antibody was derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily generate a large number of antibodies containing one or more individual germline mutations or combinations thereof. In certain embodiments, the V H Domain and / or V L Frequent in the domain In other embodiments, all framework and / or CDR residues are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., the mutated residue is found within the first 8 amino acids of FR1, or the mutated residue is found within the last 8 amino acids of FR4, or the mutated residue is found only in CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antigen-binding domain was originally derived). Furthermore, the antigen-binding domain may contain any combination of two or more germline mutations in the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residues in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues in a different germline sequence.

[0124] Biological properties of chimeric antigen receptors and corresponding antibodies The present invention provides antibodies that have high affinity (e.g., subnanomolar K D Binds to human BCMA with The present invention includes a chimeric antigen receptor having an antigen-binding domain derived from an antibody that binds the antigen.

[0125] According to certain embodiments, the present invention provides a method for the preparation of a medicament having a K of less than about 5 nM as measured by surface plasmon resonance. D A corresponding antibody that binds to human BCMA (e.g., at 25° C.) having In certain embodiments, the corresponding antibody has a K of less than about 20 nM, less than about 10 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 800 pM, less than about 700 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, or less than about 25 pM, as measured by surface plasmon resonance. D It binds to BCMA having the

[0126] The present invention also provides compounds having a dissociation half-life (t) of greater than about 10 minutes or greater than about 125 minutes, as measured by surface plasmon resonance at 25° C. 1 / 2 ) due to the corresponding antibody that binds to BCMA. In certain embodiments, the corresponding antibody has a t of greater than about 3 minutes, greater than about 4 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 110 minutes, or greater than about 120 minutes as measured by surface plasmon resonance at 25° C. 1 / 2 It binds to BCMA having the

[0127] The present invention also includes chimeric antigen receptors having an antigen-binding domain derived from a corresponding antibody that specifically binds to a human cell line expressing endogenous BCMA (e.g., NCI-H929, MOLP-8, or OMP-2) as determined by a FACS binding assay.

[0128] The present invention also includes engineered cells expressing a BCMA-specific chimeric antigen receptor that (i) are activated by cells expressing BCMA and / or (ii) exhibit inhibition of tumor growth in immunodeficient mice bearing human multiple myeloma xenografts.

[0129] Preparation of antigen-binding domains The antigen-binding domain of the chimeric antigen receptor of the present invention, specific for a particular antigen (e.g., BCMA), can be prepared by any antibody production technique known in the art. In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the corresponding antibody of the present invention are derived from a chimeric, humanized or fully human antibody. Methods for producing such antibodies are well known in the art. For example, one or more of the heavy and / or light chains can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody generation technology), high affinity chimeric antibodies against a particular antigen (e.g., BCMA) are first isolated with human variable regions and mouse constant regions. The antibodies are characterized and selected for desirable properties including affinity, selectivity, epitope, etc. As discussed herein, these human variable regions (or CDRs) can then be incorporated into the antigen binding domain of a chimeric antigen receptor.

[0130] Polynucleotides and Vectors The present invention also relates to polynucleotides and vectors encoding the chimeric antigen receptors discussed herein.

[0131] In various embodiments, the polynucleotide may comprise an expression cassette or expression vector (e.g., a plasmid for introduction into a bacterial host cell, or a viral vector such as a baculovirus vector for transfection of an insect host cell, or a plasmid or viral vector such as a lentivirus for transfection of a mammalian host cell).

[0132] In various embodiments, the polynucleotide and / or vector comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, or SEQ ID NO:89. In various embodiments, the polynucleotide and / or vector comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, or SEQ ID NO:90. In various embodiments, the polynucleotide and / or vector comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, or SEQ ID NO:100.

[0133] Methods for engineering immune cells to express chimeric antigen receptors The invention encompasses methods of preparing immune cells for immunotherapy comprising introducing into such immune cells ex vivo a polynucleotide or vector encoding one of the BCMA-specific chimeric antigen receptors described herein.

[0134] The present invention also encompasses immune cells comprising a polynucleotide or lentiviral vector encoding one of the BCMA-specific chimeric antigen receptors described herein. In some embodiments, these immune cells are used in immunotherapy (e.g., for the treatment of cancer).

[0135] The present invention also encompasses methods of genetically modifying immune cells to make them more suitable for allogeneic transplantation. According to a first aspect, for example, immune cells can be made allogeneic by inactivating at least one gene expressing one or more components of the T cell receptor (TCR) as described in WO2013 / 176915, which can be combined with the inactivation of genes encoding or regulating protein expression of HLA or β2m. Thus, the risk of graft-versus-host syndrome and graft rejection is greatly reduced. According to a further aspect of the present invention, immune cells can be further engineered to be more activated or limited attrition by inactivating genes encoding proteins that act as "immune checkpoints" that act as regulators of T cell activation, such as PD1 or CTLA-4.

[0136] Engineered immune cells An immune cell (or engineered immune cell) comprising the chimeric antigen receptor of the invention is another object of the invention. Optionally, the immune cell is an immune effector cell. Optionally, the immune cell is a T cell. Optionally, the immune cell is a T cell selected from an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte. In some cases, the immune cell is a CD8+ cytotoxic T lymphocyte.

[0137] In some embodiments, the engineered immune cells are human T cells that comprise a chimeric antigen receptor that comprises, from N-terminus to C-terminus, (a) an extracellular ligand binding domain comprising an anti-BCMA single chain variable fragment (scFv) domain comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), (b) a hinge, (c) a transmembrane domain, and (d) a cytoplasmic domain comprising a costimulatory domain and a signaling domain.

[0138] In some embodiments, the scFv domain of the engineered human T cell comprises an LCVR / HCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO: 10 / 2, 26 / 18, 42 / 34, 58 / 50, or 74 / 66. Optionally, the hinge comprises the amino acid sequence of SEQ ID NO: 97. Optionally, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 98. Optionally, the costimulatory domain is a 4-1BB costimulatory domain. Optionally, the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 99. Optionally, the signaling domain is a CD3 zeta signaling domain. Optionally, the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 100.

[0139] In various embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, or SEQ ID NO:90.

[0140] biological equivalent The invention encompasses chimeric antigen receptors and engineered cells expressing chimeric antigen receptors that have amino acid sequences that differ from those of the exemplary molecules disclosed herein but that retain the ability to bind to BCMA, activate immune cells expressing the chimeric antigen receptor in the presence of cells expressing BCMA, or inhibit the growth or proliferation of tumor cells expressing BCMA. Such variant antibodies contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit biological activity that is essentially equivalent to the biological activity of the bispecific antigen binding molecules described.

[0141] In one embodiment, two engineered immune cells expressing a chimeric antigen receptor of the present invention are biologically equivalent if there are no clinically significant differences in their safety, purity, and efficacy.

[0142] In one embodiment, the two engineered immune cells are bioequivalent if a patient can be switched one or more times compared to therapy sustained without switching between the reference product and the biological product without an expected increase in risk of adverse effects, including clinically significant changes in immunogenicity or diminished efficacy.

[0143] In one embodiment, two engineered immune cells are biologically equivalent if they both act by a common mechanism or mode of action for a condition or condition of use, to the extent such mechanism is known.

[0144] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo tests in humans or other mammals where the concentration of engineered cells is measured as a function of time in blood, plasma, serum or other biological fluids, (b) in vitro tests that correlate with and are reasonably predictive of human bioavailability data, (c) in vivo tests in humans or other mammals where the relevant acute pharmacological effects of the engineered cells (or their targets) are measured as a function of time, and (d) in vivo tests in humans or other mammals where the safety, efficacy, or bioavailability or bioequivalence of the engineered cells is established. These include well-controlled clinical trials that establish

[0145] Biologically equivalent variants of the exemplary engineered cells shown herein can be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity.

[0146] Species selectivity and species cross-reactivity According to certain embodiments of the invention there is provided an antigen binding domain which binds to human BCMA but not to BCMA from other species. The invention also includes antigen binding domains which bind to human BCMA and to BCMA from one or more non-human species.

[0147] According to certain exemplary embodiments of the present invention, there is provided an antigen binding domain which binds to human BCMA and optionally may or may not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey or chimpanzee BCMA.

[0148] Activation and expansion of engineered immune cells Whether prior to or after genetic modification of the engineered cells (e.g., T cells), the immune cells, particularly the T cells of the present invention, can be activated and proliferated independently of the antigen binding mechanism, as described, for example, in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,889,470; 6,906,680; 6,906,681; 6,906,682; 6,906,683; 6,906,684; 6,906,685; 6,906,686; 6,906,687 ... T cells can generally be further activated and expanded using methods such as those described in U.S. Patent Application Publication No. 2006 / 0121005. T cells can be expanded in vitro or in vivo.

[0149] Generally, the T cells of the present invention are expanded by contacting with an agent that stimulates the CD3TCR complex and the costimulatory molecules on the surface of the T cells to generate an activation signal for the T cells. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to generate an activation signal for the T cells.

[0150] As a non-limiting example, a population of T cells can be stimulated in vitro, such as by contact with an anti-CD3 antibody, or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. Co-stimulation of accessory molecules on the surface of T cells uses a ligand that binds to the accessory molecule. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under appropriate conditions to stimulate proliferation of the T cells. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo5, (Lonza)), which may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, 1L-4, 1L-7, GM-CSF, IL-10, IL-2, 1L-15, TGFp, and TNF-α, or any other additives for the growth of cells known to those skilled in the art. Other additives for cell growth include detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanoi. Media may include, but are not limited to, RPMI1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo1, and X-Vivo20, Optimizer, serum-free or supplemented with an appropriate amount of serum (or plasma), or a defined set of hormones, and / or cytokine(s) in sufficient amounts for T cell growth and expansion, supplemented with amino acids, sodium pyruvate, and vitamins. Antibiotics such as penicillin and streptomycin are included only in the experimental cultures, not in the cultures of cells injected into the subject. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air+5% O2). T cells exposed to various stimulation times may exhibit different properties .

[0151] In another specific embodiment, the cells can be expanded by co-culturing with tissue or cells. The cells can also be expanded in vivo, for example, in the blood of a subject after administering the cells to the subject.

[0152] therapeutic use The present invention includes compositions comprising engineered cells (e.g., T cells) expressing the chimeric antigen receptor of the present invention and a pharma- ceutically acceptable vehicle. In some cases, the engineered cells form a drug, particularly for immunotherapy. In some cases, the engineered cells are used to treat cancer (e.g., multiple myeloma). In some cases, the engineered cells are used to manufacture a drug for immunotherapy and / or for the treatment of cancer (e.g., cancer expressing BCMA).

[0153] The present invention includes a method comprising administering to a subject in need thereof a therapeutic composition comprising engineered cells (e.g., T cells) expressing a chimeric antigen receptor as discussed herein. The therapeutic composition can comprise any of the chimeric antigen receptor expressing cells disclosed herein and a pharma- ceutically acceptable carrier, diluent or vehicle. As used herein, the phrase "subject in need thereof" refers to a human or non-human animal that exhibits one or more symptoms or signs of cancer (e.g., a subject that develops a tumor or suffers from any of the cancers described herein) or that would otherwise benefit from inhibition or reduction of BCMA activity or depletion of BCMA+ cells (e.g., multiple myeloma cells).

[0154] The engineered cells of the present invention are particularly useful for the treatment of any disease or disorder in which stimulating, activating and / or targeting immune responses is beneficial.In particular, the engineered cells of the present invention can be used to treat, prevent and / or ameliorate any disease or disorder associated with or mediated by BCMA expression or activity or proliferation of BCMA+ cells.Cells expressing BCMA that can be inhibited or killed using the engineered cells of the present invention include, for example, multiple myeloma cells.

[0155] The engineered cells of the present invention may be used to treat diseases or disorders associated with BCMA expression, including, for example, cancers including multiple myeloma or other B-cell or plasma cell cancers, such as Waldenstrom's macroglobulinemia, Burkitt's lymphoma, and diffuse large B-cell lymphoma. In some embodiments, the BCMA-expressing disease or disorder is Castleman's disease, lymphoplasmocytic lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or chronic lymphocytic leukemia. According to certain embodiments of the present invention, the engineered cells are useful for treating patients suffering from multiple myeloma. According to other related embodiments of the present invention, methods are provided that include administering the engineered cells disclosed herein to patients suffering from multiple myeloma. Analytical / diagnostic techniques known in the art, such as tumor scanning, may be used to treat multiple myeloma. The methods can be used to ascertain whether a patient has multiple myeloma or another B-cell lineage cancer.

[0156] The present invention also includes methods for treating residual cancer in a subject. As used herein, the term "residual cancer" refers to the presence or persistence of one or more cancerous cells in a subject after treatment with an anti-cancer therapy.

[0157] According to certain embodiments, the present invention provides methods for treating a disease or disorder associated with BCMA expression (e.g., multiple myeloma), comprising administering to a subject a population of engineered cells as described elsewhere herein after the subject has been determined to have multiple myeloma. For example, the present invention includes methods for treating multiple myeloma, comprising administering to a patient engineered immune cells 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more after the subject has received other immunotherapy or chemotherapy.

[0158] The treatment discussed herein can be ameliorative, therapeutic, or preventive.The treatment can be either part of autoimmunotherapy or part of allogeneic immunotherapy.Autologous means that the cell, cell line, or cell population used to treat the patient is derived from the patient or a human leukocyte antigen (HLA) compatible donor.Allogeneic means that the cell, cell line, or cell population used to treat the patient is derived from a donor, not from the patient.

[0159] The cells that can be used in the disclosed method are described herein.The treatment can be used to treat patients diagnosed with pre-malignant or malignant cancer conditions characterized by cells that express BCMA, particularly cells that express excessive BCMA.Such conditions are found in cancers such as multiple myeloma.

[0160] Cancer types that may be treated with the engineered cells of the present invention include, but are not limited to, multiple myeloma, Waldenstrom's macroglobulinemia, Burkitt's lymphoma, diffuse large B-cell lymphoma, and other B-cell or plasma cell cancers. In some embodiments, the engineered cells can be used to treat diseases or disorders that express BCMA, such as Castleman's disease, lymphoplasmacytic lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or chronic lymphocytic leukemia.

[0161] The compositions and methods of the invention can be used to treat subjects who have been characterized as having cells or tissues that express BCMA, or who are suspected of having cells or tissues that express BCMA. For example, subjects who would benefit from treatment according to the invention include subjects with multiple myeloma.

[0162] Administration of the cells or cell populations according to the present invention can be carried out in any convenient manner, including aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein can be administered to patients by subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present invention are preferably administered by intravenous injection.

[0163] The administration of cells or cell populations was at 10 / kg body weight. 4 ~10 9 Cells, preferably 10 to 10, including all integer cell numbers within these ranges. 5 ~10 6 The administration may consist of administering cells / kg of body weight. The cells or cell populations may be administered in one or more doses. In some embodiments, an effective amount of cells is administered as a single dose. In some embodiments, An effective amount of cells is administered as two or more doses over a period of time. The timing of administration is within the judgment of the supervising physician and depends on the clinical condition of the patient. The cells or population of cells can be obtained from any source, such as a blood bank or a donor. Although individual needs vary, the determination of the range of effective amounts of a given cell type for a particular disease or condition is within the skill of one of ordinary skill in the art. By effective amount is meant an amount that provides a therapeutic or prophylactic benefit. The dosage administered will depend on the age, health and weight of the recipient, the type of concurrent treatment, if any, the frequency of treatment, and the nature of the effect desired.

[0164] In one embodiment, an effective amount of the cells or a composition comprising these cells is administered parenterally. This administration can be intravenous. In some cases, administration can be by injection directly into a tumor.

[0165] In certain embodiments of the invention, the cells are administered to the patient in conjunction with (e.g., before, simultaneously, or after) any number of applicable treatment modalities, including, but not limited to, antiviral therapy, treatment with agents such as cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients, or efalizumab treatment for psoriasis patients, or other treatments for PML patients, etc. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarivine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation.

[0166] In further embodiments, the cell composition of the present invention is administered to the patient in conjunction with (e.g., before, simultaneously, or after) T cell ablative therapy using either bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH; in another embodiment, the cell composition of the present invention is administered after B cell ablative therapy, such as agents that react with CD20, e.g., Rituxan. For example, in one embodiment, the subject can receive standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In certain embodiments, after transplant, the subject receives an infusion of the expanded immune cells of the present invention. In additional embodiments, the expanded cells are administered before or after surgery. In certain embodiments, any means (e.g., surgery, chemotherapy, or radiation therapy) can be used to reduce tumor burden prior to administration of the expanded immune cells of the present invention. In one embodiment, reducing tumor burden prior to administration of the engineered cells of the present invention can reduce or prevent the possibility of cytokine release syndrome or cytokine storm, a side effect that can be associated with CAR T cell therapy.

[0167] Combination therapy The present invention provides methods that include administering an engineered cell or population of cells that includes any of the chimeric antigen receptors described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that can be administered in combination with or in combination with the cells or population of cells of the present invention include, for example, anti-tumor agents (e.g., chemotherapy agents including melphalan, vincristine (Oncovin), cyclophosphamide (Cytoxan), etoposide (VP-16), doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), obendamustine (Treanda), or any other known to be effective in treating plasma cell neoplasms in a subject). In some embodiments, the second therapeutic agent includes a steroid. In some embodiments, the second therapeutic agent includes a targeted therapy including thalidomide, lenalidomide, and bortezomib, which are therapies approved to treat newly diagnosed patients. Lenalidomide, pomalidomide, and bortezomib are also used. , bortezomib, carfilzomib, panobinostat, ixazomib, elotuzumab, and daratumumab are examples of second therapeutic agents effective for treating relapsed myeloma. In certain embodiments, the second therapeutic agent is a regimen including radiation therapy or stem cell transplantation. In certain embodiments, the second therapeutic agent can be an immunomodulatory agent. In certain embodiments, the second therapeutic agent can be a proteasome inhibitor, including bortezomib (Velcade), carfilzomib (Kyprolis), ixazomib (Ninlaro). In certain embodiments, the second therapeutic agent can be a histone deacetylase inhibitor, such as panobinostat (Farydak). In certain embodiments, the second therapeutic agent can be a monoclonal antibody, an antibody drug conjugate, a bispecific antibody conjugated to an antitumor agent, a checkpoint inhibitor, or a combination thereof. Other agents that may be beneficially administered in combination with the antigen-binding molecules of the present invention include small molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or cytokine inhibitors that include their respective receptors. The pharmaceutical compositions of the present invention (e.g., pharmaceutical compositions comprising engineered cells or populations of cells as disclosed herein) may also be administered as part of a therapeutic regimen that includes one or more therapeutic combinations selected from monoclonal antibodies other than those described herein that can interact with different antigens on the surface of plasma cells, bispecific antibodies having one arm that binds to an antigen on the surface of tumor cells and the other arm that binds to an antigen on T cells, antibody drug conjugates, bispecific antibodies conjugated to anti-tumor agents, checkpoint inhibitors, such as those that target PD-1 or CTLA-4, or combinations thereof. In certain embodiments, the checkpoint inhibitor may be selected from a PD-1 inhibitor such as pembrolizumab (Keytruda), nivolumab (Opdivo), or cemiplimab (REGN2810).In certain embodiments, the checkpoint inhibitor may be selected from a PD-L1 inhibitor, such as atezolizumab (Tecentriq), avelumab (Bavencio), or durvalumab (Imfinzi). In certain embodiments, the checkpoint inhibitor may be selected from a CTLA-4 inhibitor, such as ipilimumab (Yervoy).

[0168] The invention also includes therapeutic combinations comprising any of the engineered cells or populations of cells described herein and one or more inhibitors of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the foregoing cytokines, wherein the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptibody, nanobody, or antibody fragment (e.g., Fab fragment, F(ab')2 fragment, Fd fragment, Fv fragment, Fv β ... fragments, scFv, dAb fragments, or diabodies, triabodies, tetrabodies, minibodies, and minimal recognition units). In some embodiments, the engineered cells or populations of cells of the present invention may also be administered as part of a treatment regimen that also includes radiation therapy and / or conventional chemotherapy.

[0169] The additional therapeutically active ingredient(s) may be administered immediately prior to, simultaneously with, or immediately following administration of the engineered cells of the invention (for purposes of this disclosure, such a dosing regimen will be considered as administering the engineered cells "in combination" with the additional therapeutically active ingredient).

[0170] The present invention includes pharmaceutical compositions in which the engineered cells of the invention are co-formulated with one or more additional therapeutically active ingredient(s) as described elsewhere herein.

[0171] Dosing regimen According to certain embodiments of the invention, multiple doses of engineered cells can be administered to a subject over a defined time course. The method according to this aspect of the invention includes sequentially administering multiple doses of cells to a subject. As used herein, "sequentially administering" means that each dose is administered to a subject at different times, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The invention includes methods that include sequentially administering a single primary dose to a patient, followed by one or more secondary doses, and then optionally one or more tertiary doses.

[0172] The terms "primary dose", "secondary dose", and "tertiary dose" refer to the time sequence of administration of the engineered cells of the present invention. Thus, a "primary dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the primary dose, and a "tertiary dose" is a dose administered after the secondary dose. The primary, secondary, and tertiary doses may all contain the same amount of engineered cells, but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of engineered cells contained in the primary, secondary, and / or tertiary doses differ from each other (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more doses (e.g., two, three, four, or five) are administered as a "loading dose" at the beginning of a treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis.

[0173] In certain exemplary embodiments of the invention, each secondary and / or tertiary dose is 1 to 26 (e.g., 1, 1 1 / 2 , 2, 2 1 / 2 , 3, 3 1 / 2 , 4, 4 1 / 2 , 5, 5 1 / 2 , 6 , 6 1 / 2 , 7, 7 1 / 2 , 8, 8 1 / 2 , 9, 9 1 / 2 , 10, 101 / 2 , 11, 11 1 / 2 , 12, 12 1 / 2 , 13, 13 1 / 2 , 14, 14 1 / 2 , 15, 15 1 / 2 , 16, 16 1 / 2 , 17, 17 1 / 2 , 18, 18 1 / 2 , 19, 19 1 / 2 , 20, 20 1 / 2 , 21, 21 1 / 2 , 22, 22 1 / 2 , 2 3, 23 1 / 2 , 24, 24 1 / 2 , 25, 25 1 / 2 , 26, 26 1 / 2 The phrase "immediately preceding dose" as used herein means a dose administered to a patient prior to administration of an immediately following dose in a series of multiple doses, with no intervening doses.

[0174] The method according to this aspect of the invention may include administering any number of secondary and / or tertiary doses to the patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight or more) tertiary doses are administered to the patient.

[0175] In embodiments involving multiple secondary doses, each secondary dose may be administered with the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered with the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The frequency of administration may also be adjusted by the physician during the course of treatment depending on the needs of the individual patient after clinical testing. EXAMPLES

[0176] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as the invention. While efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), some experimental Errors and deviations should be considered. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0177] Example 1: Generation of anti-BCMA antibodies Anti-BCMA antibodies were obtained by immunizing genetically modified mice with human BCMA antigen (e.g., hBCMA, SEQ ID NO: 101) or by immunizing engineered mice containing DNA encoding human immunoglobulin heavy chain and kappa light chain variable regions with human BCMA antigen.

[0178] Following immunization, splenocytes were harvested from each mouse and either (1) fused with mouse myeloma cells to maintain their viability and form hybridoma cells to screen for BCMA specificity, or (2) sorted for B cells using human BCMA fragments as sorting reagents that bind and identify reactive antibodies (antigen-positive B cells) (as described in US2007 / 0280945A1).

[0179] Chimeric antibodies against BCMA with human variable regions and mouse constant regions were first isolated. The antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, etc. If necessary, the mouse constant regions are replaced with the desired human constant regions, e.g., wild-type or modified IgG1 or IgG4 constant regions, to generate fully human anti-BCMA antibodies. The constant regions selected can vary depending on the specific application, but the high affinity antigen binding and target specificity characteristics reside in the variable regions.

[0180] Amino acid and nucleic acid sequences of the heavy and light chain variable regions of anti-BCMA antibodies: Table 1 shows the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-BCMA antibodies of the invention. The corresponding nucleic acid sequence identifiers are shown in Table 2. [Table 1] [Table 2]

[0181] Example 2: Generation of BCMA-specific chimeric antigen receptors Six anti-BCMA antibodies (mAb16711, mAb16716, mAb16732, mAb16747 and mAb21581) were synthesized using VL-VH single-chain variable fragment (ScF v) and placed into the chimeric antigen receptor (CAR) construct used for CD8α hinge and transmembrane domain, 4-1BB costimulatory domain and CD3ζ stimulatory domain. BCMA-specific CAR was cloned into (Lenti-X™ Bicistronic Expression System (Neo), Clontech Cat. No. 632181) and lentiviral particles were generated via the Lenti-X Packaging Single-Shot (VSV-G) system (Clontech Cat. No. 631276) according to the manufacturer's protocol. Jurkat cells engineered to express an NFAT-luciferase reporter (Jurkat / NFATLuc.3C7) were then transduced with the different CAR constructs according to the manufacturer's protocol using RetroNectin® pre-coated dishes (Clontech, Cat. No. T110a). After at least 2 weeks of selection in 500 μg / ml G418 (Gibco, catalog no. 11811-098), the following CAR-T cell lines were generated; Jurkat / NFATLuc cl.3C7 / BCMA 16716 VL-VH CART, Jurkat / NFATLuc cl.3C7 / BCMA 16711 VL-VH CART, Jurkat / NFATLuc cl.3C7 / BCMA 16732VL-VH CART, Jurkat / NFATLuc cl.3C7 / BCMA 16747VL-VH CART, Jurkat / NFATLuc cl.3C7 / BCMA 21581VL-VH CART. The nucleotide sequences of the CAR constructs used to generate these CAR-T cell lines are shown in SEQ ID NO:81 (mAb16711 VL / VH), SEQ ID NO:83 (mAb16716 VL / VH), SEQ ID NO:85 (mAb16732 VL / VH), SEQ ID NO:87 (mAb16747 VL / VH) and SEQ ID NO:89 (mAb21581 VL / VH), as shown in Figure 1. These six CAR-T cell lines were used to evaluate cell surface expression and activation of BCMA CAR-T cells, as discussed in Example 3.

[0182] A chimeric antigen receptor comprising an anti-BCMA VL-VH scFv, a huCD8 transmembrane domain, a 4-1BB costimulatory domain and a CD3ζ signaling domain was constructed using the VL and VH nucleotide sequences of two anti-BCMA antibodies, mAb21581 and mAb16747 (corresponding to SEQ ID NOs: 89 and 87, respectively). As a non-binding control, a similar CAR was designed using the nucleotide sequence of an unrelated scFv (CAR construct of SEQ ID NO: 91). These CARs were cloned into a pLVX lentiviral vector with an EF1a promoter and an IRES:eGFP sequence (to track CAR-transduced cells) and VSV-pseudotyped lentivirus was generated.

[0183] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs), stimulated with CD3 / CD28 microbeads + 100U / ml recombinant human IL-2, and transduced by lentivirus at MOI=5. Transduced cells were expanded with CD3 / CD28 microbeads + 100U / ml recombinant human IL-2 for 3 weeks and then cryopreserved until use in in vivo experiments. As discussed in Examples 4 and 5, these three lines of CAR-T cells were used to evaluate their efficacy in reducing tumor burden in vivo.

[0184] Example 3: Cell surface expression of BCMA CAR constructs in Jurkat cells and activation of BCMA CAR-T cells The relevant cell surface expression of BCMA CAR constructs in Jurkat / NFATLuc cells was accessed by flow cytometry. To stain, cells were plated at a density of 200,000 cells per well of a 96-well V-bottom plate in staining buffer (PBS, calcium and magnesium free (Irving 9240) + 2% FBS (ATCC 30-2020)) and stained with 10ug / ml of BCMA ectodomain fused to hIgG1-Fc (BCMA ecto-hFc) or an irrelevant protein fused to hIgG1-Fc (Fc isotype control) for 30 minutes at 4°C. After incubation with BCMA-hFc or Fc isotype control, cells were washed once with staining buffer and stained with 10 μg / ml Alexa-Flour647-conjugated secondary antibody (Jackson ImmunoResearch, Cat. No. 109-606-170) for 30 min at 4 °C. Cells were then washed and fixed using a 50% solution of BD Cytofix (BD, Cat. No. 554655) diluted in staining buffer. Samples were run on an Intellicyt iQue flow cytometer, analyzed with Flowjo 10.2, and mean fluorescence intensity (MFI) was calculated. Signal-to-noise ratio (S:N) was determined by taking the ratio of BCMA-hFc or Fc isotype control MFI to secondary antibody alone MFI.

[0185] Activation of CAR-T lines was evaluated in a CAR-T / APC (antigen presenting cell) bioassay. To perform the bioassay, 50,000 CAR-T cells were added to a Thermo-Nunc 96-well white plate (Thermo Scientific, Cat. No. 136101) in 50ul of assay medium (RPMI medium with 10% FBS and 1% P / S / G), followed by 3-fold serial dilutions of APCs (500,000 cells to 685 cells) in 50ul of assay medium. The following APCs were utilized: RAJI, Daudi, RPMI8226 (endogenously expressing BCMA) and HEK293 (BCMA negative). The cell mixture was incubated for 5 hours in a 37°C, 5% C02, humidified incubator. NFAT-luciferase activity was measured using Promega One-Glo (Cat. No. E6130) and a Perkin Elmer Envision plate reader. Generate relative luciferase units (RLU) and plot the results using a four-parameter logistic equation on the eight-point response curve using GraphPad The dose-response curves were plotted in Prism. The zero APC condition for each dose-response curve was also included in the analysis as a succession of 3-fold serial dilutions and is represented as the lowest dose. CAR-T activity was determined by taking the ratio of the highest to the lowest RLU on the curve and is represented as signal:noise (S:N) in Table 4.

[0186] Table 3 shows that 16747 and 21581 CAR-T had similar surface expression, with S:N ranging from 209 to 273, with 16716 CAR-T expressing in a background of >44-fold, whereas expression of 16732 and 16711 CAR-T was much lower, with an S:N of 13 and 4, respectively.

[0187] Table 4 shows that all six BCMA CAR-T cell lines were activated in RAJI, Daudi, and RPMI8226 cells. None of the CAR-T cell lines were activated in HEK293. The 16747BCMA CAR had the strongest activity in the CAR-T / APC bioassay, while CAR16711 had the weakest activity, regardless of BCMA-expressing APC. Finally, a correlation was observed between CAR expression (Table 3) and CAR activity (Table 4). [Table 3] [Table 4]

[0188] Example 4: BCMA-targeted CAR-T cells reduce the growth of BCMA-expressing tumors (OPM-2) in vivo in a xenogeneic tumor model To determine the in vivo efficacy of BCMA-targeted chimeric antigen receptor (CAR) T cells, xenogeneic tumor studies were performed in mice using OPM-2 human multiple myeloma cells, which express high levels of BCMA.

[0189] Xenograft tumor engraftment and measurement: On day 0, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice were cultured with 2 × 10 6 BCMAs + Mice were administered OPM-2 human multiple myeloma tumor cells intravenously. On day 21, mice were treated with either control CAR or anti-BCMA 2x10 expressing either CAR 6 T cells were injected intravenously (transduced with CAR Mice (n=5 per group) were injected with 2x10 6 Irrelevant scFc CAR T (control sc Fv CAR), 2x10 encoding 21581scFv CAR 6 Anti-BCMA CA RT, or 2x10 encoding 16747scFv 6 Anti-BCMA CAR T Tumor growth was assessed over a 61-day period by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, a group of mice (n=5) received only OPM-2-luciferase cells and no T cells. To measure background BLI levels, a group of mice (n=5) was left untreated and did not receive tumor or T cells.

[0190] Measurement of heterogeneous tumor growth: Tumor burden was measured using BLI imaging. Mice were injected IP with 150 mg / kg of the luciferase substrate D-luciferin suspended in PBS. Five minutes after this injection, BLI imaging of mice was performed under isoflurane anesthesia using a Xenogen IVIS system. Image collection was performed with a field of view at D, a subject height of 1.5 cm, and medium binning level with automatic exposure time determined by the Living Image software. BLI signal was extracted using Living Image software: regions of interest were drawn around each tumor volume and photon intensity was recorded as p / s / cm2 / sr.

[0191] BCMA + OPM-2-luciferase tumors were treated with unrelated scFv CAR T cells While tumors grew progressively in the receiving mice, CAR T cells encoding the 21581scFv CAR reduced the tumor burden to background levels in the majority of animals, and CAR T cells encoding the 16747scFv CAR reduced the tumor burden to background levels in the majority of animals. The results are shown in Table 5a below. [Table 5] [Table 6]

[0192] Mice were cultured with 2x10 6 T thin Further experiments were performed as discussed above, except that cells were injected intravenously on day 22 (instead of day 21) and tumor growth was assessed over 56 days (instead of day 61).

[0193] BCMA + OPM-2-luciferase tumors were treated with unrelated scFv CAR T cells While tumors grew progressively in receiving mice, CAR T cells encoding 21581 and 16747scFv CARs reduced tumor burden to background levels in the majority of animals. The results are shown in Table 5b below. [Table 7] [Table 8]

[0194] Example 5: BCMA-targeted CAR-T cells reduce the growth of BCMA-expressing tumors (MOLP-8) in vivo in a xenogeneic tumor model To determine the in vivo efficacy of BCMA-targeted chimeric antigen receptor (CAR) T cells, xenogeneic tumor studies were performed in mice using MOLP-8 human multiple myeloma cells, which express low levels of BCMA.

[0195] Xenograft tumor engraftment and measurement: On day 0, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice were cultured with 2 × 10 6 BCMAs + Mice were administered intravenously with MOLP-8 human multiple myeloma tumor cells. On day 12, mice were administered 2x10 6 T cells were injected intravenously (transduced with CAR) Mice (n=5 per group) were cultured with 2x10 6 Irrelevant scFv CAR T (control scFv CAR), 2x10 encoding 21581 scFv CAR 6 anti-BCMA CAR T, or 2x10 encoding 16747scFv 6 Anti-BCMA CAR T Tumor growth was assessed throughout the experiment by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, a group of mice (n=5) received only MOLP-8-luciferase cells and no T cells. To measure background BLI levels, a group of mice (n=5) was left untreated and did not receive tumor or T cells.

[0196] Measurement of heterogeneous tumor growth: Tumor burden was measured using BLI imaging. Mice were injected IP with 150 mg / kg of the luciferase substrate D-luciferin suspended in PBS. Five minutes after this injection, BLI imaging of mice was performed under isoflurane anesthesia using a Xenogen IVIS system. Image collection was performed with a field of view at D, a subject height of 1.5 cm, and medium binning level with automatic exposure time determined by the Living Image software. BLI signal was extracted using Living Image software: regions of interest were drawn around each tumor volume and photon intensity was recorded as p / s / cm2 / sr.

[0197] BCMA + MOLP-8-luciferase tumors were treated with unrelated scFv CAR T cells While tumor burden grew progressively in mice receiving 21581scFv CAR and 16747scFv CAR, CAR T cells encoding 21581scFv CAR and 16747scFv CAR reduced tumor burden to background levels in the majority of animals. The results are shown in Table 6 below. [Table 9] [Table 10]

[0198] Example 6: BCMA-specific CAR-T cells mediate cytolysis of cells expressing BCMA As discussed above in Example 2, CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs), stimulated with CD3 / CD28 microbeads + 100U / ml recombinant human IL-2, and transduced with lentivirus at an MOI of 5. Transduced cells were expanded in CD3 / CD28 microbeads + 100U / ml recombinant human IL-2 for 3 weeks before setting up a cytolytic assay.

[0199] To determine the cytolytic potential of BCMA-targeted chimeric antigen receptor (CAR) T cells, cytolytic assays were performed using expanded CAR-T cells and various tumor target cell lines expressing various levels of BCMA. On day 21 of expansion, expanded CAR-T cells were co-cultured with calcein-labeled BCMA+ target cell lines in triplicate at various ratios. Each target cell line was harvested and cultured at 2x10 6 After resuspension at a density of 1 / mL, calcein-AM dye was added at a concentration of 8uM for 3 min. Calcein was added for 35 min at 37 °C. After calcein labeling, the target cells were washed twice to remove excess calcein. T cells and target cells were then co-cultured at various ratios on 96-well round-bottom plates and incubated for 2.5 h at 37 °C when culture supernatants were harvested. For negative controls, target cells were co-cultured with T cells generated using a similar CAR designed to contain an irrelevant scFv that does not recognize BCMA. As an additional CAR negative control, non-transduced and expanded T cells from the same normal healthy donor were used. As a control for antigen-specific CAR-T cells that mediated killing, the chronic myeloid leukemia K562 target cell line was used because it is negative for BCMA expression. To determine whether calcein was spontaneously released from the H-929 and MOLP-8 target cell lines, each cell line was cultured in the absence of CAR-T cells. To determine the maximum possible release of calcein, target cell lines were cultured and lysed using Optmizer medium supplemented to contain 1% Triton™ X-114 detergent. Relative calcein levels were measured in the supernatants using a Viktor X4 plate reader, and percent cytotoxicity was calculated as ((calcein signal-spontaneous calcein release) / (maximum calcein release-spontaneous calcein release))*100.

[0200] As shown in Tables 7A-7C below, 21581 and 16747 scFv were used to produce Cultures consisting of engineered BCMA-targeted CAR+ T cells induced potent cytolysis of H-929 and MOLP-8 target cells. A lower level of cytotoxicity was observed against MOLP-8 cells compared to H-929 cells. This result is explained by H-929 expressing higher levels of BCMA antigen than MOLP-8 cells. For each BCMA-targeted CAR-T cell culture, the greatest cytotoxicity was observed against H-929 cells, and BCMA-targeted CAR-T cells engineered with 16747scFv showed the greatest cytotoxicity against both target cell lines. Both untransduced and expanded (MOI 0) T cells and irrelevant CAR-T cells (17363) did not induce cytolysis of MOLP-8 and H-929 target cells when co-cultured with target cells at a maximum ratio of 50 T cells to 1 target cell. The results indicate that cell lysis is only observed when the CAR construct contains an scFv that recognizes BCMA (e.g., from mAb21581 and mAb16747). Furthermore, BCMA-targeted CAR-T cells exhibited negligible cytotoxicity against K562 cells that lack BCMA expression, indicating that BCMA expression is required to observe cell lysis. [Table 11] [Table 12] [Table 13]

[0201] Example 7: Ex vivo cytotoxicity of BCMA-targeted CAR-T cells in bone marrow from multiple myeloma patients As discussed above in Example 2, CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs), stimulated with CD3 / CD28 microbeads + 100U / ml recombinant human IL-2, and transduced with lentivirus at an MOI of 5. Cells were expanded with CD3 / CD28 microbeads plus 100 U / ml recombinant human IL-2 for 3 weeks before setting up a cytotoxicity assay.

[0202] Upon harvest, the expanding CAR-T cells were washed and resuspended in complete medium (RPMI supplemented with 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin and 292 μg / mL L-glutamine). Bone marrow from multiple myeloma patients was thawed and resuspended in complete medium. HS-5 stromal cells were seeded at 10,000 cells per well in 96-well flat-bottom plates and incubated overnight. T cells and patient-derived bone marrow were added to the stroma-containing wells at various E:T ratios (two-fold titration starting at E:T=10:1) and cultured for 12 hours at 37°C. As CAR negative controls, non-transduced and expanded T cells from the same normal healthy donor were used.

[0203] After 12 hours, the viability of multiple myeloma blasts was determined using flow cytometry. Cells were stained with a cocktail of fluorophore-conjugated antibodies (anti-CD4, anti-CD8, anti-CD16, anti-CD45, anti-CD90, anti-CD138, and anti-SlamF7) in BD Horizon Brilliant staining buffer at 4 °C for 30 min. Cells were washed once with PBS and stained with LIVE / DEAD Fixable dead cell stain for 20–30 min at 4 °C, then washed twice with PBS and resuspended in cold Miltenyi AutoMacs buffer. CountBright beads were added to samples to quantify absolute cell numbers per well. Samples were analyzed on a BD FortessaX20 flow cytometer. Viable multiple myeloma blasts were gated as live single CD4- / CD8- / SlamF7+ / CD138+. Viability was calculated as the absolute number of viable multiple myeloma blasts in treated samples normalized to the viable multiple myeloma cells in untreated controls.

[0204] Cultures consisting of BCMA-targeted CAR+ T cells generated using mAb21581 VH / VL induced potent target-specific cytolysis of multiple myeloma blasts from two newly diagnosed and one relapsed patient. At an E:T ratio of 10:1, 87-94% of multiple myeloma blasts were lysed. Non-transduced and expanded (MOI 0) T cells lysed 34-0% of multiple myeloma blasts. This result demonstrates the ability of BCMA-targeted CAR+ T cells to potently lyse patient-derived multiple myeloma blasts in a target-specific manner. The results are shown in Table 8 below. [Table 14]

[0205] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications will be apparent to those skilled in the art and are intended to be included within the scope of the appended claims.

Claims

1. From N-terminus to C-terminus: (a) an extracellular ligand-binding domain comprising an anti-BCMA antigen-binding domain; (b) CD8-derived hinge; (c) a transmembrane domain derived from CD8; and (d) a cytoplasmic domain comprising a costimulatory domain derived from 4-1BB and a signaling domain derived from CD3 zeta; A B-cell maturation antigen (BCMA)-specific chimeric antigen receptor comprising: the extracellular ligand binding domain is an anti-BCMA single chain variable fragment (scFv) domain comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR) joined by a linker; The specific chimeric antigen receptor, wherein the LCVR comprises LCDR1-LCDR2-LCDR3 domains set forth in SEQ ID NOs: 76-78-80, respectively, and the HCVR comprises HCDR1-HCDR2-HCDR3 domains set forth in SEQ ID NOs: 68-70-72, respectively.

2. The chimeric antigen receptor of claim 1, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 93-96.

3. The chimeric antigen receptor of claim 1 or 2, wherein the LCVR comprises the amino acid sequence of SEQ ID NO: 74 and the HCVR comprises the amino acid sequence of SEQ ID NO:

66.

4. (a) the hinge comprises the amino acid sequence of SEQ ID NO:97; (b) the transmembrane domain comprises the amino acid sequence of SEQ ID NO:98; (c) the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 99; or (d) the signaling domain comprises the amino acid sequence of SEQ ID NO: 100; The chimeric antigen receptor according to any one of claims 1 to 3.

5. 2. The chimeric antigen receptor of claim 1, comprising the amino acid sequence of SEQ ID NO:

90.

6. An isolated nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 1 to 5.

7. 7. The isolated nucleic acid molecule of claim 6, comprising the nucleotide sequence of SEQ ID NO:

89.

8. A vector comprising the nucleic acid molecule of claim 6 or 7.

9. The vector of claim 8 , wherein the vector is a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

10. A cell comprising a nucleic acid molecule according to claim 6 or 7, or a vector according to claim 8 or 9.

11. The cell of claim 10 which is a human T cell.

12. An engineered cell comprising the chimeric antigen receptor of any one of claims 1 to 5.

13. (a) the engineered cell is an immune cell; (b) the engineered cell is an immune cell, and the immune cell is an immune effector cell; (c) the engineered cell is a T lymphocyte; or (d) the engineered cell of claim 12, wherein the engineered cell is a T lymphocyte, the T lymphocyte being an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte.

14. CD8 ＋ The engineered cell of claim 13, which is a cytotoxic T lymphocyte.

15. (a) providing a population of immune cells obtained from a subject; (b) introducing into said immune cells a nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 1 to 5; (c) culturing the immune cells under conditions to express the nucleic acid molecule; (d) isolating the immune cells expressing the chimeric antigen receptor on their cell surface; and A population of engineered cells can be obtained by:

16. (a) a genetically modified human T cell comprising the chimeric antigen receptor of any one of claims 1 to 5 and a pharma- ceutically acceptable carrier; (b) the engineered cell of claim 12 and a pharma- ceutically acceptable carrier; or (c) the engineered cell of claim 13 and a pharma- ceutically acceptable carrier; or (d) the engineered cell of claim 14 and a pharma- ceutically acceptable carrier. A pharmaceutical composition comprising one of the following:

17. 17. The pharmaceutical composition of claim 16 for treating a BCMA-expressing cancer in a subject.

18. The pharmaceutical composition of claim 17, wherein the subject is a human subject.

19. 17. The method of claim 16, wherein the BCMA-expressing cancer is multiple myeloma, B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin's lymphoma, leukemia and lymphoma, acute lymphoblastic leukemia, Hodgkin's lymphoma, or childhood acute lymphoblastic leukemia. Or the pharmaceutical composition of any one of 18.

20. 1. A method for engineering a population of cells to express a chimeric antigen receptor, comprising: (a) providing a population of immune cells; (b) introducing into said immune cells a nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 1 to 5; (c) culturing the immune cells under conditions to express the nucleic acid molecule; (d) isolating said immune cells that express said chimeric antigen receptor on their cell surface.

21. 21. The method of claim 20, further comprising obtaining the population of immune cells from a subject.

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